Critical Reviews in Biotechnology, 13(3):195-253 (1993)

Problems in Scale-Up of Biotechnology

Production Processes
Harold B. Reisman
Organogenesis Inc., 150 Dan Road, Canton, MA 02021

ABSTRACT: The unique nature of biotechnology processes adds to the complexity and difficulty of scale-up.
Successful scale-up means a shortened cycle to full-scale production, competitive advantage, and cost savings. The
many pitfalls as well as actual and potential scale-up problems are reviewed. Emphasis is placed on covering all
areas of concern in planning, executing, and documenting key studies. Needs in technology transfer are discussed
and regulatory requirements are incorporated into scale-up needs. A review of the recent literature is coupled with
actual case studies; problem avoidance is stressed. Problems in asepsis, in construction, and in validation are
discussed and potential solutions given. Organizational problems are noted. Finally, checklists are given for project
planning, for a safety audit, and for timely attainment of successful scale-up. Eighty-two references are included.
KEY WORDS: bioreactor, agitation-aeration, sterilization, asepsis, validation, cell culture, fermentation, project
planning, downstream processing, containment.

I. INTRODUCTION

4.
5.

It will never be known with certainty which

fermentation process occurred first in human history. There is ample evidence that beer, bread,
and wine were produced in quantity in ancient
Egypt - that is, over 6000 years ago. The likelihood is that a food fermentation occurred accidently and the resulting product was more appreciated than the starting material. The fermentation
could have occurred in a dairy product, in an early
kind of bread, or in an alcoholic ferment. Very
likely, preservation and/or taste were enhanced.
Because the mechanism was unknown, we can
presume that one of our progenitors tried - in a
purposeful way -to simulate that early fermentation process. It is likely, but by no means certain, that early attempts resulted in one or more of
the following:
1.

2.

3.

Loss of a desirable starting material (substrate)

Creation of a large volume of a useless
mixture
Foul odor or a foamy mess

Illness or discomfort to consumers

Argumentation and abuse for the perpetrator

Therefore, the first fermentation process was

quickly followed by the first scale-up problem in
a biotechnology process. Interestingly, the results
previously noted are not uncommon when a contemporary scale-up problem ensues. Of course,
today we can add one or more of such factors as
monetary loss, loss of business credibility, and
undesirable regulatory and legal consequences.
Scale-up and problems seem to go together.
This has been the norm. After reviewing the
many real and potential problems of scale-up,
one might legitimately assume that no biologic
process can be scaled up. Fortunately for modern man, this is not the case. Long lists of useful
fermentation products -made in bulk -can be
generated. We can list amino acids, antibiotics,
vitamins, biopolymers, monoclonals, vaccines,
enzymes, and physiologic proteins with dollar
values in the many billions of dollars annually.
Still, the path from research and discovery to
large-volume production has rarely been a direct
and easy one.

0738-855 1/93/$.50
0 1993 by CRC Press, Inc.

195

In this review, 1 do not separate the older

fermentation processes (commodities, bacterial,
fungal, yeast) and their inherent scale-up problems from the newer cell culture processes.
Although there are unique differences and unique
solutions (and some will be discussed), there are
many more similarities in the scale-up method, in
recognition of potential pitfalls, and in the planning of the successful scale-up effort. There are
many issues common to the various biotechnology processes and even if emphasis and approach
must vary, the overview concept might stimulate
an insight that compartmentalization would tend
to stifle.
Scale-up refers to any increase according to a
fixed ratio. Strictly, therefore, transfer of a process from a shake flask to a 5-1 fermentor is a
scale-up step. This level of scale-up is not free
from hazard, but the subject matter to follow
stresses scale-up where the fixed ratio may be
2000 or more. The scale-up problems reviewed
occur in the transfer of a research project or process through development into volume manufacturing, It should seem obvious to all concerned
that scale-up is a serious matter and involves
careful planning and careful execution. The fact
that scale-up is seldom simple indicates that the
obviousness is not clear to everyone who should
know better. There is no other explanation for the
many repeated problems that arise in the course
of scale-up. Enough is known about scale-up to
recognize the potential for probIems and, at the
minimum, to plan for problem-avoidance. Why is
successful scale-up so important?
One major requirement for commercial success is investment capital. A government publication has a terse summary that answers part of the
question note above:
Several factors have been cited for tightened availability of venture capital financing. Product development was slower than expected (e.g.. unforeseen technical problems, slow regulatory approval and patent
issuance, and difficulties in Scale-Up and in obtaining
meaningful clinical results).

Successful scale-up, almost by definition,

means that a process has been designed and built
giving a predictable increase in production capacity. It is understood that product potency is as

196

predicted and no unexpected entities are found in

the product. Although optimization may increase
yield and throughput before inauguration of large
scale production, it is hazardous to design in
process improvement.
If successful scale-up results in timely production of the desired quantity of product, another result is (hopefully) a process that can be
validated at production scale. Clinical quality and
quantity are achieved and appropriate documentation is available for regulatory purposes.
The short answer to the question of scale-up
importance consists of two words: time and money.
Most emerging companies are short of both commodities. Any delay or problem in scale-up means
loss of one and probably both needs. The longer
answer to the question involves theoretical and
empirical understanding of
1.

2.
3.
4.

5.

6.

7.

Transport phenomena in animal, plant, insect cells at various scales with various support media
Shear impact on these cells in mechanically
and/or gas-mixed systems
Genetic stability
Materials of construction (both synthesis and
isolation)
Isolation methods that are currently in lab or
small scale use and novel purification methods
Compound molecular configuration (tertiary
characteristics as relates to stability and
potency)
Control strategies for effective biosynthesis
and isolation

Successful scale-up means that a number of

issues in these various areas have been faced and
resolved. Even if complete understanding is not
achieved, a background for future development
has been established. Successful scale-up is necessary (but not sufficient) for either production
and widespread use of bioactive agent or commercial success. Often, successful scale-up precedes both achievements.
Why is scale-up an ongoing problem? To be
simplistic, every process involving living organisms is somewhat unique. The organism, often
highly selected and/or modified, interacts and

alters its environment in a very complex manner.

Changes in the environment -caused by a change
in scale - will cause unpredictable responses by
the organism that will further modify the environment at the next scale. Very often, these interactions cause unexpected results that are usually
economically unattractive. At times, results are
economically disastrous.
It is probably a truism to state that no complete set of chemical/physical conditions imposed
at a manufacturing scale will result in the same
environment that existed in shake flasks or
T-flasks. Therefore, an early and obvious premise
is that numerous compromises must be made in
the course of scale-up. Success or failure of scaleup depends on how close rate, yield, and purity at
the larger scale matches those results at the bench.
Because it is not possible to keep all chemical/
physical parameters constant on changing scale,
it is important to know what boundaries exist for
successful implementation. As one example there are combinations of agitation and aeration
that will give an equivalent result. As changes are
made in one or both of these input variables,

however, these parameters, among others, will

change: bulk mixing/turnover time, cost (both
initial capital investment and operating), foaming
characteristics, shear, pCO,, gas-liquid interfacial
area. An understanding of acceptable limits will
focus the scale-up effort and result in the most
cost-effective and easiest-to-operate production
facility.
In an overall review of scale-up, it is useful to
have some grasp of the qualitative aspects of
microbial systems involved. Although no single
table can cover the spectrum of microbial systems, general concepts can be given as a guide to
scale-up considerations. As can be seen in Table 1,
there is no overall rule or plan for scale-up that is
applicable across the board. In general, pure bacterial and fungal systems (excluding recombinant
cells for the moment) have been in use for many
decades. Sterility control, inoculum development,
strain improvement, and control strategies are
fairly well understood. Successful scale-up has
occurred. These aspects of biotechnology development and scale-up are less clear in the area of
animal and plant cell biosynthesis. Further, novelty

TABLE 1

Characteristics of Microbial and Cell Culture Systems

Bacteria

Moldlfungi

AnimaVplant cells

Doubling time
Viscosity
Medium cost

Shortest
Normally low
Low

Slowest (days)
Low
High

Shear resistance
Cost of downstream
processing
Culturing
Product concentration
Aggregation

High
Modest
(5050)
Suspension
High
Nil to low

Intermediate
Often a problem
Low to
intermediate
Often high
Modest

Product value
Cell density

Low to
intermediate
Can be very high

Clean steamMlFl
Contamination
Genetic stability

Almost never
Nil to low
Usually stableb

Suspension
Intermediate
Low to
intermediate
Low to high
Intermediate to
high
Almost never
Nil to low
Occasional
problemsb

Often low
High
(10:90)
Substrate or suspension
Can be very low
May be a problem
High to very high
Usually low
Very often
Often a problem
Sometimes a problem

Ratio of Synthesis:lsolation cost as percentages (total = 100/o).

If genetically modified, plasmid stability is often a problem.

197

in both synthesis and isolation require more careful regulatory oversight. Design requirements and
problems in process implementation flow from
the qualitative characteristics presented. It will be
seen that the problems and solutions relate quite
strongly to qualitative factors; this should not be
surprising. In fact, the sensitivity of animal and
plant cells to media constituents, to agitationaeration conditions, and to contamination means
that more lab scale experimentation must be performed and that any slight oversight in design
will result in a scale-up problem.
The qualitative review leads to a listing of
scale-up problems. Not every issue shown
(Table 2) occurs in every scale-up situation. Every
issue has the potential to arise at one or another
stage of the scale-up process, however, and in the
authors experience, each item has arisen in different biologic processes. Unfortunately, the list
is not exhaustive and new problems (process specific) arise for a new product or process. It is best
to give some attention and consideration to each
item (including subparts) shown. Assuming no
problem in a specific area is almost the same as

disregarding the potential for difficulty; such an

approach will lead to difficulty at some point.
Furthermore, if knowledge or experience is not
available in one research or development group, it
should be sought elsewhere, inside or outside the
company. To select some examples:
1.

2.

Sterility at the lab bench is not the same as

sterility in a production environment. Sterility control is often considered someone
elses problem. Operating in a laminar flow
hood is not the same as operating in a Class
10 room. Some sterile techniques are eminently suitable for small volumes; the same
technique may be very difficult or impossible on a large scale. It must be recognized
that a nonvalidated change in process (on
scale-up) may be acceptable from the view
of asepsis and be totally unacceptable for
process response.
Sterilizing a small volume (say 1 1 or less),
in an autoclave is Rot the same as sterilizing
5000 1 (or even 400 kl), especially where
heat-labile substances are present. Total heat

TABLE 2
Scale-Up Problems
Asepsis

Containment

Volume of inputs
media
water
air, other gases
neutralizing agents
Sterilization

Aerobic
flanges
connectors
pressurization
gas compressors (additives)
effluent inactivation

Time

Cleaning

Heating/cooling cycles
Holding time (stability)
Transfer time
Lag phases

Fouling
Scale formation
Equipment reuse
Equipment inspection
Agents to use (and disposal)

Volume (Vessel)

Regulatory

Turnover time (homogeneity) 10, OQ, PQ (Validation)

cGMP (Code of Federal Regulations)
Heat transfer (heat load)
FilVemptying time
Waste disposal
Local codes (especially rDNA)
Internal fittings
Finish
Material of construction

198

3.

4.

input varies as does the opportunity for media

modification.
Containment requirements are never easy,
and more stringent requirements may exist
in a laboratory than are strictly needed. The
same high level of containment in a production facility may be onerous and costly
. . . and unnecessary. Containment need
should be jointly determined with appropriately trained personnel.
The concept of time does not receive appropriate attention. Time increments that are
reasonable and meaningful in the laboratory
(such as lo min in trypsin at 35C) cannot
be scaled up indefinitely. Transfer of inoculum may take less than 30 sec in the lab; this
may translate to 30 min at production scale.
There may be no impact on the inoculum or

5.

there may be a severely detrimental effect. The time response must be verified.
Hold times may be minutes in the lab;
they may be hours in a plant. Shelf life
and stability are time-related variables.
Prediction is difficult and may be hazardous to the process.
Cleaning often receives cursory attention.
Scale formation in a small glass vessel is
solved by buying a new vessel. Hand washing is feasible in the lab. Equipment inspection is relatively easy in a lab. All these
aspects of cleaning are rendered more complex or more difficult in a production environment. Gaseous or liquid residues in an
incubator or in bioreactors may result from
commercially available, but not thoroughly
tested, cleaning/sanitizing agents. Many

TABLE 3
Scale-Up Considerations (Potential Concerns)
Potential Concerns
~~

Strain selection

Raw materials

lnoculum development

Sterilization

Selection of parameters

Materials of construction and cleaning

Monitoring

Harvest
Isolation

Purity
Stability
Mutation
Byproducts
Purity (vendor audit)
Standards
Uniformity
Interactions
Transfer time
Hold time
Number of generations
Total heat input
Method used
Fouling
Mixing (turnover)
DO, level
Homogeneity
PH ( O W
Gradients
Ionic contaminants
Corrosionlerosion
Release agents
Sensor stability
Response time
Sampling
Transfer time
Hold time (stability)
Stability
Cost (quality of control)
Regeneration

Aggregation
Viscosity
Degeneration
Phage resistance
Availability
Cost
Substrate concentration
Defoamer
Storage parameters
Number of vessels
Quality control
Maillard reaction
Hold time
Power and shear
CO, level
Oxygen transfer rate
Pressure
Temperature
Surfactants
Agents used
Surface and weld quality
Quality of control
Alarms (and response)
Cell degradation
Asepsis
Impurities
Recycle streams

199

cleaning and sanitizing agents require not

only special handling but special disposal
techniques.
A possible scenario for these potential
problems as well as others listed would be to
raise each issue at an early joint meeting involving research, development, and manufacturing personnel. Each area of uncertainty
should be assessed and any further effort (actual research work or a literature review, for
example) defined. Minutes should be kept and
a clear historical line maintained that reviews
solutions or dispositions of each potential problem.
The same sort of scenario can be extended to
a more complete listing of items that require review; here, theoretical and practical considerations
(with some detail) can be shown. In Table 3,
scale-up considerations are shown; the format is
more of a checklist. Not every consideration will
result in a scale-up problem; some points are not
relevant in certain circumstances. However, assuming no problem with insufficient information or input is definitely the wrong choice. A
clear statement (preferably supported by experimental evidence) in the technology transfer document concerning each of the issues will pay off
handsomely in subsequent scale-up and timely
accomplishment.
Another difficulty in scale-up is rather common. It is extraordinarily difficult to integrate
strain development and process development. Two
examples follow.
Assume media optimization is ongoing as
scale-up proceeds. Adjustment up or down for a
constituent listed in SOPS and documentation
available for later regulatory review presents no
problem (in most cases). If a new ingredient is
added or one is completely deleted and product
yield or rate is enhanced, however, a problem
may or may not arise. Are any new byproducts
introduced? Is product purity unchanged? Is stability impacted? There are other questions, many
of which are related to regulatory oversight. It is
clear that any genetic alternation in the producing
organism must be reviewed with great care. Although generalized mutation programs (antibiotic
biosynthesis, amino acid formation, as examples)

200

normally do not elicit concern when a superior

mutant is introduced, insertion of plasmids may
elicit concern.
A second example concerns a superior producer (at culture flask level) that - for some
reason -does not give enhanced productivity at
plant scale. What is optimal for culture 101 is
very often suboptimal for culture 125, even
though the latter always outperforms the former
in small-scale tests. It is not uncommon for the
test culture to perform below its control (lab)
level at the production scale. It requires skill,
patience, and attention to detail to pursue parallel paths. What changes should be investigated
on scale-up to mirror improved productivity?
How long should one work on a superior culture before proceeding to the next mutant culture? Other questions to consider are: Does the
new culture require enhanced mixing? Does the
new culture require a modified medium or a
modified feeding schedule? What is sensitivity
of the new mutant to changes in DO level or in
response to different CO, tension? Is the new
mutant more sensitive to variation in ion content
or concentration?
The balance of effort between the research
lab, the development group, and manufacturing
is not a simple matter, as these few examples
show.

II. PROJECT PLANNING

Later in this review, selected organizational
requirements are discussed. For a project such as
scale-up to be implemented successfully (or to
avoid serious problems), it is important to understand something about project planning and project
implementation. To avoid scale-up problems, at
least these elements should be put in place:
Establish a multidisciplinary team
Formally or informally, select or acknowledge
a product or process champion
Require early involvement by all
Develop scope of work, design criteria, and
definition of success (goals/objectives)
Establish some flexibility or alternatives in
planning

Establish a budget (or questions to be answered

so that a budget can be set in place)
Establish means of communication, both formal and informal

For a more formal planning checklist see

Table 4.This article will not give every detail
that must be checked in the course of a scale-up
project. A scale-up project may be as simple
as running a new mutant in existing equipment
with no new construction or as complex as building a new product facility with labs and offices
needed for support functions. Obviously, both
time required and funds needed will be vastly

different. In general, however, a scale-up project

will involve some amount of design or engineering, some amount of equipment/materials
procurement, some amount of construction/
modification, and some time for start up or
demonstration. Even the simplest project requires some attention to:
Process flow diagram (PFD)
Equipment list (existing, modified, or new)
Energy, utility requirements
Raw materials list and usage
Equipment layout (including personnel flow)
Regulatory impact (including health and safety)

TABLE 4
Project Planning Checklist
(Scale-Up Program)
Objectives
Scope of work (answer what? when? how much?)
Location of facility (new, expansion, or retrofit)
Schematic process flow diagram (PFD)
Need for architect? engineering consultants?
Environmental impact study
Regulatory issues (product, process, environment, personnel)
Alternatives (value engineering)
Time for construction
Time for break-in or start up
Time for demonstration
Budget and accounting procedures
Internal communications, meeting schedule, reporting
With all required inputs, develop a bid package (or request for proposal, abbreviated
RFP)
Check on time schedule (after receipt of delivery/constructiontimes)
Readjust or reevaluate Items 1 and 2 in light of responses
Develop a final schedule
Consider regulatory review of plans (consultant or government)
Identify long delivery items and place orders
Identify special utilities requirements and schedule/emergency power
Auxiliary services or inputs to program (if not included above):
Engineering
Quality Control
Regulatory
Quality Assurance
Health and Safety
MIS
Establish contractual relationship with contractors
Refine final schedule and budget
Establish construction oversight and initiate construction
Establish start-up team and start-up schedulelSOP preparation
Building permits, specialty alarms (prior to construction)
Establish document receiptldocument control function
Establish validation schedule
Reviewhefine insurance requirements
Establish inspection and testing sequence during start up
Photography (videotape) of construction and start up
Receipt of as-built drawings

201

If there is any degree of complexity (or needed

investment), provision must be made for:
Piping and instrumentation diagram (PID)
Process equipment specifications
Instrument and controls package
Although many scale-up projects are completed
successfully without any significant reference to
these many details, one should be cognizant of the
innumerable details that must be addressed to
prevent project or process problems. If a researcher
cannot explain why or how a reactor or column is
to be controlled in the lab, the success of the
scale-up project will depend on luck. One function of the planning process is to integrate instrument and control needs into the new facility or
new process. Process control is critical in the
laboratory and is just as important in the pilot or
full-scale plant. In the project planning cycle (as
discussed later), the blanks are filled in and the
key questions are answered.
Even though procurement and construction
require unique skills, I would urge members of
the scale-up team or task force to remain intimately involved during these phases. It is usual
to have research and development (R&D) personnel and operating personnel heavily involved
during the planning phase and then again during
commissioning and start up. Construction is left
to the experts, whether internal to the company or an outside general contractor. This is
nonideal. The scale-up team should be involved
at periodic progress meetings, at meetings where
alternatives or change orders are approved, and
at meetings where controls are selected and approved. Members of the scale-up team should
perform periodic inspections of the construction. Location of a gauge or a manual valve is of
more importance to the operator of the system
than to the plumbing subcontractor. Insulation
on certain lines may protect operating personnel; this could be missed in design. Clearance
for maintenance or replacements is sometimes
neglected in design; again, responsible parties
who must live with the installation will be
more critical and vigorous during reviews and
during installation. In sum, the scale-up team
should expend all necessary energy to assure

202

optimum installation. The completion of construction requires the same level of care and
vigilance. Contractors are invariably anxious to
depart once a system is in place. The transfer of
responsibility is a key event; many hidden scaleup problems exist at this point in time. It is
critically important to have joint responsibility
and joint oversight at this time (and this applies
to every key piece of equipment as well as to the
fully functional facility). Completion of construction has a number of overlapping phases
and steps:
Detailed warranty information and PM schedule
Operating manuals distributed
As-built drawings presented
Tagging valves, signage on pipes
Cleaning of piping and equipment (including
passivation)
Testing of pumps, pipelines, valves, fittings
Required hydrostatic tests (code requirements)
Instrument calibration records presented
Electrical continuity check (emergency circuits
check)
Control-point checkout sheets, control elements function
Programmable logic controllers (PLC) checkout
Software printout for all relevant controls
Alarm verification
Creation of punch lists
Special testing (as clean rooms, hoods, ovens,
autoclaves)
It is apparent that even this limited list offers a
host of opportunities for later problems. If regulatory requirements (cGMP) are included, the
potential for problems is enhanced. The scale-up
team should have sufficient and competent personnel to assure a smooth transfer of responsibility.
After completion, commissioning occurs.
Operating procedures should be in place. Normally, water batching occurs. Then all routine
sequences (sterilization, inoculation, control functions, agitation-aeration, transfers, and so forth)
are run; any correction or fine-tuning can take
place. Finally, raw materials are charged and the

planned process is initiated. Training should have

begun prior to completion of construction and is
continued and intensified at this point in time.
With this overview of the project cycle itself,
we can move to detailed analyses of specific equipment and operations. Points to consider will be
stressed with the goal being a scale-up process
that is free of delays, upsets, untoward responses,
and the concurrent disarray and financial loss.

