Accepted Manuscript

Title: Characterization of Drug Load Variants in a Thiol

Linked Antibody-Drug Conjugate Using Multidimensional
Chromatography

Authors: Jonathan J. Gilroy, Catherine M. Eakin

PII: S1570-0232(17)30454-3
DOI: http://dx.doi.org/doi:10.1016/j.jchromb.2017.06.005
Reference: CHROMB 20634

To appear in: Journal of Chromatography B

Received date: 17-3-2017

Revised date: 18-5-2017
Accepted date: 3-6-2017

Please cite this article as: Jonathan J.Gilroy, Catherine M.Eakin, Characterization
of Drug Load Variants in a Thiol Linked Antibody-Drug Conjugate
Using Multidimensional Chromatography, Journal of Chromatography
Bhttp://dx.doi.org/10.1016/j.jchromb.2017.06.005

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Characterization of Drug Load Variants in a Thiol Linked

Antibody-Drug Conjugate Using Multidimensional

Chromatography

Jonathan J. Gilroy and Catherine M. Eakin*

Department of Analytical Sciences, Seattle Genetics, Inc., Bothell, Washington 98021-3907, United States

*Correspondence should be addressed to ceakin@seagen.com

Mailing Address: Seattle Genetics, Inc., 21823 30th Drive SE, Bothell, WA 98021
ABSTRACT

The biological complexity associated with biotherapeutics such as antibody-drug conjugates (ADCs) requires
extensive characterization to ensure product quality consistency, safety, and efficacy. ADCs generated via partial
reduction of antibody interchain disulfide bonds result in a distribution of variants containing 0 to 8 conjugated
drugs. Hydrophobic interaction chromatography (HIC) is a key analytical technique used to separate drug load
variants of thiol linked ADCs and to calculate the average drug-to-antibody molar ratio (DAR). Salts present in HIC
separations are not amenable to mass spectrometry (MS) detection, therefore confirmation of HIC peak identities
have historically required offline fraction collection and subsequent MS analysis. We present a workflow
comprised of three MS compatible two-dimensional liquid chromatography methods for higher throughput
characterization of HIC peaks without manual fractionation. These methods are complementary and together
provide DAR confirmation, identification of drug-load isomers, and localization of post-translational modifications
to specific subunits. The results demonstrate an efficient mechanism for characterization of ADC HIC peaks and
provided unique insight into differential HIC retention times based on conjugation sites and glycan structure.
ABREVIATIONS:
ADC: Antibody-drug conjugate
MS: Mass spectrometry
PNGase F: Peptide-N-glycosidase F
QTOF: Quantitative time of flight
UV: Ultraviolet
RP: Reversed-phase liquid chromatography
nSEC: Native size exclusion chromatography
nrRP: Non-reduced reversed-phase
rRP: Reduced reversed-phase
Fab: Fragment antigen-binding
Fc: Fragment crystallizable region
LC: Antibody light chain
HC: Antibody heavy chain
DAR: Drug-to-antibody ratio
HIC: Hydrophobic interaction chromatography
SEC: Size exclusion chromatography
2D-LC: Two-dimensional liquid chromatography
IEX: Ion-exchange chromatography
1. INTRODUCTION

Antibody-drug conjugates (ADCs) are a complex class of biotherapeutics that combine the specificity of a
monoclonal antibody with the high potency of small molecule drugs for the treatment of cancer [1, 2]. ADCs have
proven effective in reducing the systemic toxicity typically associated with traditional chemotherapeutic
treatments since the conjugated cytotoxic agent is delivered to the tumor site by utilization of the monoclonal
antibody site specific selectivity. Once bound to the antigen target, the ADC complex is internalized and the small
molecule released upon antibody turnover [1, 3]. With the commercial availability of ADCETRIS and Kadcyla,
ADCs have clearly demonstrated a clinical benefit and interest in this class of therapeutics has increased in recent
years with at least 50 different ADCs currently being investigated in clinical trials [4].

