Case 1:18-cv-01363-CFC Document 79-10 Filed 03/22/19 Page 1 of 11 PageID #:
`9538
`
`ARTICLE
`
`Effects of Antibody Disulfide Bond Reduction on
`Purification Process Performance and Final Drug
`Substance Stability
`
`2
`
`Wai Keen Chung,1 Brian Russell,2 Yanhong Yang,3 Michael Handlogten,4
`Suzanne Hudak,5 Mingyan Cao,3 Jihong Wang,3 David Robbins,1 Sanjeev Ahuja,2
`Min Zhu6
`1
`Purification Process Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg,
`Maryland; telephone: þ1(301)398-2799; e-mail: chungw@medimmune.com
`Cell Culture and Fermentation Sciences, MedImmune LLC, One MedImmune Way,
`Gaithersburg, Maryland
`3
`Analytical Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland
`Cell Culture Sciences, Macrogenics Inc, Rockville, Maryland
`Formulation Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg,
`Maryland
`6
`Protein Science, Boehringer Ingelheim Fremont Inc, Fremont, California
`
`4
`
`5
`
`bond reduction during
`ABSTRACT: Antibody disulfide
`monoclonal antibody (mAb) production is a phenomenon
`that has been attributed to the reducing enzymes from CHO
`cells acting on the mAb during the harvest process. However,
`the
`impact of
`antibody
`reduction on the downstream
`purification process has not been studied. During
`the
`production of an IgG2 mAb, antibody reduction was observed
`in the harvested cell culture fluid (HCCF), resulting in high
`fragment levels. In addition, aggregate levels increased during
`the low pH treatment step in the purification process. A
`correlation between the level of free thiol in the HCCF (as a
`result of antibody reduction) and aggregation during the low pH
`step was established, wherein higher levels of free thiol in the
`starting sample resulted in increased levels of aggregates during
`low pH treatment. The elevated levels of free thiol were not
`reduced over the course of purification, resulting in carry-over
`of high free thiol content into the formulated drug substance.
`When the drug substance with high free thiols was monitored
`
`for product degradation at room temperature and 2–8
`C, faster
`rates of aggregation were observed compared to the drug
`substance generated from HCCF that was purified immediately
`after harvest. Further, when antibody reduction mitigations
`(e.g., chilling, aeration, and addition of cystine) were applied,
`HCCF could be held for an extended period of time while
`
`This is an open access article under the terms of the Creative Commons Attribution-
`NonCommercial-NoDerivs License, which permits use and distribution in any medium,
`provided the original work is properly cited, the use is non-commercial and no
`modifications or adaptations are made.
`Correspondence to: W.K. Chung
`Received 14 October 2016; Revision received 25 January 2017; Accepted 7 February
`2017
`Accepted manuscript online 10 February 2017;
`Article first published online 6 March 2017 in Wiley Online Library
`(http://onlinelibrary.wiley.com/doi/10.1002/bit.26265/abstract).
`DOI 10.1002/bit.26265
`
`providing the same product quality/stability as material that
`had been purified immediately after harvest.
`Biotechnol. Bioeng. 2017;114: 1264–1274.
`ß 2017 The Authors. Biotechnology and Bioengineering
`Published by Wiley Periodicals Inc.
`KEYWORDS: purification;
`stability; antibody disulfide bond
`reduction; aggregate
`
`Introduction
`
`(mAbs) are an important class of
`Monoclonal antibodies
`biomolecules that are used in the treatment of various diseases
`such as cancer, multiple sclerosis, rheumatoid arthritis, lupus, and
`respiratory diseases (Choy et al., 1998; Cobleigh et al., 1999; Haynes
`et al., 2009; Helliwell and Coles, 2009; Robak and Robak, 2009).
`During mAb process development, aggregates and fragments have
`to be removed to adequate levels due to their associated risks with
`increased immunogenicity and potential effects on drug efficacy
`(Fan et al., 2012; Rosenberg, 2006). Further, the presence of these
`product variants can also affect the stability of the product during
`storage leading to reduced shelf life.