111. SPECIAL TESTING

After reviewing all the potential problems, is
it possible to develop a definitive listing of special tests that will preclude scale-up difficulties?
Although the answer is no, it is possible to
generate a list of special tests that should be considered. If we assume there is a workable process
and that the response to key and secondary process inputs is known in general terms, special
tests should be performed. To avoid scale-up problems, the special tests should be performed at the
smallest scale that gives predictable performance.
The listing covers areas that are overlooked. (See
Table 5 ) These areas often lead to problems whenever no effect is assumed. Process responses
(cell growth, cell metabolism, product rate, and
yield) should be determined in a rigorous and
quantitative fashion. If trials are done properly,
the actual cause of a production problem might be
traced more easily, that is, an unknown cause
might be explained by a pattern of responses already seen during special testing in the course of
scale-up. Knowledge of the laboratory process
should give a very good outline of just how bad
certain upsets can be, that is, some upsets would
cause a yield reduction; others would cause a loss
of a batch. Because special testing cannot go on
for years nor can every eventuality be covered,
select those variables and tests that (1) have a
reasonable chance of occurring at large scale and
(2) have the potential for serious damage. Of
special importance are those tests of extremes
concerning the inoculum. If a production process
is to run for several years, one can be assured that
there will be problems in inoculum development.
A scale-up team should set up and run abnormal
inoculum development procedures; include stor-

TABLE 5
Special Testing at
Laboratory or Pilot Plant
Scale
Extreme (Suboptimal) Conditions
Nutrient levels
PH
Temperature
Exceed shelf life
Shear
Buffer capacity
COdHCO, levels
Defoamer
Holding time
DO*
lnoculum
Age (time in cycle)
Age (conditions of storage)
Percent inoculum
Transfer time
Refed
Reinoculation
Cross-inoculation
Media
Alternate raw materials
Resterilization
Oversterilization
Sterilization method
Water variation
Protectants
Culture Device
Materials of construction
Surface finishltreatment
Cleaning/descaling
Environment
Pressure fluctuations
Agitator failure
Control failure
Recycle (attachment agent)
Distribution piping
Lubricants (utilities)
Treatment (utilities)
Holding time (isolation)

age conditions (should the production scale batch

be held up for any reason), reinoculation, plus
potential control problems during inoculation.
Remember that inoculation transfer time is not
the same at lab or production scale. It is sometimes difficult to justify experiments that search
for suboptimal performance; however, there is
good rationale for this effort and the program
should include such testing.

203

IV. BIOREACTOR DESIGN

Once an organism is selected or modified,
one is faced with the issue of volume production. That is, either the cell mass or a product
of cellular activity is desired. Even though
smaller and smaller quantities of bioactive
ingredient are required for physiological effect, some sort of container or reactor is required. Hence, selection of a fermentor is an
early scale-up need. Whereas the very earliest
fermentations (almost always mixed cultures)
occurred in leather pouches or even in a hole
in a log or in the ground, advances in recent
decades have moved processes toward a more
controlled environment.
The design most commonly used (in the
past 50 to 100 years) is one form or another of
the stirred tank reactor (STR). The start of
antibiotic production, corresponding in time
to World War 11, was a great stimulus to use
and modification of the STR. Why was the
STR selected? There are a number of reasons,
all additive. The vessel and agitator design
were known entities, a three-phase system (gas,
solid, liquid) could be handled, the reactor
could be closed, the reactor could be built to
many sizes using different metals, heat transfer could be conveniently accomplished,
cleanability and sterilization were possible,
controls could be readily introduced, and last
but not least, the vessel could be easily converted, that is, it was a multiuse design. The
STR could also be scaled up. It is, therefore,
no surprise that this design has not been supplanted, although other bioreactors are in use.
The STR can be used in continuous fermentation (the abbreviation becomes CSTR),
semibatch or interrupted batch procedures are
possible, recycle opportunities are possible,
bottom or top drive is possible, baffles or coils
can be placed internally, and operation at an
overpressure is simple.
Although a complete review of the fermentor design is beyond the scope of this review, certain alternative designs should be considered. There may be reasons for selection of
a novel design. Alternative fermentor designs
include:

204

Air lift:

Draft tube:

Recycle:

Fixed bed:

Immobilized cell:

a tubular, tower vessel

with gas introduction to
induce circulation and
mixing
internal cylinder used to
direct circulation with or
without mechanical
agitation; gas introduction
from surface or via bottom
sparger
two vessels (equal or
unequal volume) with gas
introduction to induce flow
or external cell separation
device with return of
concentrated cell slurry
use of supports to retain
cell mass; liquid (substrate) flow through or
across bed for bioconversion
use of solid supports (may
be porous) for cell growth
and adhesion; many
applications in cell culture

Whatever system is to be used, control and

measurement of many process variables are of
critical importance. Gradients are normally undesirable and broad optima for physiologic variables are not normally found. In modem biotechnology, these factors must be considered in
fermentor design:
Materials of construction

Mechanical and
other seals
Sterility (asepsis)
Controllers/sensors
Multiple feeds
Homogeneity
Aeration (multigas)
Turnover time
Agitation (shear impact) Defoamer (additive
or mechanical)
Cleaning
Containment
Flexibility (alternate use) Internal finish
A complete listing of desirable characteristics
of pilot fermentors is available; the same list is
useful for production scale, as
A microbial system is complex in ways different from complexities related to chemical reaction. In microbial systems, not all reactions are

understood. Pathways are unclear or unknown,

even when the desired product is fully characterized. An upset to a microbial system may cause
unexpected (often undesirable) responses. The
organism to be grown is often highly selected
and/or modified; it interacts with and modifies its
immediate environment in a complex manner.
Changes in the microbial environment - sometimes caused by a change in scale - will cause
unpredictable responses by the organism. These
responses may further modify the environment at
the altered scale. Often, the results are benign. On
other occasions, the interactions cause unexpected
results that are usually economically unattractive.
On occasion, the results are economically disastrous. Therefore, the scale-up process for the fermentor has as its focus the creation of the ideal
environment for the cell mass to perform the desired transformation.
As is true in other areas of scale-up of animal
or plant cell processes, selection of the reactor is
made more complex by the nature of the organism. There was an early hope that most, if not at
all, of the newer, complex biomolecules could be
made in bacterial or yeast cells after appropriate
genetic transfers. This hope remained a hope and
the desire for product led to greater and greater
use of animal and plant cells, even with the attendant problems. Hu and Peshwa3 present both a
historical review and some projections in the design and operation of animal cell bioreactors. They
discuss the evolution from roller bottles to
microcaniers, then to hollow fiber and ceramic
systems. Scale-up considerations for the various
formats are given. Macroporous carriers (examples
of structural materials are collagen and gelatin)
are reviewed. Cell culture of some transformed
cells as aggregates has been successful. A number
of scale-up issues are discussed; the major issues
are agitation-aeration effects, oxygen supply, and
understanding of reaction kinetics. The authors
come to a conclusion that is, in the main, correct,
Despite the great advancement in bioreactor
developments in the last decade, their operation is
still largely guided by an educated guess. There
are 122 references cited in the review.
It should be noted that a not unexpected result
of the proliferation of reactor styles is the favoritism expressed by commercial interests in pictur-

ing economic advantage . . . if only a certain reactor were used. Presumably, the potential buyer is
aware of such a bias. One report discusses
scalability and comes down firmly on the side of
modularity. In this case, there are some valid
points to consider, even if some commercialism is
i n ~ o l v e dThe
. ~ most obvious advantage is matching of production capacity with demand (revalidation would probably not be necessary).
Mobility and compactness are other desirable features. Cleaning and sterilizing modular components will probably be simpler. Inoculum level is
also modular, that is, a number of small inocula
is needed rather than a single large mass. Contamination of a large batch will clearly be an
event compared with loss of one of ten modules.
Capital investment is also modular. The considerations are worthy of review prior to making a
selection.
Airlift reactors have been both used and scaled
up successfully for production of monoclonal
antibodies (MAb). Birch5 describes the selection
criteria for a system to mass produce MAb:
Unit costs
Economies of scale
Aseptic operation
Ease of process control and automation
Mixing of shear-sensitive cells
Acceptable mass transfer characteristics
Compatibility with upstream and downstream
processing
Simplicity
Apparently, vessels of 10,OOO 1 size are in use.
Problems in handling large volumes of broth are
also discussed. Techniques used include tangential flow ultrafiltration,precipitation, ion exchange
chromatography, gel filtration, and affinity chromatography. Other workers6 succeeded in volumetric scale-up from 2 to 40 1 in airlift reactors. A
murine/murine MAb was produced in both batch
and semicontinuous operation. No problems are
discussed and it seems that the scale-up protocol
went well.
Verax workers have published extensively
on their proprietary mammalial cell-fluidized bed
culture systems. One article discusses cGMP
facilities using collagen microspheres to product various biopharmaceuticals. A key feature

205

on the reactor is a recycle loop (microspheres

remain suspended in the bed and do not enter
the loop). The loop is used for pH and temperature control, DO control, additions, gas exchange, and sampling. Flow is controlled. Scaleup from 1 x 1O9 to 1.5 x 10l2cells per bioreactor
is described. Many production systems have
been altered to permit elimination of serum in
nutrient feed. Product yield for a recombinant
blood factor rose by 135-fold as scale increased
(fluidized bed volume increased 150-fold). For
tPA, a bed volume increase of 300-fold resulted in a product yield increase (units are mg/
day) of 522-fold. Of course, the problems of
scale-up are not discussed; however, the successful implementation shows what conditions
must be satisfied and what parameters require
attention. In another article from the same corporation, comparative studies using the same
hybridoma cell line were performed in different culture systems.8The systems were suspension batch, continuous (chemostat), and perfused immobilized microsphere. The article
stresses the importance of cell-specific productivity (CSP in units of pg/cell/h). Specific productivity of the hybridoma culture (at constant
specific growth rate of 0.04 h-]) was 0.30,0.90,
1.64 for batch, chemostat, and continuous perfused fluid-bed, respectively.
Alternatives do exist, although it is uncertain as to how much scale-up has actually occurred. Lazar9 has written a review on immobilization of animal cells in a fixed bed bioreactor.
Animal cells were immobilized in a polymeric
matrix of polyurethane foam. Lazar lists the
criteria for supports (true for any sort of immobi lization) :
Chemically inert, no reaction with substrate,
nutrients, products
Stable at elevated temperature
Safe for pharmaceutical products
Allow cells to grow and otherwise act normally
Retain integrity and remain insoluble
High surface area to volume ratio
Easy and simple handling
Consistent quality at an acceptable price
The highest scale described is 2.5 1.
206

An article from MercklO discusses the scaleup of a novel reactor using static mixing elements as cell attachment and cell growth surfaces. The end product was a viral vaccine. The
design criteria were fairly strict: uniform irrigation of cell surfaces, low shear, high surfaceto-volume ratio, surface/material compatibility,
CIP, SIP, monolayer growth, nonenzymatic
harvest, FDA approval, and, finally, scale-up
capability. A prototype (5 x 5 em), total volume of 103 ml, and a production model (20 x
20 cm), total volume of 7.26 1 are described.
This is an example of a nonobvious method
used to successfully achieve a number of specific goals while also allowing extension to
other cell growth systems.
An article by Kearns' compares bioreactor
systems, discusses scale-up problems, and
spends some time on integration of the extraction train. Following the article, there is a fourpage summary on fermentor and bioreactor
design, suppliers, comments, and additional
characteristics.
An interesting reportI2concerns a novel agitation system for shear sensitive cells. Two different cell systems (one insect strain and one
plant cell strain - both susceptible to damage)
were evaluated in 3- and 11-1 reactors. An engineering approach was taken and there are
plots of the impeller Reynold's number vs.
power number and k,a vs. power per unit volume. Further, k,a in various cell growth systems was compared with k,a in the novel configuration; apparently, the new design gives
two- to threefold increases in k,a. Interestingly,
surface baffles did not affect power input but
did increase both rate of mixing and 0, transfer. 0, supplementation was used in the
headspace. Tests in the new system showed
that the cell strains used were less sensitive to
agitation-aeration than in conventional reactors.
Guidelines for scale-up are also provided. The
techniques used can be followed for any agitation scheme.
Animal Cell Bioreactors, a bookJ3published
in 1991, begins with a historical overview of
animal cell bioreactors, has one chapter on scaleup (but scale-up is mentioned in many places),
and ends with a chapter on large-scale purification of clinical products derived from animal cell

cultures. Many experts contributed and although

one must search for problems, many solutions (or
potential solutions) are presented throughout.
Another book on mammalian cell culture,
Large-Scale Mammalian Cell Culture Technology, is worth reviewing.14 Once again, many experts give both reviews and details of different
technologies. There is added consideration (separate chapters) on process technology management,
process validation, and design considerations for
a manufacturing facility. These latter subjects are
critical to scale-up and those chapters are very
informative.
Two specialized chapters give details on design of cell culture reactors and scale-up of cell
culture systems.15De Bruyne discusses bench scale
reactors, shear, continuous culture, methods of
oxygenation, and design criteria for a commercial
system between 0.5 and 3 1. Griffiths reviews
scale-up and density scale-up (in the latter, process intensity is increased). A strength of the
review is presentation of alternatives. Perfusion
methods are discussed extensively. Various entrapment methods are covered. One useful table
covers scale-up factors and compares eight different systems for anchorage-dependent cells and
eight systems for suspension cells. The factors are
scale-up potential, process simplicity, mass transfer efficiency, aseptic operation, direct monitoring, high cell density, steam sterilizability,downstream compatibility, substrate reuse, area/volume
ratio, homogeneity/mixing, critical control, and
continuous process potential. Although qualitative factors are used, such an overview is critical
in process selection.
Another review article by Bliem et al.16
should be consulted prior to selection of an
animal cell reactor (although the points covered are applicable to bioreactors generally).
Selection criteria are given for various systems:
airlift reactors, fluidized and packed bed reactors, hollow fiber systems, and stirred tanks.
Scale-up pros and cons are listed in great detail
and there is an analysis of batch vs. continuous
culturing. There is a comparison (qualitative)
of various factors with three classes of systems:
batch, semicontinuous/fed batch, and continuous/perfusion. As an example, systems falling
under continuous and perfusion would have the
lowest operating expense but the highest cost for

process control, comparing the three classes. The

article is comprehensive and will prove helpful in
bioreactor selection. Strategies for the various
types are described. Seventeen characteristics of
the ideal reactor are given. No reactor class is
ideal, but every nonideality presents a potential
problem, so the table is a useful starting point.
The authors urge selection of up to six relevant
reference cell lines for use in different systems. In
this way, research results can be more readily
compared with one less key variable to consider.
An overview of reactor styles, including cell
retention techniques, is given in Table 6. All cell
culture systems (all fermentors, in general) have
areas of concern (or in the worst case, problems)
as listed at the bottom left of the table. Within
reason, pH and temperature of nutrient feeds to a
classical fermentation do not present problems.
On the other hand, control within a far narrower
range is required for nutrient feeds to a cell culture system. Sterility is a concern in any
bioprocess, but growth rate or type of product
formed in a classical fermentation may inhibit or
otherwise render contaminant entry a nonproblem.
The degree of hazard to the same entry is far
higher in a cell culture system. Substrate-attached
systems have an added set of concerns.

V. FERMENTATION/CULTURE PROCESS
Problems in scale-up can be subdivided; examples of serious issues in each subset are given.
The fermentation process consists of these key
steps:
Nutrient preparation
Inoculum development
Sterilization
Selection of parameters
Selection of process
batch
semibatch
continuous
recycle
refeed
Monitoring
Harvesting
Cleaning
207

TABLE 6
Cell Culture Overview
Culture Mode
Substrate Attached
Microcarrier (conventional)
Microporous materials
Beds

Suspension
Stirred tank
Column

Systems
Roller bottles
Stirred tanks
Airlift (internal draft tube or external circulation)
Bubble column
Hollow fiber
Ceramic annulus (with or without membranes)
Fixed bed
Rotating discs
Plate type
Cell retention
Membrane
Gravity (settling)
Rotating wire/membrane
Aggregate formation
Internal microfiltration
Dialysis
Cell entrapment in collagen
Problems
All systems (general)

Substrate attached

Homogeneity
Impeller shear
Surface finish
Temperaturea
PHa
Osmolaritya
lnoculum state
Sterility"
Scaling
Adsorption (unexpected)

Perfusion (mass transfer rates)

Shear
Equilibrium of constituents
Attachment optimization
Carrier dissolution
Cell density (homogeneity)
Blockage or channeling

Variable is for both process and nutrient feeds.

Agitation-aeration will be considered separately; this combined parameter is not only very
important, but a great deal of published information is available covering many fermentation processes.

A. Nutrient Preparation
There are a number of issues to consider in
nutrient optimization and raw material selec208

tion. General considerations (any bioprocess)

should include reputability of supplier, capability of supplier to deliver, price fluctuations,
compositional changes, need for use testing or
prescreening, potential delivery dislocations,
stability under varying storage conditions, and
contamination (both during delivery or during
storage). The latter point refers to asepsis in
certain cases but for every raw material, gross
contamination is an ever-present hazard. These

potential problems are normally not considered

in research labs; the problems are occasionally
considered in pilot plants. However, one or more
problems invariably occur at production scale.
One or two cases for each potential should suffice; it is useful to address the situation early in
the scale-up cycle.
Supplier reputability
1. Resellers may or may not stand behind certain reagents. It is embarrassing (or worse) to find that technical
specifications for a critical ingredient
have changed while the end user has
never been informed.
2. Companies merge, are bought out, or
fail. Lack of an acceptable second (or
third) source is a problem waiting to
happen.
Capability to deliver
1. Some raw materials are sourced
abroad. Regulations change, shippers
change, foreign exchange varies, and
contracts may be difficult to enforce
in a foreign court. Disruption in air or
sea traffic is common.
2. Little consideration is given to how
much material is warehoused (where?
what condition? who checks?) outside of user control. Real turnaround
time for a change in demand is essential.
Compositional changes
1. If a technical data sheet exists, it should
be clear as to who does assays, by
what procedure, and when they are
done. Dispute resolution should be
clear (this becomes obvious when
something costing thousands of dollars per gram assays low in the users
laboratory).
2. Storage conditions vary. Simple items
such as temperature and humidity must
be considered. More complex issues
(nearby storage and cross-contamination) including materials of construction (tanks, pumps, pipelines) should
be considered.

Use testing
1. An old example is the screening of
cornsteep liquor for use in penicillin
fermentation. Historically, many natural products have been screened prior
to purchase. This is common now with
animal sera. It may be necessary for
so-called pure raw materials.
2. Use testing is an added expense and is
sometimes short-circuited. Occasionally, there is no problem. More occasionally, there is.
Delivery dislocations
1. Rules exist for shipping certain products (as herbicides and pesticides) in
dedicated trucks or, at the least, in
trucks or tankcars that are thoroughly
cleaned prior to reuse. This ideal status is not always achieved, as failed
bioprocesses have testified.
2. Delivery at a preselected temperature
is often requested. What is often unknown is when and how far out of
spec a certain delivery occurred.
Extension to the other concerns noted should now
be obvious. These general considerations are
frightening enough, even though all have been
overcome (most of the time) in classic fermentation. Cell culture is more sensitive to various
upsets and so it is no surprise that problem potential is magnified for relevant nutrient preparation.
Here one must be even more vigilant. First, highly
purified materials (as hormones, recombinant proteins, growth factors) are often used in biosynthesis. Second, endotoxin - both present and induced in process - becomes a new potential
problem. Last but not least, both the quantity of
items, as well as the quality, is much higher in cell
and tissue culture. Every screening aspect - as
QC testing, shelf life, use-testing -must receive
a heightened awareness. As noted above, the potential problems never vanish; they are merely
controlled.

B. lnoculum Development
By now, the importance of inoculum development is well appreciated in bioprocessing. In-

oculum development receives as much care and

attention as the growth and product cycles; this is
as it should be. The status or health of the
inoculum has a great deal to do with the success
of the production cycle.
The problems of inoculum scale-up and inoculum development have been discussed. Some
of the potential problems will be highlighted once
again:
Age of inoculum
During scale-up, evaluate responses
when a young or old inoculum is
used.
Determine quantitative criteria for optimum transfer time or state.
2. Inoculum hold
If inoculum must be held, what are appropriate conditions? What is maximum
hold time? What are criteria for discard
of inoculum? These are a few of the questions scale-up runs should answer.
3 . Inoculum stability
Plasmid-containingcultures or highly specialized cells are often unstable. Studies
must be completed to insure minimum
variability or no unwanted differentiation,
but it is also important to find and use
quantitative limits that define good or
bad inoculum.
4. Cross-inoculation
If a fermentation is ready to start and the
inoculum is lost, it would be useful to
know when a production vessel (running
concurrently) could be used to crossinoculate the waiting vessel. Added flexibility at this point would be an economic
plus.
5 . Inoculum transfer time
Scale-up normally means greater volume
and longer time (compared with lab scale)
for process steps. Extremes in inoculum
transfer should be evaluated to set appropriate limits.
6. Number of transfers
Each transfer of inoculum presents potential hazards. If possible, inoculum
growth cycles and transfers should be
reduced. If 2% inoculum results in the
1.