ADCs are differentiable based upon three main components: the antibody, the drug, and the linker used to
covalently attach the drug to the antibody. There are now several strategies for covalently linking the drug to the
antibody [5, 6] including at lysine residues [7], interchain cysteine residues [8, 9], engineered cysteine residues [10-
12], and unnatural amino acid residues [13, 14]. Conjugation to native antibody cysteines involves reduction of
interchain disulfides, each of which generates two free sulfhydryl groups [15]. Conjugation of drug-linker to the
reduced cysteines results in a heterogeneous yet controllable mixture of ADCs with a distribution of 0 to 8 drugs
per antibody including several possible isoforms for antibodies loaded with 2, 4 and 6 drugs (Figure 1). Controlling
DAR heterogeneity during manufacturing is of fundamental importance owing to the mechanism of ADC action
with the number of conjugated drugs directly impacting cytotoxicity [16]. For ADCs employing hydrophobic drug-
linkers, clearance has also been observed to correlate with DAR where higher drug load ADCs can clear from the
body faster than lower drug loaded species [16]. The inherent heterogeneity of different drug loads, structural
complexity of positional isoforms, and the mixture of covalent and non-covalently associated antibody light and
heavy chain subunits all combine to form analytical challenges.

Heterogeneity of cysteine linked ADCs is classically monitored by hydrophobic interaction chromatography (HIC)
which separates species based on the number of drugs loaded [17, 18]. For an accurate calculation of average DAR
the HIC profile must be fully characterized to identify the DAR associated with each peak. HIC is not mass
spectrometry (MS) compatible due to non-volatile salts in the mobile phase; therefore, characterization of drug
load variants observed by HIC has required fraction collection, processing, and subsequent analysis through
orthogonal techniques such as reduced and non-reduced reversed-phase liquid chromatography (RP) and capillary
electrophoresis [9, 19]. This process is labor intensive and can subject the fractionated material to confounding
factors from sample handling such as aggregation or degradation [18]. Several native LC-MS assays have been
developed recently to assess drug distribution and have shown a strong correlation to DAR values determined
from HIC-UV based methodology. These methods include native SEC LC-MS [20] developed to maintain non-
covalently associated light and heavy chains generated upon conjugation, and native static nanospray ionization
after proteolytic cleavage of the conjugated drug [21]. Ion mobility MS has also been used for the assessment of
drug distribution and conformational characterization of cysteine conjugated ADCs [22, 23]. MS is an informative
tool for characterization, but can be operationally complex; therefore, DAR determination in quality control is
frequently monitored by HIC.

Multidimensional chromatography (2D-LC) has been recognized in recent years as a powerful tool for
biotherapeutic characterization with multiple examples including detection of impurities [24-26], degradation
products [24], glycans [27], and excipients [28]. One mode of multidimensional chromatography is heart-cutting,
where the peak of interest is diverted to a second dimension separation. For biotherapeutics, the heart-cutting
approach offers significant utility for characterization of chromatography peaks from HIC, ion exchange (IEX), size
exclusion (SEC), or RP methods which are not MS compatible. Heart-cutting peaks from these methods and
diverting to an MS compatible second dimension such as RP or SEC can establish peak identities by mass. The proof
of principle for ADC characterization with 2D-LC has been highlighted with several recent examples. The first is free
drug analysis where small molecules are separated from protein by SEC and diverted onto a second RP dimension
eliminating the need for protein precipitation or offline protein removal [25, 26]. The second is HIC
characterization of antibodies conjugated at cysteines using non-reduced RP with MS detection [29-31]. From
these analyses the drug loading of each HIC peak was inferred based on the RP and MS profiles. The third is IEX
characterization of a lysine ADC using non-reduced RP-MS in the second dimension to determine drug loading and
distribution [32].