`Many commercial manufacturing processes for mAbs involve the
`use of Chinese Hamster Ovary (CHO) cells for product expression
`and depth filtration or centrifugation for harvest, followed by
`purification, and formulation to produce the drug substance. There
`were several recently reported instances in literature whereby
`reduction of mAb disulfide (S-S) bonds is observed during different
`parts of
`the process. Hutchinson and co-workers observed
`significant mAb fragmentation of an IgG4 molecule with increasing
`
`1264 Biotechnology and Bioengineering, Vol. 114, No. 6, June, 2017
`
`ß 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.
`
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`9539
`Purification was performed at bench scale using an €AKTA
`the
`centrifugation shear conditions. They hypothesized that
`Explorer 100 (GE Healthcare, Piscataway, NJ, 18111241) systems
`mechanical forces in the centrifuge were responsible for reducing
`and Vantage L (EMD Millipore, Billerica, MA, 96220250)
`the molecule into half-antibodies (Hutchinson et al., 2006). Trexler-
`chromatography columns. Protein A capture was performed
`Schmidt and co-workers further demonstrated that antibody
`using MabSelectSuRe resin (GE Healthcare Piscataway, NJ,
`disulfide reduction can be attributed to the cell lysis and the release
`17-5438-05) followed by low pH inactivation with 300 mM
`of intra-cellular reducing enzymes (primarily thio-redoxin reduc-
`Glycine, pH 2.35. Intermediate polishing was carried out using
`tase/thioredoxin) as a result of harsh centrifugation conditions
`Super Q 650-M resin (Tosoh Bioscience, King of Prussia, PA,
`(Trexler-Schmidt et al., 2010). Hutterer and co-workers attributed
`17229) and final polishing utilized POROS 50HS resin (Thermo
`the extent of antibody reduction to be dependent on the cell line and
`Fisher Scientific, Waltham, MA, 1335908). Hold studies were
`cell culture process (Hutterer et al., 2013). Various methods of
`performed using 50 mL Flexboy storage bags from Sartorius
`minimizing antibody reduction were reported. Maintaining a highly
`Stedim. After purification,
`the samples were concentrated
`oxidative environment through air sparging was proposed as a
`through centrifugation at 4000g to approximately 120 mg/mL
`solution to shift the equilibrium of the reversible redox reaction
`using an Amicon Ultra-15 centrifugal filter unit with an
`toward oxidation (Mun et al., 2015). Addition of chemical inhibitors
`Ultracel-30 membrane, (Millipore, UFC903096). Samples were
`(e.g., cystine, copper sulfate, EDTA) to act as a competitive
`then dialyzed overnight into 10 mM histidine buffer at pH 6. The
`inhibitor, directly inhibit responsible enzymes, or remove the
`final formulation to 60 mg/mL protein in 10% (w/v) trehalose
`metal ions required in the enzymatic pathway was suggested as
`dihydrate, 0.02% (w/v) polysorbate 80, 10 mM histidine at pH 6
`means of minimizing enzymatic activity (Trexler-Schmidt et al.,
`was achieved by mixing in concentrated buffer.
`2010). However, while the manufacturing process conditions that
`cause the antibody reduction (Hutterer et al., 2013), the impact on
`the molecular structure, as well the susceptibility of various mAb
`isoforms to reduction (Magnusson et al., 1997; Wang et al., 2015)
`were well-characterized, the impact of antibody reduction on
`purification process performance and long term drug substance
`stability has not been reported.
`During process development of an IgG2 antibody, antibody
`reduction was observed in the harvested cell culture fluid (HCCF)
`and was accompanied by an increase in the aggregate levels after the
`low-pH viral
`inactivation step. The long term drug substance
`stability was also affected as a higher rate of aggregation was
`observed. This study investigated the link between antibody
`reduction in the starting HCCF material and aggregation in the
`downstream process as well as in the drug substance storage stage.
`Various reduction strategies to prevent the antibody reduction were
`tested and their impact on process performance and drug substance
`stability were examined.
`
`Antibody Aggregation Analysis
`
`The percentage of antibody aggregates was determined using a
`standard size exclusion chromatography (HP-SEC) method. An
`Agilent HPLC system (Agilent 1200 series) was used with a
`7.8 mm  300 mm TSKgel G3000SW XL column (Tosoh Bioscience,
`08541) at 1 mL/min flow rate using a mobile phase buffer of 0.1 M
`sodium phosphate, 0.1 M sodium sulfate, pH 6.8. The absorbance at
`280 nm was used to quantify the results.