210

7.

same performance as lo%, the lower level

is preferred, If frozen cells can be resuspended and used (rather than undergoing
a growth cycle), this procedure is preferred.
Draw-fill technique
If a semicontinuous process (even for a
few cycles) is possible, productivity could
be enhanced and need for fresh inoculum
reduced. A small fraction of a completed
batch (say 5 to 20% of the total) is left in
the bioreactor; fresh sterile medium is
added to start a new cycle. Such a process
does not have universal applicability.

C. Sterilization/Medium Preparation
This is a massive subject and is covered later
in this article. We discuss here the criteria for
media preparation. Every aspect covered has the
potential for problems of severe magnitude. It is
the height of folly to spend great effort and great
cost on selection and QC testing of the best raw
materials and, subsequently, mishandle the ingredients in media preparation.
The area selected for media storage, preparation, sterilization, and transfer to production should
receive the same care and consideration as the
production bioreactor or extraction train. (This is
highly desirable because the media prep area and
process receives - at the least -the same regulatory oversight as the production scheme.) The
media prep area is an integral and critical part of
the process flow so the conclusion is obvious and
logical.
Considerations for problem avoidance are
1. Ease of access, ease of cleaning, selection of

wall, flooring, ceiling tile materials

2. Level of cleanliness in environment (in some
cases, HEPA-filtered rooms are a requirement)
3. Appropriate lighting
4. Clear and appropriate storage of all needed
nutrients, glassware, plasticware, filters, protective equipment
5. Suitable instrumentation (properly certified
and routinely checked) that is cleanable

6. Well-agitated vessels, properly calibrated

7. In-process quality control (by media prep
personnel, when reasonable) in appropriate
area with proper logging and instrument
checks
8. Refrigerated or frozen storage, when required
9. Appropriate storage (properly labeled) of
retain samples. Instructions and control on
discard of retains in place
10. Suitable recording devices (if computerized,
software must be validated) and storage
11. Appropriate media transfer methodology
whether via bottle, large container, or pump.
Proper cleanability
12. All vessels, bottles, or containers used should
be of suitable quality, should drain completely, and should be easily cleanable
13. Empty raw material containers (bottles, bags,
drums, etc.) should be properly stored and
disposed of or recycled
14. Scales should be made of appropriate materials, should print results (even with computer storage of data), should be suitable for
intended use, should be readily cleanable
15. Excellent communication must be provided
to all necessary internal contacts (examples
are manufacturing group, QC personnel,
facilities management)
16. All hazardous materials must be properly
stored and handled; appropriate personnel
protection provided
17. Appropriate quality water must be provided.
A back-up supply should be available for
emergency use
18. Auxiliaries should be appropriate for intended use. For cell culture, all autoclaves,
ovens, glassware washers, steam quality
must be carefully reviewed, selected, and
installed

D. Selection of Parameters/Process
The scale-up problems that correspond to
process or bioreactor selection can be assessed
after reading a short quote by Lubbert. Unfortunately, the transport problems, microbial kinetics, and especially their interrelationship are not
sufficiently understood, particularly in produc-

tion-scale reactors. Although this statement may

be a bit harsh, it was made in a 1992 publication.
The author goes on to study gas residence time
distribution, liquid phase motion, and gives certain circulation time distributions. However, the
amount of needed information (i.e., missing data)
is stressed.
Scale-up is often related to oxygen supply
(see section on Agitation and Aeration) and a host
of problems is related to improper (too low or too
high) dissolved oxygen concentration. It is possible to review selected publications and come
away with an understanding of problems that exist
and potential scale-up solutions that may be applicable.
covers the situation
A review by Leist et
in animal cell suspension culture. Although the
direct lessons involve animal cells, the indirect
lessons have a general applicability. The authors
list the chief problems as:
Implementation strategy of the culture system
Design of the mixing system
Asepsis
Physical culture parameters
Chemical culture parameters
Measurement and control
This fairly well covers the spectrum. The morphology of animal cells, the complexity of the
genome, and the greater biochemical complexity
(compared with prokaryotic organisms) are all
responsible, but the problems look familiar even
for older fermentations. The article does go on
to compare culture methods and touches on scaleup in the various methodologies; there are 191
references cited. The authors also detail the many
scale-up problems related to serum use. Some are
obvious, others less so:
Expense
Lot-to-lot variability
Product is ill or not defined
Serum may contain growth and metabolism
inhibitors
Serum may hinder upstream processing (filters, pumps, sterilization)
Serum may hinder downstream processing
Poses infection risk (virus, mycoplasma)

211

Although serum-free media are preferred, operation serum-free is often not possible.
Another example of a potential microbial
process is given by Yoshida et al. A unique
n-alkane-assimilating bacterium was found
(Mycohacteriurn sp.) that could be grown on
methyl ethyl ketone and use phenol as a raw
material for the conversion. A membrane reactor
was run (with cell recycle) and reverse osmosis
plus low-temperature crystallization were used to
purify the product. Hydroquinone was produced
in the reactor at ca. 2 g/l. Although actual scaleup was not tried, a possible scheme (process flow
diagram) is shown with a number of useful and
inventive procedures given.
There are two other relatively new areas in
scale-up where some novelty may be required.
The problem-definition phase is well advanced
and even some breakthroughs can be claimed.
The areas are plant cell culture (PCC) and insect
cell culture (ICC). Many of the scale-up problems
are unique to the system but, once again, there is
general applicability elsewhere. Buitelaar and
TramperZodiscuss PCC in a review emphasizing
strategies to improve productivity. The recent
review states that a 75 m3bioreactor (STR) is in
use at Diversa (Germany) to produce a PCC product. They discuss alternative systems (as immobilization, gel entrapment, biofilm, absorption, foam
immobilization, and membranes) with examples
from the literature. Not surprisingly, productivity
data are sparse. The article does contain a theoretical cost comparison for production of
ajmalicine-containing biomass. LipskyZ1presents
a somewhat more theoretical approach for optimization of PCC processes. He presents various
problems and stresses the draw-fill technique for
economic optimization.
AgathoP presents both theoretical and practical considerations in ICC. The listing of considerations should be familiar by now. First, role of
nutrients and other media constituents on growth,
viability, virus infectivity, and productivity should
be determined. Then, physical requirements (as
temperature, pH, osmolarity) should be determined. Then, response to transients and stresses
should be known. Stresses include agitation, circulation time, bubbles, indirect oxygen supplementation; finally, different culture conditions

212

should be evaluated. Both upstream and downstream steps should be studied. The review does
present some of the advantages of insect cells
compared with mammalian cells. Cell line maintenance (for insect cells) is said to be easier, there
is a greater versatility in different suspension culture, cells are immortal, there is mild or no contact inhibition, gentle shaking (no trypsin) will
detach cells from substrate, and cells are less
susceptible to shock. This is quite a list of advantages and certain groups are pursuing ICC in a
serious way. It appears that on scale-up, the number of problems or the extent of response by
insect cells is less than mammalian cells. Whether
the advantages can be taken advantage of on a
broad commercial scale remains to be seen.
It is difficult to limit the number of problems
on scale-up, especially where animal cell culture
is involved. There are many potential pitfalls and
no small set can be created to cover 90% of all
potential problems. For PCC or ICC, complexity
(or lack of knowledge) rises and so does the potential for problems.
In two tables, an attempt is made to cover
areas of concern or, at the least, areas for careful
review and selection. The first (Table 7) lists what
might loosely be considered outside or environmental issues. That is, the inputs or utilities
needed for successful implementation are detailed.
A deficiency in one of the specific items listed
can create a problem. No priority can be established without some research data. Some examples:
1.

2.

3.

Some cells will not grow on plasticware

irradiated a second time; others will show
no inhibition after a number of irradiations.
Release agents in the plasticware have no
effect on some cells and a great effect on
others.
Changes in glassware washer cleaning agent
may or may not cause a later problem.
Change in a sterilizing filter may leave no
undesirable residue in a nutrient medium; it
may leave an inhibitor to a different cell
strain.

What is clear is that untested changes have the

potential to cause problems. Every change must

TABLE 7

Cell and Tissue Culture Problems (Scale-Up)

Raw Materials
quality
testing (QC)
shelf life
CO, distribution
alternate suppliers
prescreening
hold conditions

Glassware
glass type
washing
drying
endotoxin
storage
reuse
coatings

Plasticware
variability
source documents
release agents
irradiation
sterility

Water

Media preparation

Incubators

quality
testing (QC)
PM schedule
passivation of piping
records

SOPS and records

homogeneity
testing (QC)
sterility
shelf life
storage conditions
environment (HEPA)

temperature
CO, (pressure and Oh)
humidity
cleaning agents
sterility (HEPA)
logs
connections
certifications and checks

Facilities and utilities

validation
environmental testing
cleaning/sanitization
PM schedule
steam quality
data recording

be validated is not a bad rule of operations or

scale-up.
The next table (Table 8) is modified and expanded after Runstadler. Here the cell culture
environment itself is the concern. The various
input factors are detailed. Again, every item
listed is not necessarily a cause for serious concern. However, there is a wide span of considerations that must be addressed, either theoretically
or empirically.
Finally, selection of the bioprocess depends
on the microbial cell, the process itself, existing
equipment, and economics. The batch process has
the longest history and is probably the safest,
meaning that technology is fairly well known and
is simple. Continuous fermentation has the greatest theoretical advantage but also has detriments
or, at least, potential downside risks. Some are
Need for excellent control, probably with redundancy

Culture degeneration may be a problem

Specific productivity may decline (even with
no degeneration)
Asepsis is more difficult to maintain
Restart (after unexpected shutdown) is more
complex
Other feeds must be continuous (or semicontinuous)
Downstream processing must be properly sized
Usually less flexible (alternate product in same
equipment)
The positives of continuous culture are given
by H o ~ h f e l dHe
. ~ ~stresses cost effectiveness and
also notes, correctly, that continuous culture is a
powerful method for process optimization. He
notes, Scaleup of a continuous-culturebioprocess
requires detailed data from process development
and optimization studies which should be gathered with great care, since a scaleup is only as
good as the input data provided. Of course, this

213

TABLE 8
Cell Culture Environment
(Issues of Concern)
Medium
Nutrients
Hormones
Binding proteins
Attachment factors
Enzymes
Proteases
Water quality

Cell and cell products

QC
Stability
Endotoxin
Inducers
Minerals
Extracts

Cell density
Desired product
Metabolites
Inhibitory factors
Viability (stability)
Validation

Cell-cell interactions
Preservability
Safety
Transformation
Specific activity

W
I I
cell culture
environment
optimum

Matrix
Physical
characteristics
Attachment factors
Hormones
Binding proteins
Enzymes

In vivo simulation
Homogeneity
Scalability
Diffusion
Polarity
Suspending agents

Culture parameters
pH
0 2

Temperature
Pressure
Osmolarity
Shear-agitation

Redox
CO,
Humidity
Viscosity
Foaming
Homogeneity

Note: Expanded after Runstadler, P. W. et al., Continuous culture with macroporous

matrix, fluidized bed systems, in Large-Scale Marnrnalial Cell Culture Technology, A. S. Lubiniecki, Ed., Marcel Dekker, New York, 1990,366. With permission.

is true for batch bioprocesses, too. Hochfeld discusses the many scale-up factors that must be
considered. Generation number must be simulated (larger volumes mean more doublings to
reach a desired cell concentration). Maintenance
of steady-state conditions (and problems to avoid)
is reviewed. Potential problems to long-term operation are touched on: foaming, gasket and seal
replacement, wall growth, use of anti-grow-back
tubes, and use of a hooded overflow dam are
noted.
Intermediate choices involve feeding cycles
with or without withdrawal of culture broth.
These are process specific. Invariably, development time is lengthened due to the complexity of the equipment and controls. It should be
noted, however, that some of these techniques
are in use and have significantly enhanced productivity.

214

E. Monitoring
All bioprocesses require stringent monitoring; there are limited process optima and there
are severe regulatory requirements. A listing of
potential culture measurements exists.24Selection is process dependent. A listing of potential
problems that require scale-up attention follows:
1.

2.

Sterility
Instruments, in an ideal world, would be
noncontacting and not require broth recycle. In the real world, the sensor may
have to be sterilized and resterilized. Also,
entry should not improve chances for
contamination.
Stability
After repeated use (including cleaning and

3.

4.

5.

sterilization many times), does the sensor

maintain response and accuracy?
Redundancy
Some measurements are so critical (pH in
many cases, DO in others) that an incycle failure may result in a batch failure.
Should a redundant set of sensors be installed or can a portable flowcell system
(attach to sample port) be used as a temporary measure?
CentralLocal control
If centralized control is used, local backups may be required. Even with PLCs,
redundancy may be required so manual
operation might complete a batch even
after a major failure. Failure mode on
valves is important. Do valves fail open?
closed? to last control position? some
preset posit ion?
Environment
Many operations now occur in clean room
spaces. Key variables are particle count,
pressure (or pressure differential), temperature, relative humidity. It is important to establish control and monitoring
schemes, a method for data recording and
trending, a recertification program, action limits, and a program of response.

The key question is What are the critical parameters to monitor, to record, to control? One part
of the answer will relate to the process itself; the
other part will relate to the regulatory requirements for the specific product. Both are equally
important. Once the answers are written down,
the program must be accepted internally (meaning by development team) and should be reviewed
externally (with FDA representatives). Missing a
critical control point may not result in product
failure but it may result in validation failure.

F. Harvesting
Scale-up problems related to harvesting and
vessel cleaning may be summarized:
1.

Harvest time including storage (harvest tank)

The bioreactor is not normally used as a

2.

3.

4.

hold tank to feed the isolation train. Time

to harvest and hold time are both potential problems. Cell lysis is sometimes
desirable in the hold tank, most often it is
not. Proper operating parameters must be
established.
Harvest tank
The harvest tank may require sterilization and/or cleaning. Scale-up should establish minimum requirements and protocols. Special conditions (temperature,
inert gas, additives) may be required in
the harvest tank. Special mixing requirements may exist.
Harvest criteria
Not every fermentation or cell culture
proceeds in exactly the same way. Minimum criteria should be established for
harvest; if not met, the batch is discarded.
Normally, economics will be a key factor. Presence/absence of contaminants is
another key factor.
Batch discard
In the lab, loss of a batch is disturbing but
easily handled. At production scale, there
may be severe limitations on a discard.
What acceptable procedures are there for
batch discard?

G. Other Reviews
A symposium held about a decade ago covers research needs in bioprocessing.2s Surprisingly (or perhaps not so surprisingly), many
needs noted then remain as needs today. There
are generally analyses of design criteria and
some specifics to consider in downstream processing. The needs and problems (which are
true at the research scale, but also true in scaleup) include:
Pretreatments
End-product inhibition
Product recovery from dilute solution
Oxygen transfer and heat transfer
Bacteriophage
Cell recovery
Stability of immobilized cells

215

Problems directly related to scale-up are discussed

in the same review (by M. Charles):
Methods to decrease cell growth (probably
meaning reduced maintenance requirement, as
well) while driving energetics to product concentration increase
Bulk mixing, including monitoring and control
New fermentor concepts
Heat and mass transfer
Factors affecting substrate and oxygen transfer
Investigation of pathways of biosynthesis
Bacteriophage-resistant cells
Improved product separation and purification
Rheology of broths
Reliable, rational scale-up strategies and correlations
Problems specific to animal or plant cell culture
are also discussed:
Medium formulation
Factors limiting cell growth
Control of product expression
Contamination control and biohazard containment
Hardware and process design parameters
Product recovery
Although nothing noted is surprising, points are
listed for completeness.
A certain amount of published information
exists that gives actual performance in the course
of scale-up. Normally successes are given; procedures involved give insight into key factors.
Flickinger et aLZ6describe pilot fermentations at
the 120 dm3 scale for toyocamycin (an antibiotic
produced by a Streptomyces culture), The procedures detailed are used to scale the process to
12,000dm3.Sensibly enough, the primary criterion
in parameter manipulation was whether antibiotic
productivity was increased or sustained longer at
a higher level. One end result was that productivity could be predicted using oxygen-uptake rate.
Also seed tank inoculum was monitored via CO,
evolution rate and transfer time was selected based
on this parameter. Fluid mixing characteristics in
the vessels were estimated in a water system,
using a food colorant. Other process improve-

216

ments are described. The introduction of a mutant

culture is also included; it is a bit difficult to separate the key factors. The article lists a number of
references related to classic fermentation scale-up.
A careful study of both scale-up and optimization for a human hybridoma-producing immunoglobulin M (against P . aeruginosa) was performed by Swiss ~o r k e r s . 2Three
~ bioreactors were
used (0.5, 1.5, and 12 1). One of the most important considerations in any scale-up is detailed investigation of culture conditions (nutrient and
waste product concentrations and specific rates,
pH, dilution rate, temperature, DO level) and resulting impact on cell growth and product formation. In these studies, attention was paid to mixing
intensity, DO level, cell content, and viability while
optimization proceeded. Productivity and yield
were enhanced; feasibility of large-scale production was shown.
Two articles by Ozturk and Palsson2*should
be consulted for examples of necessary studies to
be undertaken prior to or during scale-up. Careful
measurements were made of 0, uptake, ATP production rates, cell growth, metabolite concentrations, antibody synthesis, as well as amino acid
consumption and production rates. Even if every
biosynthetic step is not defined, these extensive
measurements will serve as a guide for successful
scale-up. In these studies, antibody production
was not growth associated for the cell line used;
only acidic pH and high osmolarity altered the
specific antibody production rate. The authors
aim of improving productivity by understanding
the impact of process variables on cell physiology
was attained. A spectrum of process variables
was studied, and both input variables and process
responses are available for effective scale-up and
optimization. The work described is an excellent
example of methods that result in problem
avoidance.
GriffithsZ9reviews batch and continuous processes for animal cell culture. Some information
is given on culture vessels in use for production of
tPA, IFN, and MAbs. Once again the figure of
10,OOO 1 is noted as the largest unit used for
cell-derived pharmaceuticals. Some theoretical
cost analyses are given for batch vs. perfusion
culture. The numerous advantages in the use of
porous microcarriers are listed:

Cell densities 2C50 greater than use of solid

microcarriers
Supports both attached and suspension cells
Suitable for stirred, fluidized, or fixed bed reactors
Short diffusion paths into a sphere
Good scale-up potential
Cells protected from shear
Long-term continuous culture possible
3D configuration easily derived
Two reactor configurations were evaluated
by Ryll et al.3nin production of IL-2 using BHK
cells. A small (1-2 1) stirred, bubble-free agitation system was compared with a dense cell HF
bioreactor. Media were evaluated that were serum containing and also protein-free. DO was
controlled at 40% of saturation. Systems are carefully detailed. One- and two-step cell cultivation
methods were evaluated. Cost comparison is given
for various configurations (medium costs rather
than full operational). Highest cost ($1 1.56/mg)
occurred with roller bottles and the lowest ($0.751
mg) with the stirred reactor. Because tissue-like
aggregates were noted in the HF system, it is
unclear whether sufficient optimization occurred
prior to the evaluation.
Although review articles do not generally
cover problems in scale-up in a broad way, a
recent review by Werner et al.31is fairly comprehensive and discusses economic issues as well.
Biologic properties of cell culture systems are
discussed with attention to potential pitfalls. Optimum ranges for various parameters are given
as well as flow diagrams for different culture
methods. The key factors in selection of a cell
culture propagation system are capital investment, amount of material to be produced, genetic stability of the cell line, reliability of equipment and sterility control, and, finally, flexibility
desired in the production plant. The authors note
that there are established technologies for
mammalial cell culture up to the 15,000-1 scale.
Flow plans (full plant) are presented for comparing batch and continuous operations along with
potential difficulties with each format. The article is suggested reading for an overview of
issues involved in mammalial cell culture and
product isolation.

Though somewhat dated, there is a list of

fermentor manufacturers available.32 Vessel
characteristics are given. A list of manufacturers
(vessels over 100 1) is also given in Appendix C
of this article. There is a useful review on overall
design of a pilot-scale fermentation; many issues
are covered which should aid others in design and
operation of such a facility.33
An earlier book on large-scale mammalian
cell culture covers the state of the art in the early
1 9 8 0 ~Almost
. ~ ~ every one of nine contributions
relates to scale-up. The strict discipline of the
marketplace is indicated by the fact that some of
the firms represented either do not exist or are no
longer involved in cell culture. This does not
detract from the data, conceptual analyses, and
actual operational information (at large scale) presented.
Although not strictly a scale-up problem, process optimization usually becomes a correlary
problem. In the time interval between discovery
and start of pilot processing, new findings are
made that improve the process. Alternatively, in
the course of scale-up, slight alterations in inputs
are found to improve the process. The need for
kinetic data can be satisfied while process optimization occurs. Integration of process changes into
the scale-up program is another issue. One example of fermentation optimization is given by
Yang.ls Another example of optimization (production of recombinant CD4) can be found in an
article by Lazarte et a1.36
After reviewing all available processes and
equipment, one is left with a host of choices, even
after some selectivity is applied. Are there realworld examples that can be studied, preferably in
a single location? Volume 4 of Animal Cell Biotechnology3 has many useful chapters (some of
which cover newer designs) but three later chapters are reviews for specific bioproducts. One
covers growth and production of HIV, a second
covers interferons derived from human cells, and
the last reviews manufacture and use of
carcinoembryonic antigen (CEA).
Each of the reviews covers a wealth of material, describes alternatives, and the chapter on
interferons includes process flow diagrams and
protocols used for various key steps. Problems or
issues related to scale-up are summarized here:

217

HIV

Use of continuous cell lines

after adaptation
Varying levels of HIV product
with same HIV isolate
Virus variants present
Changes in cell surface markers related to
environmental changes (as
growth factors and nutrient
limitation)
Gas exchange rate
Seeding cell density
Variability in kinetics of virus
growth
Strictly controlled containment
Interferons (IFN) Low titers (leukocyte IFN)
Optimization of large-scale
production (lymphoblastoid

Im)

CEA

Concentration inducer
Scale-up of anchorage dependent cells (p-IFN)
Complexity of superinduction
(P-IFN)
Short-term culture (&IF")
Low product concentration
(high volumes to process)
Microcarrier culture 0, supply and harvest method
Packed bed nonhomogeneities
Need for low protein nutrient
media
Specificity in isolation

These problems (both general and productspecific) are covered in some detail. In earlier
sections on reactor design and process selection,
many of the issues mentioned in the three chapters cited have been discussed. Others are discussed later in this article. Real-life examples are
valuable for many reasons. Two obvious ones are
(a) problems or issues of concern force reflection
relative to one's own scale-up situation and (b)
solutions to many serious problems have been
devised.
Occasionally one can find a carefully written
article on design and construction of an actual
facility. A useful article (such as one by Srigley3*)
will give many lessons in problem avoidance and

218

will present useful examples of pay-offs related

to attention-to-detail. The article is discussed more
fully later.