To characterize ADC drug heterogeneity without fractionation we developed a workflow which uses three separate
2D-LC methods aimed at providing complete characterization of an ADC HIC profile. The first method, HIC-native
SEC-MS (HIC-nSEC-MS), unequivocally determines the number of conjugated drugs based on non-denaturing intact
mass. The second method, HIC-non-reduced RP-MS (HIC-nrRP-MS), identifies the antibody domains with
conjugated drugs and the presence of post-translational modifications. The third method, HIC-reduced RP-MS
(HIC-rRP-MS), utilizes an online reduction to localize observed post-transitional modifications to an antibody light
or heavy chain subunit. HIC-rRP-MS can also report on the extent of conjugation. Combined, these methods
complement each other and efficiently characterize drug load heterogeneity observed in ADC HIC profiles without
requiring offline fractionation and subsequent re-analysis.

2. MATERIALS AND METHODS

2.1 Antibody-Drug Conjugate. IgG1 recombinant monoclonal antibodies were expressed in Chinese hamster ovary
(CHO) cells and purified according to standard procedures. Conjugation of antibody cysteine residues to
maleimidocaproyl-monomethyl auristatin F was carried out according to established procedures to achieve a
target DAR of 4 [8].
2.2 Two-Dimensional Ultra High Performance Liquid Chromatography. Chromatographic separations were carried
out in the HIC dimension on a Waters H-Class UPLC with CM-A column heater (Waters, Milford, MA). An additional
Waters BSM pump, PDA detector and IDEX MXT715 6-port, 2-position external switching valve (IDEX ,Oak Harbor,
WA) were configured in-house to control second dimension separations (RP and SEC). A stainless steel flow cell
with 5 mm path length was used to monitor the HIC and SEC dimensions and a PTFE flow cell with 10 mm path
length was used to monitor the RP dimensions. UV absorbance at 280 nm was used for optical detection and the
second dimension was performed in-line with an Agilent 6510 QTOF mass spectrometer (Agilent, Santa Clara, CA)
for peak identification. Heart-cut timing and instrumentation control were programmed entirely within the Waters
Empower software version 3.

2.3 Mass Spectrometry. Mass spectral data was acquired on an Agilent 6510 QTOF in positive electrospray
ionization (ESI) mode. For intact analysis, parameters were similar to those previously reported [20]. Briefly, the
range was 100010,000 m/z, drying gas temperature was 360C at a flow rate of 10 L/h, and the nebulizer gas
pressure was 40 psi. The capillary, fragmentor, and octupole voltage were set at 5000, 425, and 800, respectively.
For non-reduced analysis, all parameters were the same except range of 8008000 m/z, nebulizer gas pressure of
30 psi, fragmentor voltage of 400 and octupole rf voltage of 750 were set. For reduced analysis, parameters were
the same as intact analysis except range of 8003200 m/z, drying gas flow rate of 8 L/h, nebulizer gas pressure of
30 psi, and fragmentor voltage of 400 and octupole voltage of 750 were set. The raw data was deconvoluted using
converted to zero charge mass spectra with a maximum entropy deconvolution algorithm within the MassHunter
workstation software version B.03.01.

2.4 High Load Hydrophobic Interaction Chromatography. Sample was diluted 1:1 with 2 M ammonium sulfate
(Sigma, St Louis, MO), 25 mM sodium phosphate (Fisher Chemical, Fair Lawn, NJ) pH 7.0 and separated using a
Butyl-NPR column with dimensions of 4.6 mm x 100 mm (Tosoh, Tokyo, Japan). Mobile phase A consisted of 1.5 M
ammonium sulfate, 25 mM sodium phosphate pH 7.0, and mobile phase B consisted of a mixture of 75/25% 25
mM sodium phosphate pH 7.0 / isopropyl alcohol (Acros Organic, Geel, Belgium) [20]. Isopropyl alcohol was added
to improve peak shape and recovery of ADC. Separation was obtained with a linear gradient of 2055%B over 11.7
minutes, followed by a 4.3 minute hold at 55%B, at a flow rate of 0.2 mL/min. 225 g of ADC was injected.

2.5 Native Desalting SEC. Heart-cut peaks were diverted from the high load HIC dimension in volumes less than
120 L onto a Waters Acquity BEH SEC column, containing 1.7 m particles with 125 pores, and dimensions of
4.6 mm x 150 mm (Waters, Milford, MA). Separation of ADC from the HIC salts for in-line mass detection occurred
isocratically at a flow rate of 0.2 mL/min for 17 minutes in 200 mM ammonium acetate (Fisher Chemical, Fair
Lawn, NJ) pH 7.0.

