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` REPORT
`mAbs 5:4, 608–613; July/August 2013; © 2013 Landes Bioscience
`
`Monoclonal antibody disul(cid:31)de reduction during
`manufacturing
`Untangling process e(cid:31)ects from product e(cid:31)ects
`
`Katariina M. Hutterer,1,* Robert W. Hong,1 Jonathon Lull,1 Xiaoyang Zhao,1 Tian Wang,1 Rex Pei,1 M. Eleanor Le,1 Oleg Borisov,1
`Rob Piper,2 Yaoqing Diana Liu,1 Krista Petty,1 Izydor Apostol1 and Gregory C. Flynn1
`
`1Process and Product Development; Amgen Inc.; Thousand Oaks, CA USA; 2Process and Product Development; Amgen Inc.; Seattle, WA USA
`
`Keywords: antibody disulfide reduction, free cysteine, harvest, capillary electrophoresis, CE-SDS
`
`Abbreviations: CCF, cell culture fluid; CHO, Chinese hamster ovary; DO, dissolved oxygen; DTT, dithiothreitol; DTNB,
`5,5'-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent); EOP, end of production; HCCF, harvested cell culture fluid; IAM,
`iodoacetamide; IgG, immunoglobulin G; mAb(s), monoclonal antibody(ies); MEA, micro-extractor automated instrument;
`NADPH, nicotinamide adenine dinucleotide phosphate; NEM, N-ethylmaleimide; NR CE-SDS, non-reduced capillary
`electrophoresis with sodium dodecyl sulfate; PAT, process analytical technology; PPP, pentose phosphate pathway; SDS, sodium
`dodecyl sulfate; t0, initial time point
`
`Manufacturing-induced disul(cid:31)de reduction has recently been reported for monoclonal human immunoglobulin gamma
`(IgG) antibodies, a widely used modality in the biopharmaceutical industry. This e(cid:30)ect has been tied to components
`of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the e(cid:30)ect of
`process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the
`end of production displayed large variations in the extent of antibody reduction between di(cid:30)erent products, including
`no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted
`model in which process variables could be isolated from product properties, we found that antibody reduction was
`dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin
`reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also
`light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity
`following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to
`the extent of antibody reduction during production.
`
`Introduction
`
`The target specificity, favorable pharmacokinetics and pharmaco-
`dynamics, and stability of monoclonal human immunoglobulin
`gamma (IgG) antibodies have resulted in their widespread use in
`the biopharmaceutical industry.1,2 Commercial therapeutic anti-
`body production is a complex but fairly well established process,
`typically involving expression in Chinese hamster ovary cells
`(CHO), harvesting of the secreted protein, and a series of chro-
`matography steps to remove impurities. Reduction of antibody
`interchain disulfide bonds during manufacturing operations has
`recently been the subject of much interest.3-5 This phenomenon
`is observed when extending the time that the antibody remains
`in the cell culture fluid (CCF) or harvested cell culture fluid
`(HCCF) in the “harvest” step of production. This harvest step
`includes separation of cells from the media prior to the first col-
`umn purification.
`
`*Correspondence to: Katariina M. Hutterer; Email: hutterer@amgen.com
`Submitted: 02/28/13; Revised: 04/16/13; Accepted: 04/16/13
`http://dx.doi.org/10.4161/mabs.2475
`
`Process-induced antibody disulfide bond reduction has been
`observed inconsistently at large scale processes and is not typically
`observed with standard bench-scale (up to 10 L) models.5 This
`reduction has been attributed to certain enzymes that are released
`from the intracellular compartments of lysed cells. Components
`in the thioredoxin reduction pathway, including thioredoxin
`reductase and NADPH, have been proposed as the principal
`underlying contributor for this antibody disulfide bond reduc-
`tion.3,4 Reduction has been shown to be virtually eliminated by
`maintaining dissolved oxygen (DO) levels during harvest opera-
`tions.5 In addition, the cysteine/cystine redox couple, which is
`present in the growth media, may affect disulfide bond formation,
`reduction, and rearrangement.6 Likewise, many other media com-
`ponents, such as certain metal ions and their complexes, are likely
`to affect the reduction potential during the harvest procedure.5,6
`In these studies, cell lysis and an anaerobic environment
`both promoted antibody reduction during harvest;5,6 therefore,
`
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`©2013 Landes Bioscience. Do not distribute.