VI. AGfTATlON AND AERATION

Scale-up studies are performed to determine
(with as great a degree of certitude as time and
money allows) those conditions and parameters
that will result in the same process result at a new
scale (size, mass, or volume). As it happens, one
invariably comes to agitation-aeration as a complex, intertwined, and problematic set of parameters. The variables are intertwined because obtaining the same oxygen transfer and dissolved
oxygen distribution requires consideration of both
aeration and agitation. Even if similar (not identical) conditions are satisfactory, one must consider both inputs. Further, if aeration alone is used
(bubble column), mixing must be achieved even
without a mechanical impeller. The power input
to a large vessel (however achieved) impacts
mixing (i.e., turnover time), liquid shear rates,
and gas mass transfer (i.e., the mass transfer coefficient).
It is well known that oxygen is sparingly
soluble in water and culture media at conditions
normally encountered in biotechnology applications. Growth and/or productivity of microorganisms is often controlled by oxygen availability.
Scale-up success depends on satisfying the oxygen demand all through the volume of interest. In
general, the dissolved oxygen pattern (and changes
during the cycle) should be replicated at the new,
higher scale. This is not always practical or possible. This discussion is not meant to imply that
only oxygen depletion is a mass transfer problem
on scale-up. Certain nutrients (especially those in
trace concentration) can be depleted prior to reaching a site within the organism where it is required.
Gas-liquid mass transfer is important in maintaining desired dissolved oxygen and dissolved
carbon dioxide levels in the culture broth. The
oxygen transfer rate (OTR) and the oxygen uptake rate (OUR) may be the same, but this is not
always the case. The OTR is affected by the mass
transfer coefficient (k,a), the volume involved
(V), and the driving force between 0, concentra-

tion at the gas-liquid interface (c*) and 0,concentration in the culture liquid (cL). The wellknown formula for OTR is
OTR = kLax V (c* - c,)
The mass transfer coefficient is impacted by
power input to the liquid (via impeller and gas
input), c* is impacted by pressure and pure 0,
supplementation, and c, is impacted by cellular
demand. It is often hazardous to successful scaleup to have c,, at zero or a value below that shown
to be critical in lab experiments. Both k,a and c*
are impacted by temperature but physiological
temperature optima are fairly restricted so this
effect is minimal or insignificant.
Many experimental studies have been performed to relate k,a to other fermentation or culture inputs. These inputs are power per unit volume, superficial gas velocity, volumetric gas flow
rate, number of impellers, and tank diameter.
Gassed power is different from ungassed power
and correlations for changes are also available. In
many cases, predictions incorporate a constant,
which is often a correction factor to allow data to
fit within a certain size range. A summary is
it has come to
given by H ~ b b a r d . ~ ~ Othe
v e years,
r
be understood that scale-up of agitation-aeration
by one technique may not give results comparable
with another method. That is, all critical parameters are not scaled up in the same way. Broth
density, broth viscosity, surface tension, presence
(or absence) of suspended solids all contribute to
confusing, or at least confounding, the issue. The
article noted gives a number of studies and reviews on various scale-up strategies and presents
the authors recommendation for scale-up. Generally, geometric similarity is followed and (k,a)
plant is designed to be equal to (k,,a) lab. Published correlations are used to determine air
throughput, impeller RPM, and impeller diameter. Power consumption (gassed and ungassed)
can be estimated. The manufacturers of mechanical agitators have a wealth of knowledge on scaleup methods and can generate different scenarios
(at different capital and operating costs) based on
laboratory or pilot plant data. It is probably worthwhile to contact at least two suppliers for various
estimates and technical advice.

Many studies have been performed on mixing times. Generally, at any reasonable scale,
differences (meaning nonhomogeneities) are
noted throughout the entire broth volume. Measurements on mixing time are made by having
numerous sensors (or even a single sensor) installed and by injection of a measurable entity
(salt, acid, or base) via a pulse or step input.
Change in conductivity, color, or pH with time
is recorded. An immediate problem, of course, is
that such measurements are made in an aqueous
system that may exhibit rheological properties
different from the fermentation under study.
Perturbing an actual fermentation is costly and
the input change may alter the system sufficiently to confuse the study. Past studies have
shown, however, that 10- to 50-1 fermentors
exhibit mixing times of 1 to 5 s. Vessels of 1000
to 2000 1 exhibit mixing times of 20 to 30 s. At
the scale of 100,000 to 120,000 1, mixing times
rise to 2 min and beyond. If OUR is sufficiently
high and nutrient solubility is low (as for oxygen), extended mixing times mean that oxygen
deprivation will occur in certain regions of the
fermentor broth volume. It should be noted that
along with potential or real nutrient deprivation,
there may be equally serious problems in control
of pH (nonhomogeneity due to viscosity or vessel geometry) and in control of temperature. Note
that heat-transfer area changes with the square
of vessel dimension, whereas volume changes
with the cube of vessel dimension.
The impact of volume (scale) on mixing time
can be simulated at smaller scales. Careful thought
must be given to impact of mixing and vessel
geometry on byproduct removal. In many cases,
CO, is evolved in the course of cell growth or
product formation. In a 10-1 vessel, CO, removal
is not normally a problem. In a 50-ft-tall vessel
(with overpressure), pressure at the vessel bottom
is different from that at the top. Changes in overpressure will impact c* favorably but will also
impact CO, solubility. Too high a pC0, or a CO,
level in effluent gas has had a detrimental effect
on certain biosyntheses. 0, supplementation may
enhance 0, transfer by raising c*; however, 0,
supplementation will not enhance CO, removal.
In cell culture, control of CO,/HCO,- ratios is
critical over a narrow range.

219

Hubbard et al.40have also considered scaleup of a polysaccharide fermentation. Equations

are shown that give relationships between gassed
and ungassed power, generally for Newtonian
fluids. However, for polysaccharide biosynthesis, the many effects of dissolved polysaccharides on broth properties must be known. Physical properties include viscosity, elasticity, surface
tension, density, and diffusion coefficicnt; not
all are of equal impact. Published correlations
for mass transfer in non-Newtonian systems are
reviewed. There appears to be general agreement that keeping k,a constant is a useful scaleup strategy (as noted earlier). How to achieve
this result unequivocally is not readily apparent
on scale-up. The authors give a ten-step procedure for scale-up that encompasses both
Newtonian and non-Newtonian broths. The procedure is used to scale-up a process for production of phosphomannan using Hansenufu holstii;
k,a is kept constant.
K. J. Jem4 considers scale-down techniques
as methods of generating useful information to
identify and solve potential production scale problems. Common scale-up criteria are based on tank
geometry, oxygen supply, heat transfer capability, mixing effect, and impeller power input. Equations are given relating power input, kra, tip speed,
mixing time, Reynolds number, and OUR to process parameters. As one might expect, a number
of proportionality constants are included; although
values are not given, some are available in the
literature, some are available from equipment
manufacturers, and some can be generated if appropriate testing is performed. Aside from agitation-aeration effects, the author notes that certain
biological factors that are or seem unimportant at
lab scale, can have significant effects on largescale operations. The factors listed are culture
stability, phage resistance, cell and product shear
sensitivity, pellet formation, OUR, heat generation rate, minimum DO, tolerance, and maximum
CO, tolerance. As noted, this is not an exhaustive
list. Another partial list is given by Jem concerning scale-down considerations for optimizing a
bioprocess: water quality, sterilization-temperature profile, steam quality, low-cost raw materials, serial subcultures, inoculation volumes, and
pH control agents. These are good places to start.

220

Some special scale-down techniques can be used

to detect production problems or define acceptable ranges for optimum performance. The tests
include delayed inoculation (add how to maintain
inoculum), using cross-inoculation, use of
antifoam agents (add rate of addition, time between addition, total volume, impact on isolation), changing back pressure, screening for O2
and CO, tolerance, and testing for sensitivity.
Consideration of surface aeration at small fermentor volume is noted.
Singh et aL4*discuss scale-up and optimization of 0, transfer in both Newtonian and nonNewtonian systems. Further, the article discusses
techniques for continuous optimization of 0, transfer efficiency. An Aspergillus fermentation was
run in three different fermentors (70, 3700, and
36,000 1). The authors introduce a mixedness
index (I,,,), which is
(Number_
of impellers
submerged)(
Impeller zone volume)
~
(Tank volume)

Im
=_

and further
Im

= (Constant)(Tip speed) (Tank Diameter) (T&

Diameter)-

(Tank Height)

No claim to universality of this correlation is

made. Still, actual data are used to show how the
correlation can be used. The peak OTR was 22
mmbP and it occurred at the smallest scale tested.
Correlations are given for Newtonian systems;
power input due to sparged air is included, Methods for agitation-aeration optimization (including
costs) are given. Different operating strategies are
described and a specific optimizing algorithm is
developed. The procedures are more complex for
non-Newtonian systems. In a case given, an optimization routine automatically changes aeration
and agitation conditions to achieve the highest
OTR at any point in the fermentation. Presumably, 0, supplementation can be incorporated into
the model. A cost-benefit analysis would be needed
before such a control scheme were implemented
on a large scale. It often happens that optimum
rate or yield is achieved at great expense at production scale. Value of the product is a key determinant as to whether the cost is justified.

Work has been published on animal and plant

cell systems relating aeration to hydrodynamics
and shear impact. In one such article by Jones et
al.,43 there are reviews of some of the literature
and an attempt is made to understand the interaction of microcaniers and turbulence in an airlift
fermentor and to understand the protective serum
effect. Other protective measures from the literature are reviewed. Cumulative probability distribution for velocity is shown for a 5% serum solution at a superficial gas velocity of 1.3 cm/s.
Other solutions and other gas velocities are analyzed. Unhappily, effects of serum concentration
on hydrodynamics could not be shown conclusively. Although no definitive answers are given,
the methodology is interesting and might be enhanced or expanded in the future.
The driving force for oxygen mass transfer
can be increased by raising c* (0,concentration
at the gas-liquid interface). 0, supplementation
is one method; another is raising the operating
pressure in the reactor headspace. It should be
recognized that other gases (as CO,) will have
an altered, greater solubility at elevated pressure. This latter alternative is detailed in an article by Yang and Wang.@ The article lists a
reasonably large number of references related to
enhanced 0, transfer by conventional and
nonconventional means and should be consulted
for that listing of recent publications. In the reference cited, both E . coli and Ochromonas
malhamensis (a fragile alga) were studied. For
both a chemical system and E. coli, normalized
maximum oxygen transfer rate is directly proportional to normalized pressure. For these organisms -compared with modest agitation-aeration conditions -increasing pressure (and using
pressure programming) did overcome oxygen
limitation. Cell density was enhanced in each
case. Pressure manipulation adds another dimension of maneuverability when agitation-aeration
constraints already exist. It should be noted that
many industrial fermentations are operated at or
near 2 bar. Even if the vessel could tolerate 3
bar, there may be limitations due to air compressor rating and system pressure drop. Also moving from 1 bar to 2 bar means p2/p, changes by
a factor of 2. Moving from 2 to 3 bar changes p2/p1
by a factor of 1.5. Still, as the authors note, the

procedure is usually not costly to evaluate. Also,

it is not necessary to operate at elevated pressure
(or to supplement with gaseous 0, for that matter) throughout the entire fermentation cycle. It
may be possible to program or stage the change
to correspond to intervals of low- or zero-dissolved oxygen concentration.
Stirred-tank and airlift tower loop reactors
are compared by S ~ h i i g e r lThree
. ~ ~ different antibiotics were synthesized in each system (airlift
0.06 m3 and STR 0.02 m3), and an effort was
made to optimize medium as well as inoculum
quality and quantity. Some adaptation of cell
morphology improved performance in the airlift
reactor. Results varied with the various filamentous microorganisms used; insights into potential
scale-up studies can be extracted.
Okabe et al.46used DO control to successfully scale-up an optimized fermentation for a
desired antibiotic. Streptomyces sp. produces
cephamycin C as well as the undesirable penicillin N. A complex medium was used and DO was
controlled (varying agitator speed at I v/v/min
air). After establishing conditions at the reduced
scale, the process was scaled to 1500 1. Results
indicated that cephamycin C content was increased
1.3-fold and penicillin N was not detected at the
end of the run (this was ca. 7 d, but it was not
present as early as 2 d). One interesting aspect
was use of a transfer technique, that is, after inoculation of the 1500 1 vessel, broth was transferred to a 20-1 vessel. Both were run simultaneously; in this manner, inoculum and medium
variations are eliminated.
Practice and theory are combined by Sumino
et al.47Two stirred-tank fermentors (0.2 m' and 2
m3) with two sets of 6-bladed turbine impellers
were studied. The well-known sulfite oxidation
method was used and results were compared with
published correlations. Interestingly, existing
correlations were not successful when applied to
0,-enriched air. The authors developed a new
empirical equation that gives correlation coefficients of 0.926 and 0.947 for air and 0,-enriched
air, respectively.
A scale-up problem in a Micromonospora
fermentation is described and solved (Yabannavar
et al.48).The process was run and considered optimized at the 10 to 100 1 scale. It was scaled to

221

120,000 1 scale and there was a noticeable loss of

antibiotic titer. The first thought was that CO,
level was responsible; this was shown not to be
the case. Small-scale fermentations were run at
different pressures. Control was 1.68 atm; other
tests were run at 2.03 and 2.68 atm. Cell growth
was retarded and titer reduced at 2.03 atm; the
situation worsened at 2.68 atm. The change was
not due to dissolved CO, or pH, which was kept
essentially constant. The tentative conclusion was
that the high DO level in broth was the cause of
poor growth and low titer. Fermentations started
at low aeration, reduced agitation, and reduced
backpressure (which were changed as the fermentation progressed) resulted in reduced lag. The
variables were then adjusted to reach DO levels
that exceeded the limiting value. Results were
equal to control run at reduced scale. The concept
of low initial air or agitation has been used in the
past (especially where redox levels must be kept
within an acceptable range) in industrial practice
based on empirical performance. The article cited
presents some explanatory data and the early
growth phase of every culture process deserves
attention.
Damage to animal cells in bioreactors is reviewed by pap out saki^.^^ Theoretical considerations are explained and available experimental
data are analyzed. Various interactions (bead-tobead, bead-to-reactor, agitation-aeration) are discussed. The mechanisms are poorly understood,
but responsible factors are considered frequency
of bubble breakup (including coalescence) and
severity of shear stresses resulting from entrapment in draining liquid films or bubble breakup.
Suggestions for problem minimization include
design for smaller and more rigid bubbles (i.e.,
avoid coalescence and breakup). Shear protectants prevent or reduce damage by both reducing bubble breakup and because cells are not
sheared in liquid films between bubbles. Similarities and differences between free suspensions
and microcarrier culture are reviewed and analyzed.
Oh et al.50 studied growth of mouse hybridoma cells at different DO levels and different
sparging conditions. Bubble/impeller/cell interactions are considered. With the murine cell line
studied, these areas were studied:

222

Importance of DO level
Mechanisms by which oxygenation causes cell
damage via sparging in stirred reactors
Whether and how cell damage can be reduced
Headspace oxygenation was used in some cases.
Sparger position relative to the impeller was also
studied. Pluronic F-68 (at 0.1% w/v) was used in
a limited number of runs. One basic conclusion
(for the TB/C3 cells at the scale used) is that DO
levels at 5 to 100% with no sparging have no
effect on culturing. Bubble sparging was always
deleterious at any DO level. As sparge rate was
reduced, growth approached results of the
unsparged control. Further, it was found that the
way gas is dispersed has a strong impact on cell
growth and viability. Sparge rate alone is not a
good correlator of cell damage. Pluronic F-68
addition was protective. Bubble diameter was a
key indicator (bubbles less than ca. 5 mm are
more damaging). Sparging to enhance gas-mass
transfer is feasible if sparge rate is kept low (10.02
vvm) and large bubbles (>5 mm) are formed and
not broken up by agitation. Addition of selected
and screened surfactants is also suggested.
Authors noted earlier (Yang and Wang) have
also worked in the area of cell damage due to
agitation and aeration as well as the interaction of
both. A recent article51gives results of the extensive experimentation. A fragile alga was used as
the test organism (Ochromonas malhamensis) and
a number of different cell-counting and viability
measurements were made. The MTT (blue
formazan color development) assay was used to
monitor mitochondria1damage. Vessels used were
1.1- and 10-1 volume. Analysis of the data led to
a unified bubble breakup and coalescence model;
specific cell death rate was linearly related to
specific bubble interfacial surface area. There is a
strong coupling or interaction between agitation
and aeration that results in cellular damage. Even
though empiricism normally results in designing
gas mass transfer systems for cell culture, findings in this article can be used to more rationally
design scale-up studies.
In spite of the requirement for actual testing
and evaluation of the microbial or cell system to
be scaled up, many workers continue to develop
formulations for general use, Both theory and

practice (usually ideal cases) result in correlations

of greater or lesser utility. Some recent examples
can be consulted for the methodology. Moser et
a1.52operated a deep jet system using a NaCl
tracer. The tanks-in-series model was evaluated.
An actual scale-up study involving agitationaeration studies and optimization is reported by
~ ? organism
workers at Kyowa Hakko K ~ g y o .The
is a strain of Rhodopseudomonas spheroides and
the product is coenzyme Q,, (CoQ,,). The specific mutant used provides the best product yield
under a limited 0, supply. Oxygen transfer, CO,
evolution, and oxidation-reductionpotential (OW)
were key scale-up parameters. Pilot runs (30 1)
were performed and specific respiratory rate (0,)
was plotted against the ORP and intracellular
CoQ,, level as well as cell growth rate. Not surprisingly, optimum ORP for product formation
was not the same as optimum ORP for cell growth
rate. Finally, data are given for 30 1,40 kl, and 80
kl fermentors. A close correlation is shown between respiratory rate and ORP; ORP can be used
as an effective scale-up parameter. A small sidelight of the article was potential overheating of
medium at the larger scale (apparently resolved
by medium supplementation).
In recent years, advances in impeller design
have been made. Novel designs result in enhanced
mass transfer at a constant power input when
compared with the historically used flat blade,
disc turbine. Alternatively, mass transfer could be
held at a constant value, whereas a retrofit (moving to the new impeller design) would result in
lower energy (mixing) costs. In one such design
- at a power input of 1.5 HP per 100 gal and a
superficial gas velocity of 6 ft per min -there is
an enhancement factor of about 15. Actual results
using one such design in simulated xanthan broth
are given by Galindo and N i e n ~ w . ~ ~
The importance of attention to detail and exactness in scaleup are shown in a paper by Bourne
et a1.5 Three vessels (0.045, 0.45, and 4.5 m3)
were run with an 0,-sensitive culture fB. subrilis)
producing acetoin and 2,3-butanediol.In the smallest tank, deviation in solvent ratios was noted;
this was related to DO content in the medium.
There was a lack of geometric similarity, that is,
the shaft diameter in the smallest vessel was not

properly scaled to vessel diameter. When geometric similarity was achieved, solvent ratio was
constant and independent of scale when power
per unit volume and superficial gas velocity were
held constant. No detail in scale-up can be overlooked or neglected; unexpected results will surprise the unwary.

VII. DOWNSTREAM PROCESSING

A separate review is probably required for
problems in scale-up limited to downstream processing. In a very short general review, E n d e r ~ ~ ~
describes the challenges in scale-up of biopharmaceuticals and discusses . . .the problem of maintaining (engineering through equipment design)
the same environmental conditions for all production and purification steps in the manufacturing
scale as exist in the clinical trials scale; and assuring that those conditions are in fact equivalent
(quality assurance). It is not possible to have
the same environmental conditions at every
scale, so it might be better to consider the scaleup challenge as giving assurance that the process
profiles and product profiles and purity are unchanged at production scale. That is, the focus
should be on the product rather than exactly on
environmentalconditions, although the latter constancy on scale-up is desirable. In the review
mentioned, only a few unresolved purification
issues are highlighted:
Cell disruption
Adsorption processes

Crystallization

Longer hold times result

in increased activity loss
Extended processing
times gives undesirable
side reactions
Crystal size distribution
varies at different scales.