2.6 Non-Reduced Reversed-Phase Chromatography. Heart-cut peaks were diverted from the high load HIC
dimension in volumes 120 L onto an Agilent PLRP-S column at 70C containing 5 m particles with 1000 pores
and dimensions of 2.1 mm x 50 mm (Agilent, Santa Clara, CA). Mobile phase A and B was 0.05% TFA (Fisher
Chemical, Fair Lawn, NJ) in water and 0.05% TFA in acetonitrile (Fisher Chemical, Fair Lawn, NJ), respectively.
Separation of each HIC peak heart-cut occurred with a linear gradient of 25 50%B over 20 minutes at a flow rate
of 0.1 mL/min into the mass spectrometer.

2.7 Online Reduced Reversed-Phase Chromatography. Heart-cut peaks were diverted from the high load HIC
dimension in volumes 120 L through a static U-466 mixing tee (IDEX, Oak Harbor, WA) and combined with 50
mM DTT (Pierce Biotechnology, Rockford, IL), 50 mM Tris (Sigma, St Louis, MO) pH 8.3 at a flow rate of 0.2 mL/min
to achieve a 1:1 mixing ratio. The resulting mixture was pumped onto a 500 L stainless steel loop connected to an
Agilent PLRP-S column containing 5 m particles with 1000 pores and dimensions of 2.1 mm x 50 mm (Agilent,
Santa Clara, CA). To promote reduction, the instrument flow was decreased to 0.05 mL/min after HIC peak
diversion allowing mixing with DTT at 70C within the temperature controlled sample loop. To separate in the
second dimension, mobile phase A was 0.05% TFA in water and mobile phase B was 0.05% TFA in acetonitrile. DTT
was removed by flushing the column with 25%B for 12 minutes at 0.3 mL/min. Separation of the reduced HIC peak
occurred with a linear gradient of 25 50%B over 12.4 minutes at a flow rate of 0.3 mL/min into the mass
spectrometer.

2.8 Hydrophobic Interaction Chromatography of Deglycosylated ADC. ADC was deglycosyalted by incubation at
37C for 4 hours with PNGase F (New England Biolabs, Ipswich, MA) at 1 g protein per 69 units of enzyme. The
deglycosylated ADC was then analyzed by analytical HIC. The sample was prepared for HIC as in the high load
method and separated using the same column and mobile phase compositions. Separation of ADC was obtained
with a linear gradient of 1585%B over 50 minutes, followed by a 10 minute hold at 85%B, at a flow rate of 0.4
mL/min. 40 g of ADC was injected and UV absorbance at 280 nm was used for detection.

3. RESULTS AND DISCUSSION

3.1 2D-LC Instrument Configurations

To facilitate ADC characterization and analysis, several multidimensional chromatography approaches were
developed with a self-assembled 2D-LC system. The ADC characterization workflow consisted of three individual
2D-LC methods with unique instrument configurations. For each method a single 6-port, 2-position switching valve
was used to divert the center, or heart, of each HIC peak onto a second dimension column. A heart cut approach
was selected over a comprehensive mode approach to allow diversion of larger volumes from HIC into the second
dimension, resulting in increased MS signal. Additionally, with heart-cutting the second dimension separation is
not time constrained allowing for the implementation of higher resolving methods. For these applications, a
separate pump and UV detector were employed to control and analyze the second dimension separation.
Switching valve timings for peak diversion from the first dimension onto the second dimension were controlled
with event out switches in the LC software. The valve was diverted for up to 0.6 minutes to transfer individual HIC
peaks from the first dimension onto either an SEC or a RP column in the second dimension (Figure 2). The entire
diverted sample was subsequently separated in the second dimension from the HIC mobile phases allowing for in-
line mass detection and identification of the protein species. For HIC-nrRP and -rRP separations, a sample loop was
inserted prior to the RP column to dilute the organic content in each heart-cut and improve peak shape (Figure 2B,
C). In addition, for the HIC-rRP method, a micromixer tee was installed to combine HIC heart-cuts with a DTT
solution for online reduction. After reduction, the sample was separated from DTT and HIC mobile phase by the
second dimension RP column and subsequent peaks analyzed with in-line mass spectrometry.