`
`Materials and Methods
`
`Cell Culture, Purification, and Formulation Procedures
`
`Cell culture fluid was generated using CHO cells in a 50 L scale
`fed-batch process in a stainless steel bioreactor (Applkon; Delft,
`The Netherlands) using proprietary media and nutrient feeds
`with an initial working volume of 42 L. Cell separation was
`performed by an LAPX 404 continuous centrifuge (Alfa Laval;
`Lund, Sweden). The centrate was then filtered using X0HC POD
`depth filters
`followed by SHC sterile membrane filters
`(Millipore; Billerica, MA). Storage vessels for HCCF include 1
`and 2 L PETG bottles (VWR, Bridgeport, NJ, 89096–292 and
`89095–290) for small volume aliquots and disposable sterile
`bags (Sartorius Stedim, Bohemia, NY, FXB110922) for large
`volume aliquots. For storage conditions where headspace is
`required in the sterile bags, air was introduced through a
`0.2 mm Acro 50 sterile filter (Pall, 4250) until the bag was fully
`inflated. HCCF was allowed to warm up to room temperature
`before initiation of purification if it had been stored chilled.
`
`Reduced Antibody Species Analysis
`
`Samples were diluted to 2.0 mg/mL in 1X PBS and mixed in
`non-reducing
`sample buffer
`containing N-Ethylmaleimide
`
`C for
`(NEM). All samples were heated on a heating block at 100
`2 min and the protein ladder was heated on a heating block at
`
`C for 5 min. Following denaturation, samples, and the
`100
`ladder were diluted with ultra-pure water and loaded on a
`96-well plate. The plate and a chip that contained the gel dye,
`the destain solution, and the protein express lower maker were
`placed into a LabChip GX system (Perkin Elmer, Waltham, MA,
`124582) for analysis. The GX LabChip was placed in a LabChip
`GXII analyzer (Perkin Elmer, 124582/b) and read using LabChip
`GXII software. Protein and fragments were detected by laser-
`induced fluorescence and translated into gel-like images (bands)
`and electropherograms (peaks).
`
`Free Thiol Quantitation in Harvested Cell Culture Fluid
`(HCCF)
`
`The amount of free thiol at each site of IgG from HCCF was
`determined by Lys-C peptide mapping method under non-
`reducing condition. The free cysteine was capped with NEM,
`and the free thiol per each cysteine-containing peptide was
`calculated as the percentage of NEM-capped peptide. The HCCF
`was first buffer-exchanged to phosphate buffer using a 30 kDa
`
`Chung et al.: Effects of Antibody Disulfide Bond Reduction
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`spectrophotometer. The concentration of free thiol is interpolated
`from a standard curve and the free thiol-to-antibody molar ratio is
`reported.
`
`Results and Discussion
`
`Observation of Reduced Antibody Species Formation
`
`A platform mAb purification process (Fig. 1) was used to purify the
`IgG2 monoclonal antibody that had been stored chilled in a storage
`bag with no headspace for 30 days before purification was initiated.
`As shown in Table I, Purification Run 1, high fragment levels (89%)
`were observed in the HCCF and gradually decreased with each step
`of the purification process. Aggregates levels increased by 1.0% after
`low pH viral inactivation and were removed during the subsequent
`polishing steps. As shown in Figure 2a and b, NR-GX images of the
`capture product and final chromatography polishing intermediate
`detected the presence of fragment bands with molecular weights
`corresponding to combinations of heavy chain (H) and light chain
`(L) fragments of the intact antibody that can arise as a result of
`reduction (L, H, L-L, H-L, H-H, H-H-L), showing that the inter-
`chain disulfide bonds were being reduced. As seen in Figure 2c,
`similar bands of the reduced species were also detected when the
`harvested cell culture fluid (HCCF) was analyzed by NR-GX,
`indicating that the mAb reduction phenomenon was occurring
`either in the bioreactor or at the harvest step. The cell culture
`material was clarified using centrifugation harvest and it has
`previously been reported by Trexler-Schmidt and co-workers that
`harsh centrifugation conditions can impact product quality through
`increased cell rupture leading to the release of intra-cellular host cell
`proteins such as thioredoxin and thioredoxin reductase. This can
`subsequently lead to cleavage of
`inter-chain disulfide bonds
`(Trexler-Schmidt et al., 2010). In addition to thioredoxin and
`thioredoxin reductase, other reducing enzymes present in the
`lysed HCCF including gluthathione reductase and protein disulfide
`isomerase can also cleave disulfide bonds (Guzman, 1997;
`Ikebuchi et al., 1992).