Because there are three dozen (or more) unit operations or unit processes that can be considered
part of a downstream bioprocess, there is a great
potential for scale-up problems. The number will
be some multiple of 36. Specific areas of concern
are covered in review articles. The articles should
be consulted for problems already encountered as
well as a number of solutions to some of them. A

223

list of extraction/purification processes is given in

Table 9.
TABLE 9
Downstream Processing
Separation

Concentration

Filtration

Membrane system
(RO/UF)
Extraction
Distillation
Precipitation
Crystallization
Evaporation
Ion exclusion
Stripping (vacuum or
gas)

Membrane system
Floatation
Gravity
Centrifugation
Flocculation
Disintegration

Purificatlon

Crystallization
Chromatography
Ion exchange
Carbon treatment
Electrodialysis
Adsorption
Affinity partitioning

Lyophilization
Spray drying
Tray drying
Vacuum drying

Reactionshodifications

Final product

Washing
Hydrolysis
Immobilization
Racemization

Formulation
Dosage form
Adjuvants
Sterilization

Yarmush et al.57review immunoaffinity purification. With the advent of highly specific

compounds produced at rather low concentrations, there has been a growth in use of highly
specific adsorbents. The authors give activation
procedures, review coupling chemistries, and
discuss factors that influence stability. Four factors are listed that can cause loss of column
capacity in the course of repeated operation.
These are
Nonspecific binding
Irreversible denaturation (usually related to
harsh elution)
Ligand leakage
Enzymatic or nonenzymatic proteolysis

224

Furthermore, antibody leakage could result in

product contamination. Advantages and disadvantages of the technology are discussed and
many practical problems (including some remedies) are reviewed. In spite of the cost of the
technique, its application may be enhanced by
the nature of biomolecules of interest. There are
104 references listed.
Japanese workerP present scale-up data on
hydrophobic interaction chromatography (HIC)
for purification of a fermentation derived
biomolecule. The purification process is rather
involved and includes ion exchange chromatography, HIC, hydroxyapatite chromatography, and,
finally, gel filtration chromatography. The initial scale used a 16-mm ID HIC column; the
process was successfully scaled to a 1 13-mm ID
column.
It is also possible to find successful purification schemes in the patent literature. Merck has
a European patent59application that is entitled,
Purification of recombinant Epstein-Ban virus
antigens from VERO cells, yeast cells or L cells.
The two major EB membrane antigens, gp 350
and gp 200, are synthesized and purified in preparation of vaccines. The applicants state, The
present invention is amenable to scale-up for
producing large quantities of antigen suitable for
commercially produced vaccines. One example
starts with a recombinant mammalial expression
system, proceeds to cell disruption, one or two
steps of lectin affinity chromatography, gel filtration chromatography, and certain additional
steps, which may be required. Use of protease
inhibitors is described. Other steps give various
products that might be used. Although problems
in purification are not described, there is a reasonable amount to learn from successful techniques.
It is probably a good idea to spend some
time reviewing general concepts in bioproduct
separation, that is, reflection and consideration
of alternatives should precede creation of a flow
sheet. Too early or too hasty a selection may tie
the process to techniques that later prove less
than optimum; issues of validation and/or clinical testing may preclude major changes. One
such overview is given by Fair.60 He uses a
process engineering approach and begins with a

culture broth containing less than 1 g/l of product. Separation system constraints are described,
but one or more steps consisting of conventional
filtration, ultrafiltration, centrifugation, and sedimentation can be used. Product concentration alternatives are discussed. Product isolation, using
specializedtechniques, are reviewed, as well. After
this generalized discussion, more specialized techniques - with a wealth of published information
and references - are covered in depth in a slim
volume.61There are five long chapters with major
headings on large-scale gradient elution chromatography, membranes (includingreactors and electrically driven membrane processes), aqueous twophase systems (with lists of applications), affinity
interactions, and selected precipitation. Biomolecules are discussed throughout, theory is
given, optimum strategies are discussed, and many
references are included for each subject.
Although there is not a great deal of published information and data on industrial applications of newer bioseparations, an article by
Genentech workers6*is clearly outstanding. There
is a thoughtful review of various systems with
problem analysis; finally, tangential flow filtration was selected for recovery of kilogram quantities of rt-PA. The goals were established at processing SO00 1 of conditioned medium per hour
(1.25 x 104 1 to be processed in less than 3 h);
protein yield was to exceed 99%. Actual equipment selection is described, design of the full
scale system is described in some detail, and a
photograph is included. Membrane regeneration
and sanitization is reviewed, again with some
detail. The final system is 180 m2in area; a safety
factor was included. Plots of transmembrane pressure vs. time are given. One sentence in the article
deserves stress, The determination of process
capacities as a function of flux rate under various
fluid dynamic conditions is of vital importance to
the economic implementation of large-scale processes for industrial use. Major problems in scaleup result when this point is either misunderstood
or disregarded.
It is now fairly well accepted that cost analyses of older fermentation processes followed
the rule of thumb that divided fermentationcost
and isolatiodpurification cost into a 1:1 ratio.
That is, the fermentation step cost some SO% of

overall unit product cost, whereas isolation or

extraction cost made up the other 50%. In the past
2 decades, the move toward recombinant organisms and far more exotic compounds (produced at
extremely low titers) means that the fermentation:isolation cost split is now more like 1:8
or 1:lO. That is, the culture step might incur 10%
of unit product cost, whereas isolation cost is 90%
of the total. It should be clear, therefore, that any
early choice of an isolation route (especially for a
regulated product) might well incur heavy later
production costs. There is a delicate balance that
comes into play early in the development cycle.
On one hand, there is the demand for speed this may be related to one or more of need for
patent protection, the start of animal trials, competitive pressure. Although the research scientist
has a very wide spectrum available of devices,
columns, adsorbents, and techniques, there is pressure to make product. Yield is not really critical
at this point. The tendency will be to select apparatus/procedure that worked. Not unexpectedly,
once some product is made, demand escalates.
Trying alternate routes (better rate, yield, lower
cost) is discouraged as more testing will be needed.
Once again, the tried and true is used. There may
come a time (and this may occur early in the
development cycle) when validation becomes an
issue. At this point in time, changing the purification process means revalidation. This may be a
costly and serious hurdle. Therefore, choice of an
isolation route is not a small matter. Alternative
procedures (which may or may not include usetesting) should be an early criterion. It is not an
unreasonable suggestion to consider alternate
purification paths run in parallel. A small change
in yield or purity may result; this could have
enormous impact later. Furthermore, selection of
flexible or multiuse equipment or apparatus offers potential for new products discovered in the
future. Once validation becomes an issue, having
alternate confirmed procedures or techniques
would be of great utility. Of course, development
costs would be higher. It is likely that as the
newer bioprocesses mature, final product selling
price will become smaller and smaller multiple of
final production cost. An early and thorough analysis of recovery/purification economics is an excellent idea.

225

The general problems that occur on scale-up

are those well known in unit operations; those are
changes in flow profile (alterations in ideal plug
flow pattern), temperature gradients, changes
in cycle time (excessive hold times), improper
size distribution on initial column packing,
extent of regeneration, column packing useful
life, change in velocity profile, over or under
heating on drying, and, of course, improper
entry of contaminants, be they microbial or
chemical. Time-related variables are especially
hard to fix on scale-up. If an expensive packing
is used (with or without special ligands or
adsorbents attached), how is cost per cycle to
be determined without knowledge of useful life?
Even if an ion exchange bed were to be used,
what provision should be made for complete
replacement? Number, type, and frequency of
regeneration cycles is of obvious importance.
Running successfully for 4 months or even a
year is no guarantee of process longevity. Only
actual data (perhaps gathered from a continuously running automated system) will resolve
the problem. Another problem with some
adsorbents is that the adsorbent itself may be
attacked (as by microbial or enzymatic contamination). Preservatives must be added either
during storage or, periodically, in cleaning cycles.
One can imagine the result when the preservative
is not removed prior to use or, when washed out,
the preservative is not totally removed. In purification steps, reagent and water purity are of obvious importance; simply specifying the highest
grade available may prove both costly and inappropriate. The other extreme (use what is conveniently available) is not suggested, as performance may suffer. The appropriate scale-up
evaluation with selected purity water, reagents,
and regenerants should follow thorough review
and some laboratory evaluation.
An interesting overview of biotechnology
.~~
more
scale-up is given by R o ~ e nAlthough
philosophical in tone than totally technical, a
broad-brush analysis of technology transfer from
R&D to large volume production is given. Differences and similarities to chemical processing
are covered. Properties of culture broth are detailed (each of which may lead to many diverse
problems):

226

Mechanically fragile
Temperature sensitive
Quality deteriorates rapidly

Expensive
May be corrosive
Environmental
escape

Rheologically troublesome
These are some of the factors that lead the author
to state, Working out a satisfactory separation
sequence is alone a tedious process; adding the
various economic ramifications makes it a formidable task. There is also an understanding that
scale-up involves nonideal performance and always involves a series of compromises. The technical hurdles invariably involve more time and
money that initially planned.
Another general review of biotechnology
scale-up is given by Trilli.@Each specific area of
concern is covered, even if in a terse way. Subjects include sterilization, agitation-aeration,medium ingredients, heat transfer, culture stability,
process characterization, and broth rheology. In
the agitation-aeration section, subtopics include
power dissipation, shear rate distribution, and
pumping capacity. Nine published correlations
between K,a and agitation-aeration parameters
are given. An empirical flow diagram is given for
a generalized scale-up procedure:
1. Define set of target values (experimentally

determined)
2. Compare with limit values on large scale
(lab and published data)
3. Combine these inputs to establish target
4. Define operating conditions for small pilot
fermentor
5. Perform experiments; analyze data
6. Define operating conditions for larger pilot
fermentor
7. Carry out experiments
8. Define operating conditions for production
fermentor
9. Carry out trials
10. Optimization and process evolution
11. Production
Obviously, if any result is unsatisfactory, return to an earlier step for reevaluation, a change
in operating conditions, or setting of new targets.

After a review of bioreactors, bioprocesses,

and downstream processing, it may appear that
there is an infinite potential for problems in scaleup. As one proceeds in scale-up, it still may appear that there is an infinite number of variables
to evaluate and a problem potential exists in each
one. In Table 10, some selectivity was used to
limit the number of scale-up problems. These
problems do not cover the potential spectrum but
they have occurred in the authors experience. As
such, they are probably reasonable places to start.
Should another scale-up program evolve and exhibit none of these problems, that program is off
to an excellent start. One other use of the listing
shown might be to review it with research personnel (or persons who have done early discovery or
development). Very often, one or another problem arose and was resolved; both the type of

problem and the resolution should be of interest

to the scale-up team. Either the problem may
recur or the resolution is only a temporary fix.
The same sort of discussion should occur between the scale-up team and the manufacturing
group. As noted, failure analysis is just as important as analysis of successful operation.

VIII. STERILIZATION AND CLEANLINESS

Mammalial cells or other microorganisms are
used for full or partial synthesis of desired products. Extreme controls are suggested (and sometimes mandated) or performing the biologic process and for preparation of the final product.
Throughout the process, design criteria must consider the culture (inoculum, cell banks), the pro-

TABLE 10
Scale-Up Problems
Actual Cases
Conventional systems
Byproduct formation (nonphysiologic forms)
Foaming
Cell lysis (related to hold time)
Odor
Culture degeneration (increase in doublings in inoculum buildup)
Characteristics of process water
Poor temperature control (related to heat load)
Weld problems (contamination)
Bacteriophage attack
Effect of defoamer (conventional and draft tube aeration)
Retrofitting (force fitting of process to existing plant)
Film formation (fouling)
Improper emptying of vessels (NPSH problem)
Aerosol formation

AnimaVplant cell systems

Media stability
Variation in supply of natural product
Improper cleaning agents (volatile and nonvolatile)
Materials of construction (including gaskets and seals)
Use of treatment chemicals (as steam)
Unexpected differentiation
Mycoplasma
Sterility (overall)
Recovery and recycle of suspendinglattachmentagent
Hydrodynamic shear
Product stability (both culture and purification cycles)
Waste streams (hazard)

227

cess, nearby personnel and the surrounding community. Gross contamination must be guarded
against and the contamination potential is bidirectional. Contamination refers to entry of adventitious materials, undesirable microorganisms or
their byproducts, foreign chemicals, or leakage
outward of process streams up to and including
final product.
Sterilization may be achieved by various
means. What is facile at a small scale is often
difficult or impossible at a large scale. Examples
of sterilizing methods are
Moist heat
Dry heat
Radiation

Filtration
Chemical agents
Low-temperature plasma

Problems related to sterilization studies and

scale-up are described:
1. Many culture media contain labile substances. Autoclaving (e.g., 121C for 30 min)
a 50-ml sample is routine. There is usually
a rapid heat-up cycle, the 30-min hold follows, then a relatively rapid cool down follows. The time-temperature relation gives
total heat input. Sterilizing 500 kl of the
same liquid is more complex. Batch sterilization involves a long heat up and may
involve steam injection, the 30-min hold is
the same as before, then there is a relatively
slow cool down. The time-temperature relation is very different. Total heat input is far
higher than in the small-volume sample case.
Not only can heat-labile substances be lost
(degraded) but reaction products (as from
the Maillard reaction) might form in appreciable quantities. Continuous sterilization
(so-called high temperature-short time, or
HTST) can shorten heat-up and cool-down
times and because hold temperature is normally well above 12loC, hold time is very
short (it might be measured in seconds or a
few minutes).
2. In laboratory experiments,an autoclave shutdown or cycle error normally means preparing a new batch. When large volumes of
valuable substrate are involved and untoward temperature fluctuation occurs, dis-

228

card and repreparation are wasteful. Can the

media be resterilized? It is usual to have
studies performed on exrreme cases of sterilization in the course of scale-up studies.
Resterilization can be performed (on small
scale); process responses can be studied. If
radiation is used, can culture vessels or media
containers be resterilized, whether single use
containers or recycled containers are used?
Remember that washing and recycle is different from single use modes; both should
be validated at extreme conditions (i.e.,
multiple radiation cycles or multiple autoclave cycles). It is hazardous to assume that
oversterilization (whatever the technique) is
safe. Sterility can be assured but process
performance cannot.
3. Changing from one 0.2-pm filter to another
may be harmless in lab use. (The preferred
course is to validate at every scale.j For cellculture media at production scale, this may
or may not be harmless. Testing of sterilizing filters for potential cytotoxicity is cursory by the manufacturer. Even if it were
not, the cell line and the medium used in a
new bioprocess were most likely not checked
by the filter manufacturer. Type, size, manufacturer should always be validated during
scale-up. If the filter is to be reused and
resterilized a number of times, scale-up testing should include a larger number of reuse
cycles than the production process will ever
undergo.
4. Dry heat is used for materials that withstand
elevated temperatures for extended periods.
Elimination of endotoxin is often achieved
concurrently with dry heat sterilization.The
process and equipment should be validated
for both sterility and absence of endotoxin.
In this case, somewhat less severe conditions should be tested (than those to be used
in routine processing) and validated. In this
way, a slight excursion (but within the validated range) would not constitute an out of
spec condition.
5. Loading of autoclaves, ovens, radiation
chambers is a key variable. In sterilizing
equipment validation, many different configurations are tested. Of course, a load

6.

7.

8.

9.

condition more severe than that to be used

is checked. If liquid volumes are to be
sterilized in an autoclave, surface temperature is not the key criterion. Temperature (and time at temperature) at the center of the liquid mass - or at the point in
the mass that reaches minimum temperature last - is critical. Sensors or sterility
test items (strip or vial) must be located at
a number of sites, including the key location noted.
Sterilization at extreme conditions (whatever the method) might release agents or
chemicals in the item to be sterilized. The
best method in this instance is an actual use
test. That is, sterilize the vessel, container,
tubing, plasticware at an extreme point, then
evaluate utility as well as sterility. Items to
be reused must be evaluated after a number
of cycles greater than that to be used in
practice (as noted earlier).
Membrane filters are in common use in large
volume air filtration. These filters must be
installed properly and must be sterilized
under carefully controlled conditions.
Prefiltration is also desirable. In some cases,
condensation is undesirable. Therefore, air
temperature must be well above the dew
point.
If a chemical agent is to be used for sterilization, processing should occur at concentrations and times greater than, and less than,
the design criterion, an acceptance range
should be created that ensures:
Complete sterility over the range
No degradation or reaction of the desired
product
Complete loss, elimination or degradation of the sterilizing agent (absence in
finished product)
No byproducts that are harmful or unexpected
If steam contacts a process stream (or surfaces that contact the process/product
stream), the purity of the steam must be
considered. There are requirements for process steam in food, dairy, and pharmaceutical facilities. Commercial equipment is available for generation of clean steam. In a

retrofit facility, quality of steam to be used

is a critical point.
10. Phage infection may not be a problem in a
lab or pilot plant. Routine processing at production scale (large number of batches,
higher volumes, aerosol formation) creates
the ideal environment for phage magnification and infection. There are at least two
counters: design to prevent phage attack and
have phage-resistant cultures available. For
cell strains, mycoplasma is an ever-present
danger. Extensive screening (at every step
of cell bank preparation) and prompt destruction of mycoplasma infected banks are
countermeasures.

A. Aseptic Design
Aseptic design is a massive subject. References are available that cover the large number of
details to be considered.6s67
Problems in scale-up are both diverse and
complex. Some of the problems can be summarized:
1. All pipes to be used in an installation should
be sloped to drain. Standing liquid is an
invitation to problems.
2. All piping leading into or out of a fermentor
should be steam sealed; for example, they
can be maintained under positive steam pressure when not in use (121C minimum). It
may be necessary to cool the pipe prior to a
transfer in or out.
3. If possible, use a magnetically coupled agitator seal (no stuffing box). If an agitator
seal is to be used, a double mechanical seal
is suggested. Seal faces should operate at
high temperatures or have a high-temperature lubricant (examples are steam or sterile
defoamer). If a hazardous culture is under
study, the mechanical seal should be encased in an outer, emergency seal assembly
(failure of the inner agitator seal will not
cause hazardous spray or discharge).
4. Sensor inserts should be of fail-safe design;
an inner seal or O-ring failure is not catastrophic.

229

5. If necessary, off gas must be rendered harmless by filtration or incineration.

6. Valves should not have rising stems; preferred mode is to have no path between the
process stream and the environment.
7. Inoculation should be affected without exposure of fittings to the nonsterile environment. Ideally, any connection made should
be sterilized prior to use.
8. For clean rooms: Proper design and construction to preset quality level, appropriate
testing and validation, appropriate training,
and monitoring.
9. The gas exhaust line of a fermentor must be
carefully designed. Entry of contaminants is
possible at this point. Viewports should be
provided, sloping is essential, provision for
cleaning and/or sterilization of the effluent
gas line are all useful considerations.
10. Positive pressure should be established and
maintained in a sterile vessel, a sterile
transfer line, or a sterile space (as a clean
room). Development of reduced pressure
(as a partial vacuum due to steam condensation) could allow entry of undesirable
organisms.
11. There should never be a permanent connection between sterile and nonsterile parts of a
system. Temporary connections, as for cleaning, are permitted but only when a specific
function is being performed; otherwise, disconnect.
12. All parts of a sterile system should be designed for ease of cleaning and ease of inspection. A periodic inspection schedule is
recommended.
13. All instrument holders or vessel entries
should be sloped to drain. Prevent formation
of dead spaces or pockets by proper design.
14. Manifolding is permissable but not desirable. Redundant systems or check valves
should be used to prevent improper transfer
or cross-contamination.
Sterile filtration is used more and more as cell
culture systems are scaled up. There are many
filter manufacturersand many different filter media
now available. Some systems permit reuse; others
do not. Quality control on purchased units is critical. If possible, select systems that have under-

230

gone a pressurization test (to assure absence of

pin-holes or breaks) as well as outside sterilization. It is useful to perform a pressure test inhouse after a filter has been used. If the used filter
fails the postuse test, media filtered should be
refiltered or discarded. As noted elsewhere, any
change in filter supplier or design or method of
sterilization should stimulate revalidation.
C h i s P gives details on design and operation
of bioreactors with sterility in mind. Actual valve
sequencing (with appropriateflow diagrams given)
is given to assure complete flushing and heating.
Inoculum transfer and sampling are covered. The
author recommends against use of flexible transfer piping (need for elimination of air pockets and
condensate pools). In my experience, Teflon@lined, stainless steel braided flexible hose can be
used routinely if proper fittings and procedures
are utilized.
Two other areas will be touched on. The first
concerns validation of cell banks. When products
for human use are produced by mammalial cells
or when human cells themselves are used (as in
tissue engineering products), it is necessary to
fully validate the cell strains. Whether procedures
are performed in-house or at outside laboratories,
work should be conducted under good laboratory
practice (GLP) regulations of the FDA. Suggested
tests include:
Tumorigenicity in athymic nude mice
Mammalial cell growth potential in soft agarose
Evaluation for adventitious viruses (in vivo)
Evaluation for adventitious viruses (in vim)
Evaluation of HIV-1 (antigen capture ELISA
technique)
Karyotyping and species identification
Evaluation of cytomegalovirus status (in v i m )
Retrovirus particle detection by electron microscope
Detection of Hepatitis B surface antigen (enzyme
immunoassay )
Detection of xenotropic retroviruses (mink S+Lfocus-induction)
Bulk sterility
Mycoplasma assay
Detection of Epstein Barr virus DNA sequence in
cell genome
Detection of retroviruses (reverse transcriptase
and DNA polymerase activities)