3.2 ADC Drug Load Identification

The 2D-LC system was utilized to determine the drug load of ADC species separated by HIC using HIC-nSEC-MS. The
SEC column functions to separate ADC from HIC salts. In addition, the SEC mobile phase is MS compatible and non-
denaturing allowing for the preservation of non-covalently associated subunits present in ADCs conjugated via
interchain disulfide bonds. HIC-nSEC-MS therefore enables direct DAR confirmation of each HIC peak based on the
observed intact mass [20]. The peaks chromatographically separated by HIC were putatively assigned DAR values
from 0 to 8 based on retention times and published ADC characterizations (Figure 3A) [19, 20]. To confirm the
putative DAR assignments, each HIC peak was individually heart-cut and diverted to nSEC-MS. The deconvoluted
mass analysis from DAR regions 2-6 identified major species at 150,550 Da, 152,398 Da, 152,399 Da, and 154,251
Da (Figure 3C-F). These masses are within 24 ppm of ADC containing 2, 4a, 4b and 6 conjugated drugs, respectively.
The close agreement between theoretical and observed masses for peaks corresponding to DAR2 thru DAR6
agrees with the accuracy of other intact mass methodologies [20, 22] and directly confirms the putative
assignments of these HIC peaks. In addition, only masses for the expected DAR were detected indicating
chromatographic resolution of the individual species by HIC. The observation of two peaks with masses
corresponding to 4 drug loads, DAR4a and DAR4b, suggests separation of ADC structural heterogeneity by HIC
(Figure 3A, D, E). Additional characterization is needed to further define these isoforms. The quality of the mass
spectrum was poor for the HIC heart-cuts in the DAR0 and DAR8 regions. A dominant mass was not observed for
DAR0, while the DAR8 region did have a weak mass of 156,116 Da which is 81 ppm from the theoretical mass of an
ADC with 8 conjugated drugs (Figure 3A, B, G). The data for minor peak regions eluting between the major peaks
were also too low in intensity to make positive identifications. This highlights the known sensitivity limitations of
nSEC-MS relative to denaturing methods due to the decreased ionization efficiency of the mobile phase [20]. As a
result, for these lower abundant species (<6% peak area) there was insufficient mass quality to report a definitive
DAR. Based on HIC elution time the DAR0 and DAR8 species are putatively identified but confirmation required
additional characterization.

3.3 Identification of ADC Positional Isomers

A 2D-LC HIC-nrRP-MS method similar to those previously reported [29, 30] was implemented as a more sensitive
and orthogonal approach to further characterize the two HIC peaks corresponding to an average DAR of 4, and to
definitively identify the lower level putative DAR0 and DAR8 peaks. ADCs with masses consistent with the same
drug load but chromatographically resolved by HIC may be positional isomers, ADCs with the same DAR but with
conjugation of drugs to different antibody cysteines [29]. In order to determine if this was the case for the ADC
analyzed here, nrRP-MS was utilized in the second dimension of HIC heart-cuts. nrRP operates under denaturing
conditions, which dissociates non-covalently associated subunits for subsequent identification by in-line mass
spectrometry [29-31]. The presence of non-covalently associated subunits is a feature of ADCs conjugated via
native cysteines due to the reduction of interchain disulfide bonds followed by conjugation with drug. For
example, under nrRP conditions the positional isomers shown for a DAR4 (Figure 1) would be expected to
dissociate to form a heavy-heavy chain subunit with 2 drugs (HH+2D) and two identical light chain subunits with 1
drug (L+1D), or two identical heavy-light chain subunits with 2 drugs (HL+2D), or a heavy-heavy-light chain subunit
with 3 drugs (HHL+3D) and a L+1D. The predominant isoform in each HIC peak can then be determined by piecing
the observed subunits back together.