`Based on the hypothesis that the loss in product quality was
`driven primarily by the release of intra-cellular reducing enzymes
`(either from dead cells in the bioreactor or through harsh
`centrifuge conditions) disrupting disulfide bonds in the mAb
`molecule, controls were put in place to slow down the enzymatic
`
`reaction by the temperature of the HCCF to 2–8
`C. In this study,
`(Table I, Purification Run 2), storage of the material in a vessel
`with air-containing headspace was also employed in an attempt to
`provide a more oxidative environment. It was reported that
`providing an oxidative environment can allow for re-oxidation of
`reduced disulfide bonds (Mullan et al., 2011; Mun et al., 2015;
`Wang et al., 2015). As a rule of thumb, the volume of the storage
`vessel employed was twice the volume of HCCF (e.g., 2 L PETG
`bottle were used to store 1 L of HCCF, storage bags were filled to
`half
`the recommended maximum volume and inflated with
`filtered air). In addition, the HCCF was purified within 3 h after
`harvest
`to minimize the exposure of
`the mAb to reducing
`enzymes. As re-oxidation of reduced species can occur across the
`duration of the purification process, multiple 1 mL aliquots of
`purification process intermediates were made as soon as they
`
`Figure 1. Schematic of platform mAb purification process.
`
`MW cut-off centrifugal device. Prior to digestion with a serine
`protease, sample was mixed with NEM and guanidine to cap
`the free cysteine and denature the protein. Following protease
`digestion, half of each reaction mixture was reduced by
`the addition of 1,4-dithiothreitol (DTT). The reduced and
`non-reduced digests were both separated by a 1.7 mm,
`2.1  150 mm Acquity UPLC HSS C18 column (Waters,
`176001126) and analyzed by a tunable UV (TUV) detector
`and an Orbitrap mass spectrometer. The mobile phase A was
`0.02% trifluoroacetic acid (TFA) in water and the mobile phase
`B was 0.02% TFA in acetonitrile. The peptides were eluted at a
`flow rate of 0.2 mL/min with a gradient of mobile phase B from
`0% to 95% over 90 min.
`
`Colorimetric Free Thiol Quantitation in Purification
`Process Intermediates
`
`the disulfide
`The free thiol assay evaluates the integrity of
`connections in a protein by measuring the levels of free thiol groups
`on unpaired cysteine residues. Samples are incubated under native
`0
`-dithiobis-(2-nitrobenzoic
`and denatured conditions with 5, 5
`acid (DTNB) that binds to free thiol and releases a colored thiolate
`ion. The colored thiolate ion is detected with a UV-visible
`
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`AggregatelevelsweredeterminedbyHP-SEC.FragmentlevelsweredeterminedbyHP-SECandnon-reducedGX(NR-GX).FreethiollevelsintheHCCFandsubsequentprocessintermediatesweredeterminedbyLC-MSandacolorimetric
`
`assay,respectively(NA,notanalyzed).