One should review the FDA document Points to

Consider in the Characterization of Cell Lines,
dated 1987. Preparation of a cell bank for production use is time-consuming and complex in any
event; the added time and cost in the validation
effort means that every precaution -taken earlyon -will be well worth the effort. The validation
testing may take 3 to 4 months after test samples
are delivered, and full testing will cost between
$40,000and $50,000 (1992). Failure in one of the
final tests will mean not only waste of a great
amount of resources but also delay in scale-up or
production.
The other issue concerns final product testing. The full scope of this subject cannot be covered here, but it should be obvious that product lot
analysis is an area that must receive careful and
specialized attention. A short review article69gives
a list of relevant documents issued by the Center
for Biologics Evaluation and Research of the FDA,
by the European Community and by the Canadian
Ministry of National Health and Welfare. A shortflow diagram gives seven general subject headings; each step is both costly and time consuming.
Each step must receive detailed attention and a

time table for all intermediate steps should be

established. Any one of the many substeps has the
potential not only to create a scale-up problem but
to derail the entire development sequence. The
seven general steps are preliminary cell line screening, characterization of master cell bank (MCB)
and manufacturers working cell bank (MWCB),
in-process testing, validation of the purification
process, characterization and testing of final bulk
product, studies on toxicology and pharmacokinetics, clinical trials. It should be obvious that the
seven steps could very well be doubled or tripled;
the important point is that an overall project schedule be established that encompasses all required
testing.
Selection of level of containment required is
an important criterion from both a cost and an
operational viewpoint. Table 11 lists various design points or associated points to consider. There
is a short description for the highest containment
level required, that is, not every rDNA or cell
culture production facility requires satisfaction of
every design criterion. An analysis of the organism and the process will probably lead to unequivocal answers to many, or even most, poten-

TABLE 11
Containment
Highest containment level
Process separated from environment
Exhaust gases
Seal design
Valve design
Transfer (in or out)
Harvest
Spill or leak
Ambient pressure
Safety

Access
Washing/decontamination
Input and exit air (facility)
Facility
Effluent treatment
Air testing
Training
Samples, sinks, showers, drains

Absolute - use closed system

Complete treatment - incinerate or filter (full validation)
Totally enclosed - failure alarm
Totally enclosed - bellows or alternate
Prevent release - double pipe
Total cell inactivation by chemical and/or physical means
Containment - emergency response and inactivation
Lowest pressure (negative to rest of structure)
Posted areas, trained emergency response team
Airlock entry and exit
Special hoods and garments
Severely restricted - testing of personnel
Essential facilities with required showering
HEPA filtered
Sealable for total disinfection
Full containment, complete inactivation
Routine testing in limited access area, rest of building, and
surrounding environment
Complete and documented with periodic
retraining
Collection and complete inactivation

231

tial problems. The few uncertainties should be

resolved with the aid of experts in the field. It is
probably safe to say that the more cost is deemed
the key to a judgment call, the more likely regulatory problems (or worse) will ensue. This is not
a plea for overdesign; it is a suggestion that experience and expertise (with regulatory input, if required) be the keys to an appropriatejudgment on
level of containment to be installed.
There is a federal guideline on level of containment applicable to good industrial large-scale
practices (GILSP) with emphasis on use of recombinant DNA-derived industrial microorgani s m ~A. ~table
~ (Appendix K, pg. 33175) compares requirements for 21 design and operating
criteria. Many design criteria are detailed; the
guidelines are not onerous and should be reviewed if a new facility is planned or a major
retrofit is to be undertaken. Many of the points
cover personnel entry and personnel protection;
these points, if followed, could protect the process and product as much as they protect individuals.

materials compatible with the sterilization

process?
Is there proper inventory and production flow
control to prevent mixups between sterile and
nonsterile products?
Is packaging adequate to maintain sterility
during acceptable shelf life?
Are chemical and/or biologic sterility indicators in use? Are they appropriate for the process and product?
Is there meaningful testing of product and packaging after sterilization?
Is resterilization permitted? Are adequate
records maintained regarding reason for and
results of resterilization?
Are cleaners and sanitizing agents approved to
assure compatibility and effectiveness? Are
residues measured?
Is there a proper investigative method to follow up on sterility failures?

The process and product to be scaled up

must pass a host of internal hurdles. Sterilization - at one point in the process or another
(and in many cases, at multiple points) - is
only one of these internal hurdles. If a healthcare
product is involved, government regulation is a
given. This is true in most countries around the
world. Sterilization is one (albeit very important) part of the codes of good manufacturing
practices (GMP).71
In the US.,the Food and Drug Administration has umbrella GMPs that cover various medical products including drugs and devices. Although
many of the concerns involve the actual manufacturing facility, actual equipment, and production
personnel, it is possible to select certain questions
that are relevant to the laboratory scientist and the
scale-up team. Examples are

These are only a limited number of examples. If answers to these and other questions
were to await planning of a manufacturing facility, valuable time would be lost. A great deal
of experimentation would have to be redone. If
these issues are faced in the laboratory and at
the initiation of scale-up, valuable information
can be gathered for both internal use and for
regulatory purposes. Further, design of the
manufacturing facility is simplified. It is never
too early to establish guidelines for sterilization. Once established and documented, regulators will have detailed proof that an effective,
controlled, and reproducible sterilization process exists. Performance qualification with the
actual process and product involves (1) product
functionality evaluation, (2) equipment performance evaluation, and (3) microbial challenge.
It is usual to have three identical runs performed. Under present regulations, the ultimate
responsibility for effectiveness of the sterilization method resides with the manufacturer. This
does not obviate the need for organized and
detailed information in these areas:

Is the environment (especially where pilot quantities are made) controlled to prevent undesirable product contamination from any source?
Are materials of construction and packaging

1. Type of process used and impact on product, including measurement of resulting

impurities and residues
2. Average bioburden and alarm limits

B. Regulatory Aspects

232

3. Product load configuration and relevant testing

4. Sterilization cycle parameters (and records)
5. Sterilization equipment used, including personnel training
6. Recertification and preventative maintenance
7. Type, number, location, measurement parameters (destruction rate) of indicators
used
8. Incubation media and methods for biologic
indicators used (include negative and positive controls)
9. Reference to approved procedures used
10. Safety level or inactivation level attained
11. Packaging materials used and labeling
12. Completely described validation methodology
13. Reprocessing
It is not an overstatement to say that if the
sterilization procedure is altered during scale-up
or if information (as noted) is lacking, there is a
good chance that a separate sterilization scale-up
program will be required. The complexity of sterilization coupled with its critical importance to
product utility indicate that serious consideration
be given this operation early on in the scale-up
process.
Of course, terminal sterilization is only one
method of assuring product free of adventitious
microorganisms. If the finished product contains labile materials or living cells (terminal
sterilization not possible), then sterile processing is a permitted alternative. In some ways,
sterile processing requires all of those concerns
listed and more; every process step presents a
potential contamination hazard. All ingredients
entering the process (media, cells, plasticware,
substrate, packaging) must be shown to be sterile, all surfaces contacting any of these inputs
must be shown to be sterile, any intervention
(human or robotic) must be shown to cause no
problems, and multipoint sterility testing for
both environment and process streams must go
on routinely. Bioburden (other than desired
cells) cannot be low; it must be zero. Many new
scale-up issues enter the picture. Elimination of
steps and process simplification - before and
during scale-up - may be critical for commercial success.

IX. VALIDATION
Generic issues of validation are of obvious
importance in any scale-up. Everyone involved
should be interested in assuring that the production process will result in a product/process attaining specified levels of yield, rate, and purity.
When a potential human or animal drug is involved, however, regulatory issues are involved.
Therefore, validation (involving one or more centers of the FDA) must have a higher level of
priority.
It is my contention that the later in the development and scale-up process that validation issues are considered, the more likely there will be
a time delay in achieving desired goals. Further,
the later in the cycle that a serious problem is
found, the more costly will be the resolution.
Although this is not a call for every researcher to
become a regulatory specialist, it makes good
sense for the members of the scale-up team to
become conversant with FDA language, terminology, and guidelines. The guidelines - while
serving a regulatory function - are sensible for
those involved in any process scale-up. This conclusion will become clear after reviewing many
of the concepts and suggestions presented by the
FDA.
One short document (ca. 10 pages) is worth
consulting for a general overview.72The FDA
definition of process validation is
Process validation is establishing documented evidence which provides a high degree of assurance that
a specific process will consistently produce a product
meeting its pre-determined specifications and quality
characteristics.

Further,
There shall be written procedures for production and
process control designed to assure that the drug products have the identity, strength,quality and purity they
purport or are represented to possess.

Although these are eminently sound practices,

they are more than philosophical in nature. Process validation is a requirement of cGMP (Current Good Manufacturing Practices Regulations
for Finished Pharmaceuticals [2 lCFR, Parts 210
and 2111). The guideline stresses the need for
documented procedures to monitor output and

233

validate performance of the manufacturing processes that might cause variability in characteristics of in-process materials and the product itself.
Documented procedures must be in place to prevent microbial contamination and validation of
any sterilization process is required. The guidelines document speaks directly to the issue of
scale-up:
During the research and development (R&D) phase,
the desired product should be carefully defined in
tenns of its characteristics, such as physical, chemical, electrical, and performance characteristics. It is
important to translate the product characteristics into
specifications as a basis for description and control of
the product. Documentation of changes made during
development provide traceability which can later be
used to pinpoint solutions to future problems.

An FDA investigator has written that process

validation may include development data that
describe the limitations and efficiency of the proC ~ S S . ~A narrative covering the manufacturing
process should describe and define the purpose of
each stage and expected quality at each stage.
Experimental or development batches made by
R&D personnel determinethe operations described
and supply raw data for process validation. Obviously, then, laboratory and pilot plant notebooks
and records may be reviewed by regulatory personnel. Full-scale production batches should have
been run to ensure consistency and to detail why
and how the scaled-up process is the same as that
desired and developed.
If any further evidence is needed to conclude
that process validation is (or should be) an integral part of the scale-up process, it can be found
in another FDA p~blication:~
In early developmental work, every step would usually have been examined (at least for extent of reaction) and every intermediate at least partially characterized with some estimate of purity. As experience is
gained with the synthesis, the critical reaction steps
and intermediates to be monitored are selected. At the
time of NDA submission, in-process control points
should have been selected and appropriate specifications and tests established to meet the requirements of
the regulations. This whole operation is part of the
process validation of the synthesis. The basis for selecting control points and intermediates should be
explained, and the adequacy of the specifications and
tests to control the synthetic process demonstrated.
The ranges for the operating parameters in the written

description of the synthesis should be chosen in light

of the controls (specifications and tests).

Although these requirements are aimed at

chemical transformation, the projection to biosynthesis is clear and obvious. Fermentors make
up only one set of equipment that must undergo
review and evaluation. Sterilization is only one
operation (albeit a critical one) in the fermentor.
Product and impurity profiles are important. In
simple terms: there must be fully documented
methods and procedures, there must be complete
analytical procedures, complete records, procedures for making changes and assurance (via
written documents) that consistency in producing
product having predetermined specification is
achieved. There must be a complete paper trail
from start to finish. Not surprisingly,perfection is
rarely achieved.
Although the regulatory requirements are
sufficient incentives for following appropriate
guidelines, it should be clear by now that the
correct approach will aid the scale-up process.
Because validation applies to the entire spectrum
of systems, process, documents, and programs
that support the production process, successful
implementation must include all or many of the
requirements of the validation process. For a product that will end in human use, the requirements
are mandated. It is probably never too early to
consider validation activity; at the latest, these
issues should be brought to the fore at the end of
the preliminary design phase. Of course, validation activity continues through the design phase,
through scale-up, and, finally, through construction and start-up. It is obvious that a premature
selection of culture vessel, separation technique,
or purification method will mean that revalidation
might be required if a significant change in the
process is made. During a well-planned validation effort, programs are put in place to provide
mechanisms for evaluating changes and their
impacts on a validated process or piece of equipment. The evaluation is multidisciplinary; one
person or a group alone cannot make the determination.
There are many steps in a well-executed validation program. Some definitions follow and these
are both applicable and useful for scale-up planning:

1.

2.

A Master Plan should be prepared. The

Master Plan will include the foundation
(rationale) for the entire validation program.
It will describe the project, detail training
schedules, outline basic procedures, set
schedules and resource planning, acceptance
criteria for equipment, table of SOPS, and
methods involved in actual validation, and,
in general, set the tone for the effort. The
Master Plan defines quality standards for all
critical production parameters and details
the efforts needed to support the manufacturing process.
Installation Qualification (IQ) is a process
that determines that the system, as installed,
meets requirements of the design drawings
and the written specifications. Included are
checks of purchased equipment and parts,
installation checkout documents, and the
actual installation itself. Records (such as
boroscope readings, passivation, radiographic analysis, test coupons) must be made
at the appropriate time. IQ auditing includes
reviews of calibration records, documentation and maintenance schedules. (See Table
12.)

TABLE 12
Installation Qualifications
Description of the process and its intended
purpose
Identification of items requiring calibration
Identification of items requiring maintenance
Establishment of the maintenance frequencies
and procedures
Cleaning procedures
Operating procedures; including how to adjust
the process
Trouble shooting guide
Basic procedures for monitoring and control of
the operation
A spare-parts list to assure that appropriate parts
will be available
Documentation that all appropriate manufacturing,
maintenance, and QC personnel have been
properly trained to control this operation

3.

Operational Qualification (OQ) determines

that each piece of equipment (individually
and in concert) operates as specified and

4.

meets design criteria and requirements for

the control of critical operating parameters.
OQ is used to establish a performance level
or baseline that provides clear assurance that
the system can do what it is designed to do.
When properly documented, OQ can serve
as a guide to ongoing troubleshooting.
Performance Qualification (PQ) determines
that equipment consistently meets appropriate design and QC specifications. Equipment, as installed and controlled, must provide satisfactory performance throughout the
accepted range of operating conditions. Process Qualification is not exactly synonymous with Performance Qualification. Normally, three batches of product are run;
production parameters should be varied (if
at all possible) to span the proposed operating range. PQ is required for critical utilities
(such as clean steam generation, water for
injection, certain gases). After Performance
Qualification is complete, Process Qualification is achieved after analysis of the three
(or more) batches, including isolation of
product.

At some early point in the validation process,

but after the Master Plan in complete, it might be
a good idea to review the plan and schedule with
a nearby FDA office. Because there are many
rules and changes are made fairly often, an early
exchange of views will be useful in establishing
appropriate emphasis. There is no requirement
for such a meeting but early correction is far more
efficient than a midcourse correction. The validation effort should also encompass review of SOPs
(compliance with GMP), emergency shutdowns
and reaction routines, preventative maintenance,
equipment calibration and recertification, cleaning and sanitation, and creation of indexed and
accessible files. If software is involved (monitoring, recording, and/or control), it, too, must be
validated.
A great deal of effort is involved in the course
of construction. The validation group is involved
in vendor audits, compilation of preinstallation
documents,and monitoring of construction.Whenever testing is performed, appropriate documentation must be prepared. Sensors must be shown
to operate within acceptable limits; when neces235

sary, traceability to a NIST standard should be

documented. Whenever necessary modification,
repair, or recalibration is required, the work
must be monitored and documented. Finally,
the prequalification acceptance testing is assumed to be complete and the system or plant
is turned over to the validation team. The validation team can be internal or external (consultants) or a combination of both. The key,
however, is that the validation team must report
to a high enough level within the company so
that needed changes can be made in a timely
manner. The validation effort is time consuming and costly; recommendations must be acted
on to avoid later and larger waste of time and
money.
With the plant complete, IQ, OQ, and PQ
are completed. The calibration program is executed and instrument calibration is completed
before PQ steps. A validation report can be
prepared at this time. All validation test results
are included, analyses of test data are given,
and the acceptability of the system or plant to
consistently perform within predetermined acceptance criteria is stated. Process Qualification follows (or may be included in the final
report). The regulatory guidance concerning
validation concludes that there are a number of
factors that could render the process out-ofcontrol. For a regulated product, the process is
considered out-of-control i f
1.

The process has never been validated or

has been validated improperly.
2. One or more parts of the process have
been altered to an extent that the new
process cannot be reasonably expected to
perform as it was designed (or as it was
once validated).
3. Instrument or sensor recalibration has not
been performed in a competent manner on
a routine basis.
4. Operating controls are not being followed.
This short review of the validation program
should show its importance to scale-up. Even
without regulatory involvement, there are excellent reasons for following a rigorous and

236

systematic protocol in moving from the lab to

full-scale production. The rule, What can go
wrong, does go wrong is universally applicable. No part of the validation effort can be
left out without introducing risk. Furthermore,
when problems do arise or when needed plant
modifications are to be made, one should have
a baseline to work from; proper validation provides this background or documentation and
recorded experience. Because validation covers the spectrum of procedure and equipment,
there are myriad problems that might ensue. In
Table 13, there are a number of issues or problems that recur. Although many of these problems have been discussed elsewhere in this review, they can be considered validation issues
in that documentation - addressing these issues - should be prepared. If approached at
the beginning of the validation effort (corresponding to the end of the preliminary design
phase, at the latest), there is a good chance that
some accommodation or adjustment can be
made to prevent an issue from becoming a
problem.

X. COMPUTER USE
Scale-up procedures, at best, require relatively large inputs of manpower and money.
Any equipment or technique that would reduce
experimental variability (batch-to-batch variation), improve utilization of personnel, create
useful documentation, and speed the process
would be welcome. Use of computer control in
pilot operations adds value in all these areas. It
would be beneficial if computer control were
available at full production scale, but this addition is not critical. For reasons to be detailed,
computer use at laboratory and pilot scale is
valuable.
An entire book has been published on computer control in fermentation processes.75There
are individual chapters on specially designed
and installed systems at Eli Lilly and Company, Merck and Company, Bristol-Myers,
Hoffmann-LaRoche, and Smith Kline and
French. There is more than enough information
to initiate plans for such a system or to modify

TABLE 13
Validation Issues/Problems
Documentation
Personnel training
Exclusion of personnel
Complies with filings
Alternate suppliers
Process or SOP changes
Self-audits
Production monitoring
Improper corrections
Lack of policy/procedures
Improper (or no) approvals
Simple file retrieval
Process variations not included
GLP not followed
Software
Labels
Complaint form and response
Organization chart
Incompletely filled forms
Lack of flow diagrams
No revision number or date
Limits of performance not stated
Excessive use of red lined documents
Facilities and equipment
Cross-contamination
Microbial contamination
Endotoxin
Particulates
Personnel flow
Environmental control
Erosion and corrosion
Key variable(s) control
Solvent reuse
Deficient engineering drawings
Water
Change orders (construction)
Validation program
Quarantine area
Product flow
Utilities (additives)
Inadequate maintenance
Cleaning
Recycle (product, air, reagents)
Dosage form
Excipient changes
Stability
Bioavailability
Impurities
Assay specificity
Deficient test methods
Record retention
Rework

an existing system if cost-benefit analysis points

in that direction.
The characteristics and values of a properly
designed computer system are detailed below
with some expansion of specific details.
1.

2.

3.

4.

5.

Monitoring and control (direct ties to sensors)

Key variables are either monitored, controlled, data recorded, alarm points set
-either one or all of these functions can
be programmed and modified.
Allowance for improvement/modification
The system should allow for novel or
changing instrumentation, changing processes, changing raw material inputs, and
a changing regulatory environment. Addition of new instrumentation-to-computer interfaces should be facile and not
excessively costly.
Networking
The system should allow relatively simple
linking of intraplant and interplant computers. The system should permit use of
different devices (multiple manufacturers) to take advantage of specialty units
and various PCs. It would be useful to
have elements of standardization between
the pilot system and the full-scale manufacturing computer system. Addition of
printers, graphics capabilities, plotters,
tape, or alternate storage devices should
be simple.
Calculation and control of indirectly measured parameters
We can presume that rapid measurement
of multiple inputs and outputs is possible.
Rapid calculation of indirectly measured
quantities should be possible; further,
some change in input (i.e., some element
of control) should be achievable. Indirect
parameters include respiratory quotient,
heat load (energy balances), biomass,
mass transfer coefficient, and economic
optimization, Data analysis is designed
into the system; statistical analysis would
be a useful adjunct.
Complete documentation
A hierarchy of security levels must be

237

6.

7.

8.

provided. A complete record of changes

to the software must be available, as must
be a complete record of changes to control points and ranges. The software must
be documented. Process responses must
be documented. A complete record of
activities should be provided. Clear and
complete documentation (delineating interactions between hardware and software) should be provided.
Automatic and repetitive process control
Certain process sequences or process
control strategies should be included;
others may be added as the need arises.
One example is vessel or equipment sterilization, another is inoculum transfer, a
third might be oxygen supplementation
under defined conditions. Harvest and
transfer can be controlled as well as
equipment cleaning.
Internal system checks
The computer system should perform
internal system checks and appropriate
failure modes should be provided. A
system failure should not cause a process failure; some redundancy (such as
local control) should be provided.
Transferability (Standardization)
If a corporate computer is in place, a
link between the pilot plant computer
and the corporate computer is desirable.
The transferability to a production computer control system has already been
noted. Standardization allows for fewer
people required to maintain systems and
allows technology transfer to occur more
easily and more rapidly (same hardwaresoftware-operations interface).

The current upgrades and future developments

in computer controlled biotechnology processes
are also important. Scale-up will be facilitated,
optimization will be speeded, and regulatory concerns will be alleviated.
1.

2.

238

Documented audit trails will be available

for both product and process.
Interfacing to plant-scale or corporate level
computers will be simplified and communi-

3.

4.