Analysis of the DAR4a and DAR4b peaks by HIC-nrRP-MS show different non-covalently associated subunits which
confirm these peaks are isomers differing in the sites of conjugation. nrRP-MS of the DAR4a peak showed two
major subunits with masses of 24,806 Da and 102,785 Da which are within 7 and 19 ppm of a L+1D and HH+2D,
respectively (Figure 4A, B, D, E). These subunits are consistent with an ADC conjugated at the light chain and heavy
chain in each fragment antigen-binding (Fab) region. An inherent property of this ADC is chromatographic co-
elution of L+1D and HH+2D by nrRP, however, the different species are readily detected in the raw and
deconvoluted mass data. The DAR4b peak contained one major subunit with a mass of 76,199 Da which is 4 ppm
from the expected mass of HL+2D (Figure 4A, C, F), indicating an ADC with four drugs conjugated in the hinge of
the fragment crystallizable (Fc) region. The minor species observed by nrRP were determined by mass to be
subunits corresponding to the other isomer. These minor peaks result from impure heart-cuts due to the lack of
baseline resolution between DAR4a and DAR4b in HIC. By combining the HIC-nSEC-MS and the HIC-nrRP-MS results
the drug load and the positional isoform of the DAR4a and DAR4b peaks were unambiguously determined.

The HIC-nrRP-MS method was also used to characterize the predominant isoforms for the other HIC peaks. The low
abundance of DAR0 and DAR8 prevented peak confirmation by HIC-nSEC, however, the identity of both species
were confirmed using HIC-nrRP-MS (Table 1). For DAR0, a mass consistent with intact unconjugated antibody was
observed, while for DAR8 two subunits were observed, L+1D and HC with three drugs (H+3D), thus confirming
these HIC peaks as DAR0 and DAR8. The DAR2 peak resulted in L+1D and HHL with one drug (HHL+1D), consistent
with two drugs conjugated on one Fab arm. Three subunits were observed for the DAR6 peak; L+1D, H+3D, and
HL+2D. This suggests the DAR6 peak is composed primarily of an ADC containing four drugs conjugated in the Fc
and two drugs in a Fab region. The mass accuracy of the nrRP fragments from the HIC peaks were all observed to
be <25 ppm, which is in good agreement with other HIC- nrRP-MS analyses (Table 1) [29, 30]. The isomers
identified here by 2D-LC are consistent with previous characterization of cysteine linked ADCs but did not require
offline fractionation and thus afforded a significant time savings [9].

3.4 Differential HIC Retention from Glycan Structure and Conjugation Site

Minor species in region A eluting between DAR2 and DAR4a were also characterized by HIC-nrRP-MS (Figure 5A).
Region A was found to contain unconjugated HL, HH+2D, L+1D, and HL+2D all within 31 ppm of their expected
masses. From the observed subunits, the HH+2D and L+1D could form the DAR4a species, and two HL+2D could
form the DAR4b species. The DAR species corresponding to the unconjugated HL subunit is unknown. The
presence of DAR4a in region A can be accounted for by an impure heart-cut, however, DAR4b is well resolved from
region A thus its presence cannot be explained by co-migration. Notably, the DAR4b HL+2D species found in region
A contained two additional predominant masses. These masses corresponded to 228 Da and 1445 Da lighter than
HL+2D (Figure 5A, B). Antibodies contain an N-linked glycan in the heavy chain Fc region [33] and the masses
observed here are consistent with the presence of a mannose 5 or aglycosylation [34, 35].