`
`Chung et al.: Effects of Antibody Disulfide Bond Reduction
`
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`
`Biotechnology and Bioengineering
`
`NA
`
`NA
`
`0.0(%frag)
`1.0(%agg)
`99.0(%mon)
`0.2(%frag)
`99.8(%mon)
`0.1(%frag)
`1.5(%agg)
`98.4(%mon)
`
`NA
`
`0.0(%frag)
`0.5(%agg)
`99.5(%mon)
`
`NA
`
`NA
`
`substance
`
`Drug
`
`0.05(mol/mol)
`0.7(%frag)
`99.3(%mon)
`0.0(%frag)
`0.8(%agg)
`99.2(%mon)
`0.0(%frag)
`100.0(%mon)
`0.0(%frag)
`1.0(%agg)
`99.0(%mon)
`0.9(%frag)
`99.1(%mon)
`0.0(%frag)
`0.4(%agg)
`99.6(%mon)
`13.4(%frag)
`87.6(%mon)
`1.3(%frag)
`0.5(%agg)
`98.2(%mon)
`
`Finalpolishing
`
`NA
`
`0.7(%frag)
`99.3(%mon)
`
`NA
`
`0.0(%frag)
`
`100.0(%mon)
`
`0.1(%frag)
`3.6(%agg)
`96.3(%mon)
`0.6(%frag)
`99.4(%mon)
`0.0(%frag)
`2.8(%agg)
`97.2(%mon)
`
`NA
`
`1.2(%frag)
`2.8(%agg)
`96.0(%mon)
`
`polishing
`
`Intermediate
`
`0.14(mol/mol)
`0.7(%frag)
`99.3(%mon)
`0.0(%frag)
`2.2(%agg)
`97.8(%mon)
`0.2(%frag)
`99.8(%mon)
`0.1(%frag)
`4.0(%agg)
`95.9(%mon)
`0.7(%frag)
`99.3(%mon)
`0.0(%frag)
`2.8(%agg)
`97.2(%mon)
`36.5(%frag)
`63.5(%mon)
`1.1(%frag)
`3.2(%agg)
`95.7(%mon)
`
`inactivation
`
`Post-lowpHviral
`
`0.1(mol/mol)
`0.7(%frag)
`99.3(%mon)
`0.0(%frag)
`1.8(%agg)
`98.2(%mon)
`6.9(%frag)
`93.1(%mon)
`0.1(%frag)
`2.0(%agg)
`97.9(%mon)
`1.0(%frag)
`99.0(%mon)
`0.0(%frag)
`2.3(%agg)
`97.7(%mon)
`86.9(%frag)
`13.1(%mon)
`1.6(%frag)
`2.2(%agg)
`96.2(%mon)
`
`Capture
`
`Purificationprocessintermediate
`
`(intra-chainHC)
`
`30.8%
`
`(intra-chainLC)
`
`5.4%
`
`2.1%(inter-chain)
`
`Freethiol
`
`13.4(%frag)
`87.6(%mon)
`
`NR-GX
`
`Cstoragein2Lbottlewithheadspace
`
`(iii)purificationafter2weekhold
`(ii)2–8
`spike
`
`
`
`NA
`
`16.5(%frag)
`83.5(%mon)
`
`NA
`
`12.2(%frag)
`87.8(%mon)
`
`NA
`
`93.6(%frag)
`6.4(%mon)
`
`NA
`
`HCCF
`
`HP-SEC
`
`PurificationRun4PurificationprocesswithHCCFsubjectedto:(i)0.8mMcys/gmAb
`
`NR-GX
`
`HP-SEC
`
`NR-GX
`
`HP-SEC
`
`NR-GX
`
`HP-SEC
`
`analysis
`Purity
`
`Cstoragein50Lbagwithheadspace
`
`(iii)purificationafter4dayhold
`(ii)2–8
`(i)0.4mMcys/gmAbspike
`
`
`
`PurificationRun3PurificationprocesswithHCCFsubjectedto:
`
`Cstoragein50Lbagwithheadspace
`
`(ii)immediatepurification
`(i)2–8
`
`
`
`PurificationRun2PurificationprocesswithHCCFsubjectedto:
`
`Cstoragein50Lbagwithoutheadspace
`
`(ii)purificationafter30dayhold
`(i)2–8
`
`
`
`PurificationRun1PurificationprocesswithHCCFsubjectedto:
`
`Purificationconditions
`
`no.
`PurificationRun
`
`TableI.Comparisonofaggregateandfragmentlevelsinthepurificationprocesspre-andpost-processoptimization.
`
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`
`Figure 2. Non-reduced GX (NR-GX) electropherograms of purification Run 1 (Table I) process intermediates (a) capture product, (b) final polishing product, (c) harvested cell
`culture fluid (HCCF). Black trace: sample; gray trace: reference standard; L: light chain fragment; H: heavy chain fragment.
`
`were available, flash-frozen (by immersion into a solution of
`ethanol containing dry ice) and stored at 80
`
`C. When thawed,
`the aliquots were analyzed immediately in order to get a
`representative readout of the product quality at each step of the
`purification process. The approach of sample handling was
`applied to Purification Run 2 and all subsequent purification
`Runs.
`As seen in Figure 3 and Table I, NR-GX analysis of the
`intermediates showed no reduced species (fragments) present
`across the purification process. In contrast to Purification Run 1
`which started with 86.9% fragment in the HCCF and ended with
`13.4% at the final polishing step, Purification Run 2 had fragment
`levels of 12.2% and 1.0% in the HCCF and final polishing step,
`respectively. Aggregate levels increased by 0.5% (from 2.3%
`
`pre-low pH treatment to 2.8% post-low pH treatment), which was
`lower than the 1% increase observed in Purification Run 1.