5.

cation enhanced. Connectivity to manufacturing/inventory control software will be

enhanced, as well, allowing improvements
in scheduling and resource allocation. Economic optimization will be part of the scaleup process with a company-wide perspective rather than a single-process viewpoint.
Artificial intelligence (use of expert systems)
is common in certain applications; greater
use will be made of these capabilities in
biotechnology processes. Smart alarms using complex strategies - will further reduce the need for human intervention.
Novel control strategies will be developed.
Complex interactive systems will be created
as ever more complex sensors are introduced
and complex calculations are made that better understand the process. Solutions of
multivariable equations are now possible but
will be applied to biologic systems to a
greater degree.
Prediction of (as well as response to) process upsets will be facilitated by computer
control. With appropriate inputs, the computer system and its affiliated controls can
be programmed to give an appropriate response to an anticipated problem. The system response to these preventative measures
will be used to moderate or magnify the
corrective, but before-the-fact, response.

These advances can be further developed in a

pilot plant environment and thus can be both the
result of scale-up studies as well as major stimuli
to more efficient and speedier scale-up procedures.
There are recent publications that describe
up-to-date applications of computer logic and
control to fermentation systems. Shi and Shimi~u~
utilize neuro-fuzzycontrol in novel experimental control systems that relate DO level, substrate
feed rate, and ethanol production. Pokkinen et
al.77create a knowledge-based system to act as a
supervisory system for a computer control system. The expert system consists of a shell (user
interface, reasoning control mechanism, mechanism for automatic fault detection, and a tool to
construct a knowledge base) that handles uncertainties by fuzzy reasoning both in measurement

design alternatives will result in a facility that

includes few blind spots and fewer undesirable
compromises. At the very least, the jargon that
the different groups use will become understandable at an early stage and individual concerns will
be voiced. Although not insuring success in scaleup, these exchanges will allow common discourse.
If a novel process is to be scaled-up, test
procedures (asepsis, in-process control, QC) may
be novel. Early design intervention will allow
unique requirements or unique facilities to be
designed in prior to costly changes. A few examples will suffice:

and knowledge. The system was tested with real

data; it is said that the expert system could determine phases of the fermentation and detect faults
not deducible from off-line measured data. Early
correction of faults was possible. The model system was the lactic acid fermentation. The speed
with which advances in control theory are being
applied to bioprocesses is remarkable.
Brasker et al.78describe a system that has
been designed and installed that includes process
automation and up-to-date data collection methods. Recombinant fermentation processes are
stressed but design features are universally applicable.
Yang et al.79present information on computer-controlled cultivation for the production of
recombinant human interferon. The authors established a nutrient feeding profile that improves
both plasmid stability (recombinant E. coli used)
and overall interferon productivity. All supervisory control tasks and calculations were implemented via computer. The optimized process was
said to increase interferon productivity by 12.9fold with a 3.7-fold increase in specific productivity compared with the manual process.

4.

XI. CONSTRUCTION CONCERNS

5.

It is not a normal procedure to have research

personnel involved in the planning of a large
biotech manufacturing complex. Normally, research personnel are involved in laboratory planning and engineers or development personnel are
involved in pilot plant design. There is normally
some overlap between development and manufacturing groups in the design of a large, production plant. This division of labor is workable but
research personnel might be profitably engaged
in construction details. There are many areas of
design and many opportunities for cost saving
where a research group that has performed the
work might have soft information to aid in
making appropriate design compromises. Alternatively, design difficulties might lead to pointed
experimentation that would clarify or lead to correct choices in planning. The interaction between
those who have experienced research problems in
synthesis and isolation and those who can offer

Construction of any clean-room space entails

its own set of specific requirements. Although
there is a federal standard relating to final certification, there is no federal standard (as yet) pertaining to the protocols needed in clean-room
construction. As the clean-room space is built and
enclosed, there is, or should be, a construction
requirement for increasing levels of protection,
that is, one may begin with booties and bunny
suits and progress to face masks and, finally, to
full operational gowning. As an example, HEPA

1.

2.

3.

Which utilities should be manifolded because there are a number of points-of-use?

Is emergency power needed? Where and
how much? The size of an emergency generator is a key design input.
It is a simple matter to require proportional
control, recording plus alarms for every
variable. Can some variables be monitored?
Can some variables be controlled without
recording? Costs are impacted by the choices.
What sort of external and internal security
system will suffice? Is round-the-clock coverage required?
What level of cleanliness is required? Again,
it is very simple to call for clean-room conditions everywhere;cost would be quite high.
Degrees of cleanliness must be defined for
the various stages of processing. Appendix
B contains one section concerning Construction. This is a starter list for construction concerns before and during scale-up;
the same (and new) concerns arise prior to
and during construction of a full-scale plant.

239

filter installation should be accompanied by use

of hoods, gloves, bunny suits, face mask, and
booties. Final as-built certification requires full
operational gowning. It goes without saying that
dedicated tools should be used and, at some point,
tools and instruments - once cleaned - should
remain in the clean room until all tasks are completed. There must be written guidelines for each
construction stage, and the monitor for compliance cannot be a representative of the subcontractor doing the actual work. The reason is obvious.
Once a clean room is shown not to satisfy design
criteria, it is a bit late to fix blame and find fault
with construction protocols or lack of adherence
to them. Every precaution must be taken before
and during construction to insure clean installation; correction after the fact is onerous and usually costly.
An article*O covering construction concerns
relevant to biotech facilities is worth reading, by
all parties involved in commissioninga new manufacturing facility. The concerns are relevant to a
retrofit facility as well. The major concerns are
divided as follows:

Cleanliness
Sealing of equipment and piping (precleaned
offsite) prior to shipment
Protective package integrity and maintenance
on-site
Cleanlinessfor manufacturingand clean-room
areas
Product/process contamination during a retrofit
Movement of workers and equipment
Documentation
Process validation documentation
Validation master plan
Installation qualification (IQ)
Proper certifications and test results
Proper sign-offs
Civil/architectural
Cleanability criteria
Wash/cleaning materials and agents allowed
Final wall, floor, ceiling finishes
Pass-throughs, piping chases, expansion ca-

240

pability
Allowable fuels, solvents, paints
Ease of inspection
Spare pass-throughs (especially in clean
rooms)

Pipindwelding
Materials of constructionlsegregation and
control
Welding specifications
Validation (permanent records)
Supports and insulation
Quality control and daily inspection of finished welds
Equipment
Connectibility and coordination drawings
Accessibility - inspection and maintenance
Sealing (no pockets or traps)
File history
HV ACfductwork
Leak testing
Filter testing
Sealants
Mapping (location)
Proper drawings and monitoring
Electrical/instrumentation
Lighting
Facilities management
Requirements
Use of universal solenoids
There are innumerable issues involved in creating appropriate equipment, material, and personnel flows. Criticality of equipment, inventory
of parts, redundancy, potential failure modes, and
hazards analysis are but some of the factors that
must be addressed by research personnel in concert with designers and manufacturing personnel.
Overdesign is not only costly at the start; high
maintenance and replacement costs will continue
to be incurred. Design effort, construction cost,
and instrumentation requirements must be centered on areas that have the greatest potential for
gain or loss in routine operation of the biotech
process; only input from the process designers

(research and development personnel) will result

in the optimum facility when built.
A good deal of technical detail is given by
21 in a book published in
S r i g l e ~in~Chapter
~
1990. The discussion covers design and construction of a mammalian cell-culture plant for production of therapeutics. For mammalian cell culture, the key design factors concern in-process
and final-product sterility (and dosage form), handling of medium and serum, process equipment
and facility design, and potential for biohazards.
Cell-culture technology is reviewed and differing
requirements are related to plant-design criteria.
The emphasis of the chapter is on the Invitron
plant and on actual solutions to real or perceived
problems. As an example, the number of air
changes ranges from 30 per h (less critical rooms)
to over 50 per h in rooms used for aseptic operations, Room air pressures, temperature, and humidity of individual rooms are monitored and
controlled by a central, microprocessor-based
control system. Very early- and regular ongoingreviews with the FDA are suggested to agree on
design criteria. The more novel the process or
product, the earlier the initial review. A point was
made concerning picture taking (coded lines and
other critical items covered in the course of construction) and use of video taping. These techniques were used to give a visual and easy-tounderstand record for later review and certification.
It is never too early to consider personnel
safety and environmental safety. Happily, there is
a very useful book that covers many areas of
concern.81Facility considerations are covered in
six separate sections; each is written by knowledgeable people. The need for early intervention
(before the facility design is fixed) is clear as is
the need for expert opinions. Biosafety practices
are covered from different perspectives, and one
section covers a medical surveillance program.
Four separate chapters cover considerations that
would interest and involve regulatory agencies.
(Also see Appendix D.) This entire book is geared
toward problem avoidance. There are many ways
safety and good practice can be violated; resultant
costs -in harm, money, bad publicity, and legal
ramifications - are always high. There are far
fewer correct methods and procedures; it is worth

studying what has worked and what is permitted

and follow those practices. The book previously
noted is a treasury of valuable procedures and
practices.

XII. ORGANIZATIONAL PROBLEMS

Although this article covers a great number
of technical considerations, there are organizational or psychological issues that should be recognized as well. Technical problems are most
often soluble; however, a structural problem that
is allowed to persist will delay implementation,
lengthen the scale-up cycle, and will cause ongoing irritation and economic loss. Further, the
subjective issues often weigh more heavily on
whether or not scale-up is successful in a short
time frame. Structural problems that are allowed
to fester will have a negative impact on every
example of scale-up, even if technical competence is assumed.
Not every reader will be in a position to direct
resolution of structural problems. There are many
reasons to understand the situation, however.
Knowledge of a problem may allow individual
input to alleviate it. Further, there may come a
time when you can have a major impact on resolution. The organization/structul problems of
scale-up or technology transfer can be summarized:
Inadequate documentation by Research
Inadequate sensitivity analysis
Inadequate staffing by Manufacturing
Inadequate training or a heightened perception
of sensitivity
Misunderstanding of Manufacturing schedules
or equipment
Lack of a joint (defined) Development team
Lack of high level sponsorshiplcorporate interest
Lack of defined objective, whether it be commercial or otherwise
Table 14 lists both some of the attributes of
structures that function well (promoters) and
some of characteristics that cause problems up to
and including failure (barriers). Studies have
been made of what works. Successful compa-

241

TABLE 14
Transfer from R&D to Manufacturing
Promoters
Bilateral champions
Manufacturing involvement (from design onward)
Joint selection of vendors
In-plant demo (bilateral)
Manufacturing dedicated personnel
Joint implementation plan
Past success

Barriers
Inadequate manufacturing staffing
Inadequate retrofit
Perception of technology
too complex
too fragile
too variable
Disruption of existing schedules
Unclear benefits
Past failures

nies (meaning those that exhibit smooth technology transfer and timely scale-up) have experienced product and process histories that fall in the
promoters column. Characteristics are easily
recognizable. Unfortunately, the obverse is also
readily apparent. The barriers to success need
concerted and directed effort; expectation alone
will not cause them to disappear.
There is no single key to success, but if one
were forced to order the promoters and select the
most critical, I would opt for Manufacturing
involvement from design onward. If there are
persons from the manufacturing group selected
to assure success (however that is defined) and
if there is an investment of time and energy
(duly noted in objectives and performance appraisal), the odds for successful implementation
become more favorable. Design, planning, implementation, scheduling, and criteria for success
must be jointly set and the manufacturing group
must buy in to all these concurrent programs.
The need for involvement at the earliest stage is
obvious.
In my opinion, the greatest barrier to success lies in a misperception of the technology
(in this instance, the process itself). When one
group is given objectives or results achieved

242

and established by another group, there is a

natural resistance to accede. If established by
fiat, resistance is maximized. Early disclosure
of potential problems and of variation in performance can clear the air at the outset; a willingness to accept joint resolutions and joint
inputs (from the onset of joint reviews) will do
a great deal to improve the perception of the
robustness of the technology.
In an introductory section to this review,
project planning was discussed. Potential pitfalls
were reviewed in outline form. There are ways to
avoid these pitfalls, and methods have evolved to
enhance changes for success. Project management techniques are well known; these methods
concern all aspects of implementing a novel technology. Factors such as organization structure,
technical requirements, cost, and timing are considered and addressed. Communication channels
are established and a formalized system is established to stimulate cooperation and to correct
deficiencies in information flow before the project
is impacted. It is important that the scale-up project
be carefully defined and the reason for implementing the project is detailed. Once these definitions are in place, organization and planning can
be set, detailed planning can begin, and appropri-

ate resources allocated. It is surprising that these

seemingly obvious steps are not always taken
or that answers to the questions Why?,
What?, and When? are absent or vague. If
these definitions and planning steps are unclear
or open to varying interpretation, the seeds for
future discord and delay are sown. After initial
steps and project initiation, a systematic procedure is put in place to check specific and detailed milestones. Both time and money must
be tracked; the key question is, Do project
results conform to goals, plans, and specifications? Note that this question can be put to the
design/construction of a physical facility (pilot
or full-scale plant) or to achievement of defined development results (rate, yield, purity,
throughput, unit cost). Of course, once tracking
methods are in place, a parallel function is
project control. The need for full and open communication and channeled cooperation is now
obvious. Unexpected deviations should not result in fire-fighting and creation of ad hoc groupings; correction mechanisms should be in place
prior to need. Personnel responsibilities should
be established for the overall project and also
established for deviations and untoward responses. Finally, a definition for the termination of the project is necessary. A scale-up
project has a start, middle, and end. If the end
is ill-defined, the project will continue with
mixed interest or support; no one will be able to
assess achievement or make needed adjustments.
One result will be a waste of manpower and
money. Another result will be a psychological
barrier to future multidisciplinary projects. In
summary, project management methods cover
these various areas:
Overall goal as established in multiple detailed
objectives
Personnel and managerial organization and
interactions
Method of implementation (timing included)
Budget
Responsibility assignments
Communication flow (including meeting minutes and reviews)
Required approvals

Support functions (internal or external to organization)

Response to deviations
Definition of success (project termination)
The management of process technology is
critically important. Even with a superbly designed process, it is possible to build a costly,
defective, and unreliable facility. The many required details (and, by inference, the many potential pitfalls) are carefully listed by Bergmann
(Chapter 15 in Reference 14). Because the general process is cell-culture manufacturing and
because the information is so detailed and meticulous, this chapter is highly recommended reading.
A general study on technology transfer from
R&D to manufacturing highlights the many pitfalls.82Although any sort of technology transfer is
discussed, anyone who has been involved in biotechnology tech transfer will be able to recognize
the applicable difficulties.

XIII. SUMMARY
Just as it is difficult to cover all problems in
biotechnology scale-up, it is as difficult to summarize the requirements for problem avoidance
resulting in successful scale-up. First and foremost, the project team should understand what
sequence of steps is involved. In an accompanying table (Table 15), I attempt to show the
iterative process that covers successful scale-up
and successful process development. The starting
point would be a laboratory process or an improvement concept. The sequence of steps with only major headings shown - must be repeated as often as necessary until the agreed-on
goal is achieved. Rarely is the process once
through, that is, one proceeds sequentially and
arrives - in linear fashion - at a full-scale operation. More often, there is a repeated cycling
(with diversions and alternate paths) until one
approaches the goal. Also, the steps overlap and
not every company group is heavily involved at
every step. The key requirements for successful
scale-up (technical competence is assumed) are

243

TABLE 15
Scale-Up
Research Summary
Technology Transfer Document
Manuals and Procedures
Quality Control

Improvements

L
L

ir

Optimization
Full scale operation
Final documentation
Audits (by R&D)
Comparison to expectation
Economics
Acceptance of process

Joint planning
Scheduling
Capital equipment
Build or retrofit
Cultures/cell banks
Performance criteria

L.

Identify and correct deficiencies

Newhodified equipment
Training of operators
Multiple runs
Yield, rate, purity (statistics)
Long-term contracts (purchasing)

Initial runs
Preparation of batch sheets
Validation
Quality assurance
Protocol corrections

Involved at every step:

Regulatory affairs
Maintenance/calibration

ttt

Internal audits
Training

attention to detail, patience, training, mutual understanding of the shared goal, and some appreciation of the iterative process involved.
Even if difficult, one feels obliged to
present a personal list of the requirements and
details for successful scale-up (Table 16).
These are obviously mere headings or, perhaps, titles for specialized reports or summaries. Each of the twenty separate items could
stand as the subject of an article; some have
been the subjects of books. It may be that
successful scale-up can be achieved while to-

244

Purchasing
MIS

tally disregarding one or more of the subjects.

However, insufficient or no attention to one of
these areas merely lengthens the odds for success in an already complex situation. Should
the reader feel that the task is insurmountable,
there are some heartening statistics. In 199 I , it
is estimated that 6.3 billion gal of beer were
produced in the U.S.; the author estimates that
50 to 100 kg of tPA was produced in the same
year in the U.S. The clear conclusion is that
the scale-up of biotechnological processes is
both feasible and possible.

TABLE 16
Details for Successful Scale-up
Appropriate management structure
Joint development team
Multidisciplinary inputs
Complete process documentation
Research, process development, manufacturing
Raw materials supply
Complete set of specifications
Defined QC testing and acceptance criteria
Validated cell banks
Multiple storage sites
Facility design
Meet all internaVexterna1 requirements
Preventative maintenance
Complete records, logs, calibrations
Materials of construction
Impact on process
Ease of cleaning and inspection
lnoculum development
Process optima
Understanding of trends
Sensitivity analysis
Process and environmental monitoring
Sterility control
Appropriate pressurization
Alarms, responses, records
Agitation-aerationconditions
Stability
Media, cell banks, culture broth, intermediates, cell culture,
product
Scheduling and planning
Loss prevention programs - hazards analysis
Plant support organizations
Responsibility assignments: technical services, engineering, QC
Training and retraining programs
Internal audit program with detailed corrective measures
Redundancy in process design, key equipment, utilities
Effluent or discharge precautions with special treatments
Defined communications path including emergency response

REFERENCES
1. Biotechnology in a Global Economy, Congress of the
United States Office of Technology Assessment,
Washington D.C., October 1991, 6.

2. Reisman, H., Economic Analysis of Fermentation

Processes. Table 13, CRC Press, Boca Raton, FL,
1988, 37.
3. Hu, W. and Peshwa, M., Animal cell bioreactors recent advances and challenges to scale-up, Can. J.
Chem. Eng., 69, 409, 1991.
4. Honvath, B., Mammalian cell culture scale-up: is
bigger better?, BiolTechnology, 7, 468, 1989.
5. Birch, J., Cells sell, Chemtech, 17, 378, 1987.

6. Cortessis, G., Proby, C., and Petrossian, A., Airlift

bioreactors for production of monoclonal antibodies,
Biopharm. Manuf., 1, 30, 1987.
7. Runstadler, P. and Young, M., GMP production of
biopharmaceuticals using high density, fluidized-bed
cell culture technology, in Animal Cell Culture and
the Production of Biologicals, Sasaki, R. and Ikura,
K., Eds., Kluwer Academic Publishers, Dordrecht,
Netherlands, 1991, 103.
8. Runstadler, P., Ozturk, S., and Ray, N., An evaluation of hybridorna cell-specific productivity: perfused
immobilized, continuous suspension, and batch suspension cultures, in Animal Cell Technology: Basic
and Applied Aspects, Murakami, H. , Shirahata, S.,

245

and Tachibana, H., Eds., Kluwer Academic Publishers, Dordrecht, Netherlands, 1992, 57.
9. Lazar, A., Immobilization of animal cells in fixed
bed reactors, Biotechnol. Adv., 9, 411, 1991.
10. Paul, E., Design and scale-up of an anchorage dependent mammalial cell bioreactor, Ann. N.Y. Acad. Sci.,
589, 642, 1990.
11. Kearns, M., Integrated design for mammalian cell
culture, BiolTechnology, 8, 409, 1990.
12. Kamen, A., Chavarie, C., Andre, G., and
Archambault, J., Design parameters and performance of a surface baffled helical ribbon impeller
bioreactor for the culture of shear sensitive cells,
Chem. Eng. Sci., 47, 2375, 1992.
13. Ho, C. and Wang, D., Eds., Animal Cell Bioreactors,
Buttenvorth-Heinemann, Stoneham, MA, 1991. Especially Rhodes, M., Gardiner, S., and Broad, D.
Scaleup of animal cell suspension culture, chap. 10.
Glacken, M. Bioreactor control and optimization, chap.
15.
14. Large-Scale Mammalian Cell Culture Technology,
Lubiniecki, A., Ed., Marcel Dekker, New York, 1990.
15. DeBruyne, N., The design of bench-scale reactors,
chap. 6, and Griffiths, J., Overview of cell culture
systems and their scale-up, chap. 7, in Animal Cell
Biotechnology, Vol. 3, Spier, R. and Griffiths, J., Eds.,
Academic Press, San Diego, 1988, p. 142 and p. 179.
16. Bliem, R., Konopitzky, K., and Katinger, H., Industrial animal cell reactor systems: aspects of selection and evaluation, in Advances in Biochemical EngineeringlBiotechnology,No. 44,Bioreactor Systems
and Effects, Fiechter, A., Ed., Springer-Verlag, Berlin
Heidelberg, 1991, 1.
17. Liibbert, A., Advanced methods for bioreactor characterization, J . Biotechnol., 25, 145, 1992.
18. Leist, C., Meyer, H., and Fiechter, A., Potential and
problems of animal cells in suspension culture, J .
Biotechnol., 15, 1, 1990.
19. Yoshida, S., Yoshikawa, A., and Terao, I., Microbial production of hydroquinone, J . Biotechnol., 14,
195, 1990.
20. Buitelaar, R. and Tramper, J., Strategies to improve the production of secondary metabolites with
plant cell cultares: a literature review, J . Biotechnol.,
23, 11 1, 1992.
21. Lipsky, A., Problems of optimization of plant cell
culture processes, J . Biotechnol., 26, 83, 1992.
22. Agathos, S., Production scale insect cell culture,
Biotech. Adv., 9, 51, 1991.
23. Hochfeld, W., Scaleup of continuous-culturefermentation for production of biodrugs, Genet. Eng. News,12,
8, 1992.
24. Reisman, H., Economic Analysis of Fermentation
Processes, Table 3, CRC Press, Boca Raton, FL, 1988,
15.
25. Fink, D., Curran, L., and Allen, B., Eds., Research
Needs in Non-Conventional Bioprocesses, Battelle
Press, Columbus, OH, 1985.