To confirm that the observed mass differences were indeed associated with glycan differences on the heavy chain,
a 2D-LC HIC reduced RP-MS (HIC-rRP-MS) method was developed. Peaks were heart-cut from HIC followed by
online reduction and separation by RP in the second dimension [24]. The reduction was assessed as complete
based on the absence of covalently bound subunits in the RP dimension. Separation of the light and heavy chain by
reduction allows for the assignment of the observed mass changes to a particular subunit. Additionally, HIC-rRP-
MS can serve as an orthogonal technique to determine predominant DAR isoforms. Analysis of the major HIC
species confirmed the assignments determined by HIC-nrRP-MS (Table S-1). HIC-rRP-MS analysis of HIC region A
identified unconjugated LC and HC, L+1D, H+1D and H+2D. As observed by HIC-nrRP, mass losses of 230 and 1447
Da were observed on the DAR4b H+2D subunit localizing these modifications to the HC suggesting they are from
smaller glycan species (Figure 5A, C). The smaller glycans made up approximately half of the glycan population in
both the HIC-nrRP-MS and the HIC-rRP-MS methods suggesting that the intact DAR4b species migrating in region A
is composed of one HC with a canonical glycan and one HC with a smaller glycan species. Combined, these results
suggest that glycosylation may impact HIC retention time with smaller N-glycans eluting earlier. Region A of the
HIC profile has been characterized for a different thiol linked ADC as the location of odd-loaded species with no
chemical modifications reported [30]. This suggests that the species eluting in this area may be specific to
individual ADCs.

To determine the impact of antibody glycosylation on HIC retention time N-linked glycans were removed from the
ADC by enzymatic treatment with PNGaseF and the ADC analyzed by analytical HIC. The deglycosylated ADC
maintained a similar retention time and peak intensity for the DAR0, DAR2 and DAR4a species, but had an increase
in region A intensity along with a corresponding decrease in DAR4b peak intensity. DAR6 and DAR8 peaks also
appear to shift to earlier elution times (Figure 6). The DAR4b, DAR6 and DAR8 species all contain 4 drugs in the
antibody Fc region and all have a change in retention time upon deglycosylation. In contrast, the retention time of
ADCs conjugated in the Fab region (DAR2 and DAR4a) were unaffected by the removal of N-glycans. To identify the
species that increase in region A upon deglycosylation, HIC-nrRP-MS was utilized. Analysis of region A from
glycosylated and deglycosylated samples were found to contain the same species (HL, HH+2D, LC+1D and HL+2D).
However, upon deglycosylation, the level of HL+2D increased 2-fold in comparison to the other peaks, which were
relatively unchanged. The HL+2D subunit is a unique identifier of the DAR4b species and is not present in the other
closely eluting peaks (DAR2 or DAR4a) suggesting that, for this ADC, region A contains DAR4b with small or no N-
glycans on at least one heavy chain. The shifting of the DAR6 and DAR8 species to earlier retention times upon
deglycosylation was also confirmed by the observation of the expected subunits for each of these species.
Combined, these observations suggest that glycosylation status can impact the HIC retention time for ADCs
conjugated in the Fc region while ADCs conjugated in the Fab region are not impacted. HIC separates proteins
based on hydrophobic differences, yet the interaction between protein and stationary phase is localized to amino
acid residues on the protein surface. Changes in protein conformation or hydrophobicity can therefore be detected
by differences in HIC retention time [36, 37]. The observation of glycans impacting HIC retention when the Fc
region is conjugated suggests a difference in hydrophobicity between aglycosylated and canonically glycosylated
ADC which is not detected with conjugation in the Fab region. The proximity of the Fc conjugation sites to the N-
linked glycosylation site may contribute to this retention time difference.

4. CONCLUSIONS

During ADC development HIC characterization of drug load variants are necessary for accurate DAR determination.
Multidimensional chromatography has emerged as a powerful tool for rapid characterization of non-MS
compatible methods, such as HIC [29-31], IEX [32] or SEC [25, 26]. Here, we evaluate an ADC HIC profile using a
suite of 2D-LC methods aimed at identification of DAR, positional isomers, and post-translational modifications
that influence HIC retention time. HIC-nSEC-MS was developed to provide intact mass data to unequivocally
determine the DAR of HIC peaks without inference from subunits generated upon denaturation. Further
characterization of predominate structural isoforms and post-translational modifications was then achieved
through the use of HIC-nrRP-MS and HIC-rRP-MS methods. As a whole, the workflow was able to quickly deliver a
comprehensive characterization of an ADC HIC separation without a large demand on material or personnel. The
time savings afforded by these 2D-LC methods make them attractive options to characterize hydrophobicity
profiles of ADCs generated with different drug-linkers or conjugation approaches. Further improvement to the
workflow throughput could be achieved by decreasing second dimension cycle times, using multiple loops to
capture several HIC peaks per injection, or employing comprehensive mode conditions. In addition, the ease of 2D-
LC analysis opens up opportunities for identification of potentially unknown peaks in samples not routinely
assessed by offline techniques, such as forced degradation and early process development samples. In this work,
we focused on ADC HIC peaks; however, 2D-LC can largely be considered a modular technique where first and
second dimension methods can be mixed and matched as needed. For instance, the masses of monomer or
fragments observed in an SEC profile could be characterized through 2D-LC with nrRP-MS in the second dimension.
The 2D-LC methods presented here represent a foundation that could be leveraged throughout development for
direct identification of distinct attributes such as size, charge, and chemically modified variants of proteins,
antibodies, and ADCs.