`Hence, it is likely that prevention of reduction in the HCCF also
`helped in minimizing aggregate formation during low pH
`treatment.
`for reduction through the
`is possible to control
`While it
`combined approach in Table I, Purification Run 2 (chilled HCCF, air-
`containing headspace, immediate processing), its implementation
`imposes severe limitations on manufacturing flexibility. Chilling of
`the HCCF requires increases processing time. Head space aeration
`potentially becomes less effective as product surface area to volume
`ratios decrease with increasing processing scale. Larger product
`hold tanks may be required and lower capacity limits may have to be
`set due to the need for adequate headspace. While alternatives like
`
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`
`Figure 3. NR-GX images of purification process intermediates from purification
`Run 2 (Table I). HCCF subjected to chilling, headspace aeration, and immediate
`purification after harvest (L: light chain fragment; H: heavy chain fragment—disulfide
`link between fragments).
`
`air sparging in the hold vessel may be a solution, these may not be
`available at every facility. Immediate purification of the HCCF
`becomes challenging when column size limitations
`in the
`manufacturing plant may require multiple cycles of the capture
`step to be performed.
`
`Impact of Cystine Addition on Reduction Control
`
`In order to allow for extended HCCF holds to assure manufacturing
`flexibility without impacting product quality, L-cystine spikes into
`the HCCF were evaluated. L-cystine was reported to be a potential
`competitive inhibitor of the reducing enzymes or to act as a
`surrogate substrate for the enzyme in place of the mAb product
`(Trexler-Schmidt et al., 2010).
`As part of an initial assessment of cystine levels on reduction
`control,
`the level of L-cystine in the harvested HCCF was
`adjusted to 0, 0.4, and 0.8 mM L-cystine per gram of mAb (mM
`Cys/g mAb) through the addition of L-cystine into the HCCF
`immediately after harvest. To provide a worst case scenario for
`antibody reduction (low oxidative environment),
`the HCCF
`aliquots were sealed in flexboy storage bags without headspace
`
`and held for 2 weeks at 2–8
`C before being analyzed. Figure 4
`shows reduced species were not detected at the start of the hold
`
`Figure 4. NR-GX images of harvested cell culture fluid (HCCF) at t¼ 0 and
`2 weeks, containing 0, 0.4, and 0.8 mM cystine per gram mAb. During 2 week hold, HCCF
`C in air-tight bags. (Note: a separate aliquot of the same HCCF at t¼ 0
`
`was held at 2–8
`
`C in a
`was spiked to 0.4 mM cystine per gram mAb and purified after a 4 day hold at 2–8
`vessel with headspace and is listed as purification Run 3 in Table I).
`
`study, but by the end of the hold study, bold bands representing
`H-H-L fragments and faint bands of H-L fragments were
`identified in the samples containing 0 (no cystine) and 0.4 mM
`Cys/g mAb while the sample containing 0.8 mM Cys/g mAb still
`showed no signs of reduction. Decrease in band intensity with
`increasing cystine levels also demonstrates that 0.4 mM Cys/g
`mAb slowed down the rate of reduction during the 2 week hold,
`but 0.8 mM Cys/g mAb was required for complete prevention of
`reduction. Disulfide mapping mass spectrometry (MS) provided
`further evidence that supports the hypothesis of 0.4 mM Cys/g
`mAb being inadequate for complete reduction mitigation
`(Fig. 5). Higher levels of inter-chain free thiol were observed
`in the 0 and 0.4 mM Cys/g mAb samples by the end of the hold
`but not in the 0.8 mM Cys/g mAb sample nor in the control
`(HCCF þ 0 mM cys,
`t ¼ 0). The
`starting material
`largest
`increase in free thiol content occurred at the hinge cysteines,
`followed by the inter-chain cysteines (LC Cys 216, HC Cys134).
`The intra-chain cysteines on the light chain of the mAb showed
`smaller increases in free thiol than the inter-chain cysteines.
`Little or no increase in free thiol was detected on the intra-chain
`cysteines of the mAb heavy chain.