246

26. Flickinger, M., Greenstein, M., Bremmon, C., and

Conlin, J., Strain selection, medium development
and scale-up of toyocamycin production by Streptomyces chrestomyceticus, Bioprocess Eng., 5, 143,
1990.
27. Schurch, U., Cruz, S., and Lang, A., Scale-up and
optimization of culture conditions of a human
heterohybridoma producing serotype-specific antibodies to Pseudomonas aeruginosa, Appl. Microbiol.
Biotechnol., 37, 446, 1992.
28. Ozturk, S. and Palsson, B., Growth, metabolic and
antibody production kinetics of hybridoma cell culture. I. Analysis of data from controlled batch reactors. 11. Effects of serum concentration, dissolved
oxygen concentration and medium pH in a batch reactor, Biotechnol. Prog. 7, 471, 1991.
29. Griffiths, J., Animal cell culture processes -batch
or continuous?, 1.Biotechnol., 22, 21, 1992.
30. Ryll, T., Lucki-Lange, M., Jager, V., and Wagner,
R., Production of recombinant human interleukin-2
with BHK cells in a hollow fibre and a stirred tank
reactor with protein-free medium, J . Biotechnol., 14,
377, 1990.
31. Werner, R., Walz, F., N d , W., and Konrad, A.,
Safety and economic aspects of continuous mammalial
cell culture, J . Biotechnol., 22, 51, 1992.
32. Knight, P., Fermentation report, BiolTwhnolngy. 6,
505, 1988.
33. Kennedy, L., Boland, M., Janssen, D., and Frude,
M., Designing a pilot-scale fermentation facility for
use with genetically modified microorganisms, in
Fermentation Technologies: Industrial Applications,
Pak-Lam Yu, Ed., Elsevier Applied Science, London,
1990, 383.
34. Large-Scale Mammalial Cell Culture, Feder, J. and
Tolbert, W., Eds., Academic Press, Orlando, FL,
1985.
35. Yang, X.-M., Optimization of a cultivation process
for recombinant protein production by Escherirhiu
coli, J . Biotechnol.. 23, 271, 1992.
36. Lazarte, J., Tosi, P.-F., and Nicolau, C., Optimization of the production of full-length rCD4 in
Baculovirus-infected Sf9 cells, Biotechnol. Bioeng..
40, 214, 1992.
37. Animal Cell Biotechnology, Vol. 4, Spier, R. and
Griffiths, J., Eds., Academic Press, San Diego, CA,
1990, especially Greenaway, P. and Farrar, G., The
growth and production of human immunodeficiency
virus, 379, Mizrahi, A., Lazar, A., and Reuveny, S.,
Interferons derived from human cells, 414, Epstein,
N., Reuveny, S., Friedman, J., and Ariel, N., The
manufacture and use of a colon cancer antigencarcinoembryonic antigen, 446.
38. Srigley, W., Design and construction of manufacturing facilities for mammalian cell-derived pharmaceuticals, in Large-Scale Mammalian Cell Culture Technology, Lubiniecki, A., Ed., Marcel Dekker, New York,
1990,567.

39. Hubbard, D., Scaleup strategies for bioreactors containing non-Newtonian broths, Ann. N.Y. Acad. Sci.,
506, 600, 1987.
40. Hubbard, D., Harris, L., and Wierenga, M., Scaleup
for polysaccharide fermentation, Chem. Eng. Prog.,
85, 55, 1988.
4 1. Jem, K., Scale-down techniques for fermentation,
BioPharm., 2, 30, 1989.
42. Singh, V., Fuchs, R., Hensler, W., and
Constantinides, A,, Scaleup and optimization of oxygen transfer in fermentors: Newtonian and nonNewtonian systems, Ann. N.Y. Acad. Sci., 589, 616,
1990.
43. Jones, G., Erickson, L., and Glasgow, L., Effects of
microcarriers and serum on local hydrodynamics
within an airlift column, Ann. N.Y. Acad. Sci., 589,
431, 1990.
44. Yang, J. and Wang, N., Oxygen mass transfer enhancement via fermentor headspace pressurization,
Biotechnol. Prog., 8, 244, 1992.
45. Schiigerl, K., Comparison of the performances of
stirred tank and airlift tower loop reactors, J .
Biotechnol., 13, 251, 1990.
46. Okabe, M. et al., Preferential and high-yield production of a cephamycin C by dissolved oxygen controlled fermentation, J. Ferment. Bioeng., 73, 292,
1992.
47. Sumino, Y., Sonoi, K., and Akiyama, S., Oxygen
transfer rate in stirred-tank fermentors under the supply of oxygen-enriched air, J. Ferment. Bioeng., 73,
175, 1992.
48. Yabannavar, V., Singh, V., and Schaefer, E., Effect
of pressure on an aminoglycoside fermentation mediated by dissolved oxygen, J. Ferment. Bioeng., 73,66,
1992.
49. Papoutsakis, E., Fluid-mechanical damage of animal cells in bioreactors, Trends Biotechnol., 9,427,
1991.
50. Oh, S., Nienow, A., Al-Rubeai, M., and Emery, A.,
Further studies of the culture of mouse hybridomas in
an agitated bioreactor with and without continuous
sparging, J. Biotechnol., 22, 245, 1992.
51. Yang, J. and Wang, N., Cell inactivation in the presence of sparging and mechanical agitation, Biotechnol.
Bioeng., 40, 806, 1992.
52. Moser, A. et al., Mathematical models for mixing
in deep-jet bioreactors, Part 1 Analysis, Part 2 Calculation of parameters, Bioprocess Eng., 7, 171,
1991.
53. Sakato, K., Tanaka, H., Shibata, S., and
Kuratsu, Y., Agitation-aeration studies on Coenzyme Qlo production using Rhodopseudomonas
spheroides, Biotechnol. A p p f . Biochem., 16, 19,
1992.
54. Galindo, E. and Nienow, A., Mixing of highly viscous simulated xanthan fermentation broths with the
Lightnin A-315 impeller, Biotechnol. Prog., 8, 233,
1992.

55. Bourne, J., Zurita, E., and Heinzle, E., Bioreactor

scale-up for the oxygen-sensitive culture Bacillus
subtilis: the influence of stirrer shaft geometry,
Biotechnol. Prog., 8, 580, 1992.
56. Enders, J., Current issues and challenges in the scaleup of biopharmaceutical products, Genet. Eng. News,
12, 6 , 1992.
57. Yarmush, M. et al., Immunoaffhity purification: basic
principles and operational considerations, Biotechnol.
Adv., 10, 413, 1992.
58. Ishida, S. et al., Scale-up of hydrophobic interaction
chromatography for purification of antitumor antibiotic SN-07, Bioprocess Eng., 4, 163, 1989.
59. Merck and Co., Inc., Rahway, NJ 07065 Patent
Application (Europe), Purification of recombinant
Epstein-Barr virus antigens from VERO cells, yeast
cells or L cells, Publ. no. 0312164, date: 19.04.89.
60. Fair, J., Commercially attractive bioseparation technology, Chem. Eng. Prog., 85, 38, 1989.
6 1. Advances in Biochemical EngineeringIBiotechnology
No. 47, Bioseparation, Tsao, G., Ed., Springer-Verlag,
Berlin, 1992.
62. van Reis, R., Leonard, L., Hsu, C., and Builder, S.,
Industrial scale harvest of proteins from mammalian
cell culture by tangential flow filtration, Biotechnol.
Bioeng., 38, 413, 1991.
63. Rosen, C.-G., Biotechnology: its time to scale-up
and commercialize, Chemtech, 17, 612, 1987.
64. Trilli, A., Scale-up of fermentations, in Manual of
Industrial Microbiology and Biotechnology, Demain,
A. and Solomon, N., Eds., American Society for Microbiology, Washington, D.C., 1986, 277.
65. Sterile Pharmaceutical Manufacturing, Applications
f o r the 1990s, Vols. 1 and 2. Groves, M., Olson, W.,
and Anisfeld, M., Eds., Interpham Press, Buffalo
Grove, IL, 1991.
66. Sterilization of Medical Products, Vol. V, Morrissey,
R. and Prokopenko, Y., Eds., Polyscience Publications, Montreal, Canada, 1991.
67. mug, I., Wet-heat sterilization, including both the
design of the process and equipment used to sterilize
product, in Sterilization of Medical Products, Vol. 4,
Gaughran, E., Momssey, R., and You-sen, W., Eds.,
Polyscience Publications, Montreal, Canada, 1986,
40.
68. Chisti, Y., Assure bioreactor sterility, Chem. Eng.
Prog., 88, 80, 1992.
69. Schiff, W., Moore, A., Brown, J., and Wisher, M.,
Lot release-final product safety testing for biologics,
BioPharm, 5, 36, 1992.
70. Federal Register, Department of Health and Human
Services, NIH, Recombinant DNA Research: Actions
under the guidelines, Vol. 56, No. 138, July 18, 1991/
Notices, p. 33174.
71. Nygard, J., Good manufacturing practices - overview, in Sterilization of Medical Products, Vol. 111,
Harris, L. and Skopek, A., Eds., Johnson and Johnson,
Australia, 1985, 195.

247

72. Guideline on General Principles of Process Validation (An FDA Document), Center for Drugs and
Biologics and Center for Devices and Radiological
Health, Rockville, MD, May, 1987.
73. Avallone, H., GMP inspections of drug substance
manufacturers, Pharm. Tech., 16, 46, 1992.
74. Guidelines for Submitting Supporting Documentation
in Drug Applications for the Manufacture of Drug
Subslances (An FDA Document), Center for Drug
Evaluation andResearch,Rockville,MD, Febmary, 1987.
75. Computer Control of Fermentation Processes,
Omstead, D., Ed., CRC Press Inc., Boca Raton, FL.
1990.
76. Shi, Z. and Shimizu, K., Neuro-fuzzy control of
bioreactor systems with pattern recognition, J. Ferment. Bioeng., 74, 39, 1992.
77. Pokkinen, M., et al., A knowledge based system
for diagnosing microbial activities during a fer-

248

78.

79.

80.

81.

82.

mentation process, Bioprocess Engineer., 7, 33 1,

1992.
Brasker, J., Smith, M., and Zavela, D., Batch process automation applied to recombinant fermentation
systems, BioPharm, 4, 28, 1991.
Yang, X.-M., Xu, L., and Eppstein, L., Production
of recombinant human interferon-a, by Escherichia
coli using a computer-controlled cultivation process,
J. Biotechnol., 23, 291, 1992.
Odum, J., Construction concerns for biotech
manufacturing facilities, Pharm. Engineer., 12,
8, 1992.
Biohazards Management Handbook, Liberman, D. and
Gordon, J., Eds., Marcel Dekker, New York, NY,
1989.
Souder, W. and Padmanabhan, V., Transferring
new technologies from R&D to manufacturing, Res.
Technol. Mgrnt., SepVOct 1989, p. 38.

APPENDIX A
TYPICAL REVIEW AREAS (SAFETY AUDIT)
1.
2.
3.
4.
5.

6.
7.
8.

9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.

Hazard classification and HAZOP study

Volume of hazardous material (flow plan)
Containment practices - all streams
Analysis of failures (controls, earthquake, utilities)
Training (new employees, ongoing recertification, emergency responses)
Facility procedures and practices/Response teams
Raw material storage, sample storage, hold tanks, spill control plan
QA/QC/Sterility control
All effluents and aerosols (location, quantity, tests)
Waste treatment, including potential for breakthrough
Disposal (plasticware, gloves, equipment, etc.)
Certification and validation (especially frequency)
Documentation (process and procedures) and JSA
Emergency procedures/Monitors/Drills/FirstAid
Environmental testing and monitoring/Alarms
Vaccination program/Serum sampling
Medical record keeping and surveillance
Self-audit with corrective measures (feedback)
Accident and near-miss reports (with follow-up)
Conformation to regulatory standards (EPA, OSHA, TSCA, FWRA, USDA, FDA)

APPENDIX B
DETAILED QUESTION LIST
(Scaleup Check1ist)
Process
1. Is there a reproducible process?
2. Are impacts of upsets known?
3. What is the projected production rate?
4. Are critical control points known?
5. Has a cost of goods analysis been made?
6. Do SOPS exist for the process?
7. Have Research, Development, QA/QC, Engineering, Manufacturing personnel signed off on
process documents?
8. Is all equipment in place to produce material? Are spares or standby units in place?
9. Are cGMP procedures in place (if required)?
10. What is the percent running (or on) time?
11. What is expected reject rate?
12. Has a hazards analysis been performed?

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13. Are cleaning procedures known and documented?

14. Is inoculum development optimized, understood, reproducible?

15. Are all effluentdwastes (gas, solid, liquid) detailed and provisions for handling clearly understood?
16. Is an effective process control and monitoring system described?
17. Are operations sequenced and scheduled appropriately?
Raw materials
1. Are all raw materials and suppliers known?
2. Are alternative sources known?
3. Do QC specs exist on all raw materials?
4. Are purity requirements known?
5. Are purity and sterility requirements known for biological items?
6. Do contracts exist (if needed)?
7. Are supplies of raw material sufficient (inventory, market conditions)?
8. Any special precautions or special tests needed?
9. Are any use tests required?
10. Do adequate areas exist for storage of raw materials?
11. Can inventory of raw materials be monitored and controlled easily?
12. Are expiration dates known (shelf life)? Can materials be rotated properly?
13. Does each material have its appropriate sterilization procedure?
Quality controllquality assurance
1. Are all quality control tests (including in-process) known and documented?
2. Are all QA procedures known and documented?
3. Is equipment in place and validated?
4. Are cGLP procedures in place (if required)?
5. Is documentation sufficient to satisfy regulatory needs?
6. Are there special (controlled) areas for Hold, To be Tested, Approved for Use, Approved
for Shipment, Reject for raw materials, WIP, finished goods?
7. Are time sequences known for analytical effort to clear raw materials, WIP, product?
8. Are trained personnel available to perform assays?
9. Is there a documented complaint-handling procedure?
10. If outside testing is needed, is time provided? Are costs known?
1 1. Are documentation and record-keeping procedures in place?

Documentationlvalidation
1. Is process documentation complete? System for changes including approvals?
2. How are documents maintained? How are batch records checked and maintained?
3. Who performs certifications? Schedule of preventative maintenance and recertifications?
4. Are internal audits in place? Scheduled?
5 . Is all paperwork (or data storage) easily accessible for regulatory review? Complete trail of process
and product?
6. Are all appropriate permits and licenses in place and available for review?

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Construction
1. Are designers and contractors aware of facility requirements? Are they prepared to modify usual
techniques and protocols to fit aseptic and/or cleanroom design?
2. Are persons designated to check deliveries of pipe, tubing, ductwork, valves to be sure that (a) end
caps or other protective closures are used, (b) items are stored in a protected location, (c) items
are moved without losing integrity of protection?
3. Are strict rules in place (and enforced) for limiting areas for smoking, eating, drinking?
4. What design details have been incorporated to limit dust and debris and, more important, to limit
introduction of foreign items into hidden areas of the new construction?
5 . Is the construction sequence in place to achieve final design criteria? Do pipes slope correctly? Is
cleanability considered at each step? Are protuberances limited or eliminated? Are openings
sealed or caulked (sanitary and cleanable sealer)?
6. Are appropriate surface finishes (floor, ceiling, walls, insulation, ducts, passthroughs) selected with
care for the intended use? intended cleaning? antimicrobial properties?
7. Does design account for heat and humidity generated?
8. Are welding specifications sufficiently detailed? Are weld tests (including visual records) in place?
Are welds checked daily?
9. Are appropriate supports in place? Are they cleanable? Are they accessible? Are utility inputs
accessible?
10. Is emergency power available? Are all critical utilities or components correctly connected?
11. Are all HVAC filters prevalidated? Are all HEPA filters prevalidated? Is location of every filter
(every one numbered) known? Are all tests documented?
12. Are all fixtures, fittings, conduits designed to minimize or eliminate contamination and permit
sanitization?
13. Are documentation procedures in place? Are appropriate certifications required? Are tests defined
(and who performs tests)? Are certified as-built drawings required?
14. Are all pressure vessels properly coded? Are appropriate relief devices in place? Design drawings
properly stamped?
15. Is there provision for complete sanitization (or sterilization, where appropriate), for access for
replacement or for maintenance, for inspection by regulatory persons?
16. Have the designer, contractors, or subcontractors designedbuilt biotech facilities? Can these
facilities be inspected?
17. Are special company personnel (meaning company paying for facility) selected and trained to
inspect work as it is proceeding?
Are
local, state, and federal regulations known and understood? Are all regulatory issues under18.
stood at the outset? (Examples are emissions, fume hood operation and use of radioactive
compounds, hazardous waste storage and disposal, alcohol storage, narcotic storage, handling of
blood or tissue, flammable reagents, special alarms, OSHA rules, boiler operation, recombinant
organisms, transformed or transfected cells, TOSCA rules).
19. Are pipes, tubing, valves, breakers, instrument lines properly labeled, tagged, or identified in a
uniform and consistent manner?
20. If double containment is required, is the design both functional and economic? Are appropriate
sensors in place?
21. What control points or environmental conditions are alarmed? What is alarm function, messaging,
and proposed response?

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APPENDIX C
MANUFACTURERS OF FERMENTORS
(OVER 100 LITERS)
APV Crepaco
8303 West Higgins Road
Chicago, IL 60631

LSL Biolafitte, Inc.

719 Alexander Road
Princeton, NJ 08540

Applikon Lnc.
1165 Chess Drive
Foster City, CA 94404

Lee Industries Inc.

P.O. Box 688
Phillipsburg, PA 16866

Artisan Industries
73-T Pond Street
Waltham, MA 02254

New Brunswick Scientific

44 Talmadge Road
Edison, NJ 088 18

B. Braun Biotech
999 Postal Road
Allentown, PA 18103

Paul Mueller Co.

P.O. Box 828
Springfield, MO 65801

Chemap, Inc.*
111 D Corporate Boulevard
South Plainfield, NJ 07080

Pope Scientific
N90 W14337 Commerce Drive
Menomonee Falls, WI 5305 1

DCI Inc.
600 North 54 Avenue
St. Cloud, MN 56303

Porton Instruments/LH Fermentation

3942 Trust Way
Hayward, CA 94545

Endotronics (AcusystTM)
8500 Evergreen Boulevard
Minneapolis, MN 55433

Precision Stainless
P.O. Box 668
Springfield, MO 65801

Engineered Technologies Corp.

222 Metro Center Boulevard
Warwick, RI 02886

Sulzer Biotech*
230 Crossways Park Drive
Woodbury, NY 11797

Feldmeier Equipment Inc.

6800 Town Line Road
Syracuse, NY 13211

Walker Stainless Equipment Co.

618 State Street
New Lisbon, WI 53950

Henderson Industries
45-T Fairfield Place
West Caldwell, NJ 07006

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MBR BioReactor AG (Sulzer) and Chemap AG (Tetra Laval) merged as of November 1, 1992.

APPENDIX D
STATUTES AND NOTICES THAT MAY BE APPLICABLE TO SCALEUP
OF BIOTECHNOLOGY PRODUCTS
Federal Food, Drug and Cosmetic Act (FDCA)
21 U.S.C. $8301-92
Federal Register, Department of Health and Human Services, NIH,
Vol. 51, No. 88, May 7, 1986 Notices with Additional Actions of
Aug. 24, 1987
July 29, 1988
Oct. 26, 1988
Mar. 13, 1989
Mar. 1, 1990
Sept. 12, 1990

July 18, 1991

Oct. 15, 1991
Nov. 21, 1991
Jan. 28, 1992
Apr. 22, 1992
Aug. 26, 1992

Public Health Services Act (PHS), 42 U.S.C. 5526263.

Toxic Substances Control Act (TSCA), 15 U.S.C. 552601-29.
Occupational Safety and Health Act (OSHA), 29 U.S.C. 55651-78.
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA)
7 U.S.C. $5136-136y.
Clean Water Act (CWA), 33 U.S.C. 55466-466g.
Federal Clean Air Act (CAA), 42 U.S.C. 597401-7642.
Resource Conservation and Recovery Act (RCRA)
42 U.S.C. $56901-87.
Virus, Serum and Toxin Act (VSTA), 21 U.S.C. 55151-58.
National Environmental Policy Act (NEPA), 42 U.S.C. 554321-70.
Comprehensive Environmental Response, Compensation and
Liability Act (CERCLA), 42 U.S.C. 559601-57.
Other acts relate to meat and poultry inspection, plant pests, weeds and plant quarantine. This listing
refers only to Federal jurisdiction and is NOT meant to be complete. Involved Federal agencies
include FDA, FSIS, APHIS, EPA, NIH, and OSTP.

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