`
`reported in some scaled-up, but not bench-scale, processes.5 This
`effect of scale may be attributed to the maintenance of oxygen
`in small-scale harvests, which may preserve disulfide bonds.
`Typically, bench-scale experiments are open to the air, which
`allows more efficient oxygen transfer than typical manufacturing-
`scale (15,000 to 20,000 L) cell culture production. Bench-scale
`experiments also use different centrifuge equipment, introducing
`the possibility of different degrees of cell shearing during removal
`of debris. To facilitate harvest reduction experiments, a small-
`scale model, similar to previously described models,5 was devel-
`oped. A “worst case” reduction model of cell culture extract was
`generated by mechanically shearing 2 L of whole cell culture fluid
`(CCF) used for production of an IgG1κ mAb (mAb A), trans-
`ferring the sheared CCF into a 3 L bioreactor, and sparging the
`resultant slurry with nitrogen to simulate the anaerobic environ-
`ment of the commercial scales. Samples were taken at 0, 0.5, 1,
`2, 4, 8, and 24 h and immediately frozen at −70°C. Non-reduced
`capillary electrophoresis with sodium dodecyl sulfate (NR
`CE-SDS) was performed on all samples to measure the degree of
`interchain disulfide bond breakage. Representative electrophero-
`grams of a partially reduced antibody, a properly disulfide-linked
`antibody, and a blank are shown in Figure 1. This figure shows
`that the peaks in the pre-peak region of the electropherogram
`increase in intensity relative to the main, properly disulfide-
`linked, peak. These pre-peaks have been shown to be light chain
`(L), heavy chain (H), and combinations of the two chains (HL,
`HH, HHL).7 Because size exclusion chromatography indicates
`that reduction does not result in disassembly of the antibody
`chains, the NR CE-SDS pre-peaks represent properly assembled
`antibodies with one or more broken interchain disulfide bonds.
`The relative area associated with the pre-peaks and main peak
`were used to monitor interchain disulfide reduction in a series
`of harvest experiments. Results showing antibody reduction in
`mAb A for up to 24 h after cell shearing in the small-scale model
`are shown in Figure 2. An increase in the percentage of pre-peaks
`over time is observed, from 9% at the initial time point to ~45%
`at 8 h. This increase in the percentage of pre-peaks replicates
`previously published results5 and demonstrates that the small-
`scale model is capable of inducing and monitoring disulfide bond
`reduction. It is worth noting that the pre-peak level decreases
`after 8 h, and it is only ~12% by 24 h, indicating that disulfide
`bonds can reform. This observation is consistent with previously
`published results.5
`Product, cell line and process. Partial disulfide bond reduc-
`tion behavior was probed with multiple Amgen therapeutic
`antibodies and cell lines. Three products, an IgG2λ (mAb B),
`an IgG2κ (mAb C), and mAb A, the IgG1κ discussed above,
`were tested by shearing end of production cells and subjecting
`the lysed CCF to nitrogen sparging in the small-scale model at
`25°C. Figure 3 displays the relative amount of intact antibody for
`each of these products as a function of time. Although the IgG1κ
`results demonstrate that an interchain disuflide can be reduced in
`this antibody type using this small-scale model, no changes were
`seen in mAb C (IgG2κ) or mAb B (IgG2λ). This lack of reduc-
`tion under these reduction promoting conditions has not been
`previously reported, and indicates tight controls of air sparging
`
`Figure 1. NR CE-SDS Electropherograms. Partially Reduced mAb (top),
`Puri(cid:31)ed mAb (middle), and Blank (bottom).
`
`Figure 2. Reduction of mAb A in Small Scale Reduction Model as a
`Function of Time. Pre-peaks (circles) and Main peak (squares).
`
`it is clear that adequate process understanding and control is
`necessary to minimize or eliminate disulfide bond reduction
`induced by manufacturing procedures. In addition to variation
`due to manufacturing processes, differences between products
`were observed.5 Because cell cultures, cell lines, and the prod-
`ucts themselves can vary in cell cultures expressing two differ-
`ent antibody products, the underlying causes for these reduction
`differences could not be determined. The study presented
`here explores the relationship between reduction and process
`variables, separating the influence of process and products to
`demonstrate that CHO cell line or cell culture process can dra-
`matically influence reduction during harvest operations and
`that the antibody class and light chain type also influences the
`extent of that reduction.