`To assess the impact of cystine addition on purification
`process performance, HCCF was spiked to 0.4 mM cystine/g mAb
`(same material as used in the cystine hold study above) and
`
`purified after a 4 day hold at 2–8
`C in a vessel with headspace
`(Table I, Purification Run 3). At the start of purification, higher
`levels of fragment were observed in the HCCF (16.5% frag) as
`compared to Purification Run 2 (HCCF: 12.2% frag). After
`low pH treatment, a 2% increase in aggregate was observed. In
`comparing Purification Runs 1, 2, and 3 in Table I, a trend is
`observed wherein higher levels of fragment in the HCCF results
`in higher levels of aggregate after low pH treatment. Given that
`antibody disulfide bond reduction was observed for Runs 1 and 3
`but not Run 2, it can be hypothesized that reduction in the HCCF
`is related to the higher levels of aggregate formation during
`low pH treatment.
`The purification performance of Run 3 coupled with the
`observation of reduction for the same HCCF during the cystine
`hold study indicates that chilling, provision of headspace in the
`hold vessel and spiking of cys to 0.4 mM/g mAb was insufficient
`in the prevention of antibody reduction during the HCCF hold.
`To better understand the contributions of headspace provision
`to preventing antibody disulfide reduction, a 0.75 mL aliquot of
`
`the HCCF from Run 3 was stored at 2–8
`C in a 1.5 mL
`Eppendorf tube for 2 weeks before being analyzed by NR-GX.
`Fragment bands were detected in the sample at the end of the
`2 week hold but not at
`the beginning indicated that
`the
`provision of headspace was playing a less significant role to the
`prevention of antibody disulfide bond reduction as compared to
`spiking adequate levels of cystine. Although it was not evaluated
`in this study, it is possible that agitating the HCCF (for large
`volumes) through storage of HCCF in vessels with mixing
`impellers could make the provision of an oxidative environment
`a more effective strategy in preventing antibody disulfide bond
`reduction due to improved oxygen transfer into the HCCF as
`compared to the small scale systems where constant agitation is
`difficult to achieve.
`
`Chung et al.: Effects of Antibody Disulfide Bond Reduction
`
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`Figure 5. Mass spectrometry quantification of change in free thiol levels for HCCF held for 2 weeks in the presence of 0, 0.4, and 0.8 mM cystine per gram of mAb.
`
`Impact of Reduction During HCCF Hold on Aggregation
`During Low pH Treatment
`
`When the capture product from Purification Run 2 was held in
`low pH buffers ranging from pH 3.2 to 3.6, an increase in free
`thiol content was observed with decreasing pH (Fig. 6a). As
`reducing enzymes such as thioredoxin reductase, glutathione
`reductase, and protein disulfide isomerase generally exhibit
`decreased enzymatic activity with decreasing pH (Guzman,
`1997; Ikebuchi et al., 1992; Xia et al., 2003), the increase in free
`thiol is unlikely to be due to enzymatic action but rather due to
`exposure of the antibody to low pH.
`An increase in aggregate content was also seen with decreasing
`pH (Fig. 6b), indicating that there could be a correlation between
`free thiol content and aggregate content. Franey et al. (2010) have
`reported similar findings that an increase in free thiol level leads to
`a corresponding increase in aggregate formation for monoclonal
`antibodies, with the impact on IgG2 molecules being more severe
`compared to other IgG formats due to higher number of inter-chain
`disulfide bonds. It has been established earlier that reduction
`during the HCCF hold leads to the generation of reduced species
`along with increased free thiol. The reduced species with heavy
`chain subunits (H-L, H-H, H-H-L) are likely recovered in the
`protein A product along with intact monomer and undergo low pH
`treatment. With a higher starting level of free thiol in the protein A
`product, coupled with further free thiol formation during low pH
`treatment,
`increased aggregate formation occurs. Although the
`exact mechanism was not investigated in this study, Buchanan and
`co-workers have demonstrated through site-directed mutagenesis
`that having unpaired cysteines on the surface of a mAb can lead to
`significantly increased rates of aggregation (Buchanan et al., 2013).
`Hence, a larger increase in aggregate content was observed during
`
`low pH treatment for Purification Runs 1 and 3 (which showed
`reduction in the HCCF), but not Purification Run 2 (no reduction in
`HCCF due to immediate purification after harvest).