ACKNOWLEDGMENTS

The authors would like to thank Oscar Salas-Solano and Kevin Anderson for their helpful discussion, review, and
support of this work.

FUNDING

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit
sectors.

AUTHOR CONTRIBUTIONS

The manuscript was written through contributions of all authors. All authors have given approval to the final
version of the manuscript.

APPENDIX A. Supplementary data (Table S-1)

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Table 1. Predominant subunits identified from HIC-nrRP-MS analysis for the major HIC peaks. The difference from
theoretical mass is shown in ppm. The ADC isomer for each DAR is inferred from detected subunits.

Figure 1. Schematic of a cysteine conjugated ADC containing 0, 2, 4, 6 and 8 drugs per antibody. Positional isoforms
are shown for DAR2, DAR4 and DAR6 species. Antibody light and heavy chains are depicted, with the shorter
subunit corresponding to light chain.

Figure 2. Switching valve states during 1st and 2nd dimension separations denoted in 1, and heart-cut diversion to
the 2nd dimension column denoted in 2. Flow paths and valve configurations are shown for three different second
dimension separations (A) HIC-nSEC-MS (B) HIC-nrRP-MS, and (C) HIC-rRP-MS.

Figure 3. ADC analyzed using HIC-nSEC-MS. (A) HIC chromatogram of an ADC with DAR peak assignments shown.
(B-G) Deconvoluted mass spectra from nSEC-MS of HIC heart-cuts DAR0, DAR2, DAR4a, DAR4b, DAR6, and DAR8,
respectively. Asterisks denote ADC glycoforms.

Figure 4. Identification of DAR4a and DAR4b positional isoforms by HIC-nrRP-MS. HIC chromatogram with dotted
lines showing heart-cut regions of two DAR4 peaks (A). Second dimension nrRP chromatograms of the DAR4a (B)
and DAR4b (C) regions. Deconvoluted mass spectra from the nrRP dominant peak. DAR4a contained HH+2D (D) and
L+1D (E). DAR4b contained HL+2D (F). Asterisks denote heavy chain glycoforms and circles denote ammonium
adducts.

Figure 5. Analysis of region A by HIC-nrRP-MS and HIC-rRP-MS. (A) HIC chromatogram with dotted line showing
region A heart-cut. (B) Deconvoluted mass spectrum of HL+2D peak from region A by nrRP. (C) Deconvoluted mass
spectrum of H+2D peak from region A by rRP.

Figure 6. HIC chromatograms of (A) glycosylated and (B) deglycosylated ADC. Region A heart-cut for analysis by HIC-
nrRP-MS is shown in the dotted box. The denotes DAR4b, the denotes DAR6, and denotes DAR8.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Table 1

Presumed
HIC
Predominant Subunits Observed ADC
Peak
Isomer

DAR0
LHHL
20 ppm

DAR2
L+1D HHL+1D
21 ppm 24 ppm

DAR4a
L+1D HH+2D
13 ppm 10 ppm

DAR4b
HL+2D
1 ppm

DAR6
L+1D H+3D HL+2D
9 ppm 3 ppm 2 ppm

DAR8
L+1D H+3D
9 ppm 9 ppm