`
`Effect of Increased Cystine Levels and HCCF Hold
`Conditions on Product Quality
`
`As shown above, spiking of HCCF to 0.4 mM cys/g mAb in
`combination with low temperature hold and provision of headspace
`was only able to slow down the rate of reduction and not prevent
`reduction completely. This resulted in high free thiol levels (as
`determined by disulfide mapping MS) which caused increased
`aggregation during low pH treatment. In contrast, 0.8 mM cys/g
`mAb seemed to provide better reduction mitigation and minimize
`aggregate formation. This hypothesis was evaluated through the
`study outlined in Figure 7. To one sample, no L-cystine (0 mM
`cystine) was spiked into the mixture; to the other sample, the
`mixture was spiked to 0.8 mM cystine/g mAb. Half of each sample
`mixture was purified and formulated to 50 mg/mL immediately,
`while the other half was held in vessels with headspace for 2 weeks
`
`at 2–8
`C before purification and formulation. After formulation,
`all four lots were monitored for aggregation stability for up to 17
`
`
`months at 2–8
`C and 1 month at 25 and 40
`C.
`Table I, Purification Run 4 shows the purification performance
`for the HCCF lot that was spiked to 0.8 mM cystine/g mAb and
`stored chilled in a vessel with headspace for 2 weeks before being
`purified. Fragment levels in the HCCF (13.4% frag) were lower as
`compared to Run 3 (16.5% frag) even though the material was held
`for a longer duration under similar conditions. After low pH
`treatment, aggregate levels only increased by 0.4% (comparable to
`Run 2), further supporting the theory that prevention of reduction
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`Case 1:18-cv-01363-CFC Document 79-10 Filed 03/22/19 Page 8 of 11 PageID #:
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`
`at 2–8
`C in the absence of cystine. In contrast, HCCF that was
`spiked to 0.8 mM cys/g mAb and held under similar conditions
`showed no change in free thiol levels. Similar trends in free thiol
`levels were also observed in the subsequent process intermediates
`(Fig. 8b). Capture product generated from HCCF held for 2 weeks in
`the absence of cystine showed the highest free thiol level. Free thiol
`content increased for all aliquots after low pH treatment but the
`capture product with the highest starting free thiol level also showed
`the largest increase during low pH treatment. Though it was not
`investigated in this study, existing levels of free thiol in a sample can
`potentially determine the rate of increase during low pH treatment.
`Free thiol
`levels decreased for all aliquots between the
`low pH treatment and final polishing chromatography process
`steps. There are two possible explanations for this observed
`decrease: (i) fragments and aggregates containing free thiols
`were removed by the polishing chromatography steps; and (ii)
`exposure to the atmosphere during purification allowed for
`disulfide bond formation (i.e., re-oxidation) between free
`thiols
`leading to the
`reformation of
`larger
`fragments,
`monomer, and aggregates. The intermediate polishing unit
`operation consisted of an anion exchange (AEX) chromatog-
`raphy step that is operated in flow-through mode which does
`not retain any fragment, monomer, or aggregate. The final
`polishing unit operation consists of a cation exchange (CEX)
`chromatography step (operated in bind and elute mode) that
`was designed for aggregate removal. Shown in Supplementary
`Figure S1a and b are CEX chromatograms from Purification
`Run 1 (showed the highest degree of antibody disulfide bond
`reduction and high fragment levels) and Run 2 (showed no
`signs of antibody disulfide bond reduction and low fragment
`levels), respectively. In Purification Run 1, a step yield 48%
`was obtained when the column was loaded to a capacity of
`21g/L. In contrast, purification Run 2 gave a step yield of 63%
`for the same column loaded to a capacity of 16g/L. Protein
`breakthrough did not occur in the loading and re-equilibration
`steps, indicating that all loaded protein was collected back in
`the elution and strip pool. Compared to Run 2 which showed
`the typical CEX chromatographic profile for the process, the
`elution peak (usually composed of monomer) in Run 1 had a
`lower peak height with significant tailing, while the strip peak
`(usually composed of monomer and aggregate) was larger than
`expected. Despite the lack of peaks in the load and re-
`equilibration steps, fragment levels by NR-GX fell from 36.5%
`at the end of low pH inactivation to 13.4% at the end of CEX.
`These observations supports the hypothesis that re-oxidation
`of the free thiol
`leading to the format