`
`Results
`
`Small scale model. Harvest-related disulfide reduction has been
`reported as highly dependent on process scale and has been
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`Figure 3. Reduction Behavior of Di(cid:30)erent Products and Cell Lines over
`time. mAb A (triangles), mAb B (circles), and mAb C (squares).
`
`the cell culture process differences, either indirectly, by influenc-
`ing expression of thioredoxin, expression of thioredoxin reduc-
`tase, and utilization of the PPP, or directly, by differing levels of
`redox active media components such as cystine/cysteine and cop-
`per. In the case of mAb C, the measured thioredoxin reductase
`activity is lower than that for mAb A (Table 1); however, this
`could be due to the lower cell density. In contrast, the cell density
`is similar for mAb B, and therefore the cell line and process can
`be determined to have a significant effect on reducing power. The
`presence of substantial thioredoxin reductase activity in these
`cell lysates is not unexpected because some apoptosis, which will
`release intracellular contents, inevitably occurs during cell cul-
`ture. This means, however, that the difference in reducing power
`cannot be attributed to thioredoxin system activity alone because
`the thioredoxin reductase activity was higher in the mAb B lysate
`than the mAb A lysate. Therefore, either the other redox active
`components of the system have a major affect or substantial dif-
`ferences in the availability of NADPH exist. Taken together, the
`results suggest that the cell line (clone) or cell culture process play
`a key role in harvest-related reduction.
`Product properties. As described above, the reconstituted
`extract model, demonstrates that striking differences exist in
`susceptibility to reduction among antibody products. Previously
`published studies have shown that antibody sub-classes differ
`in sensitivity to disulfide bond reduction.8 Differences in reduc-
`tion susceptibility due to light chain type have not previously
`been observed for thioredoxin catalyzed reduction, but have been
`shown using chemical reductants.9-11 A chemical model system
`was developed to investigate antibody type (IgG1 and IgG2) and
`light chain type sensitivity to thioredoxin catalyzed reduction.
`As illustrated in Figure 4, reduction sensitivity is dependent on
`both antibody class and light chain type. Reduction sensitivity, in
`decreasing order, is IgG1λ, IgG1κ, IgG2λ, IgG2κ. This trend held
`true for all of the additional molecules we have tested, and for dif-
`ferent stoichiometric ratios of the reagents and antibody (data not
`shown). Sensitivity to antibody subclass has also been reported for
`other reductants, such as DTT.9,10 The reduction sensitivity trend
`
`or cell shearing are not necessary for all antibody production pro-
`cesses. This comparison of end of production CCF shows stark
`differences in behavior, but does not distinguish between the
`effects of product, cell line, or cell culture process. While the
`mAb C (IgG2κ) titer and cell density are fairly low, and that
`might account for the difference, both the mAb A (IgG1κ) and
`the mAb B (IgG2λ) have relatively high titers and cell densities,
`as shown in Table 1. IgG2s, such as the mAb B, are known to
`be less susceptible to reduction by thioredoxin;8 however, there
`could also be differences between the cell lines or processes that
`could contribute to these observations.
`Direct comparison between cell lines is complicated due to the
`differences that may arise through the transfection process. Both
`copy number and insertion site can vary from clone to clone, and
`both of these parameters may also affect cell growth, viability,
`productivity, and metabolism.12,13 Therefore, to partially disen-
`tangle the effect of product, cell line and process on the level
`of reduction, end of production cells from these three products
`were lysed, and the original product was removed via Protein A
`affinity to create soluble cellular component material. The reduc-
`tion activity was shown to be maintained through this type of
`processing by Trexler-Schmidt et al.5 The results in Table 2 illus-
`trate the difference in NR CE-SDS % Main peak between the
`t0 and 8 h samples for several combinations of cells and purified
`products. To determine whether this material remained active,
`purified mAb A was spiked back into its own soluble cellular
`components and held under nitrogen overlay for 8 h. When mAb
`A sheared cell broth was reconstituted in this manner, the differ-
`ence in NR CE-SDS % Main peak was ~45%, identical to the
`small-scale model results, which indicates that the reducing activ-
`ity was preserved through this processing step. This is consistent
`with the experiment performed by Trexler-Schmidt et al.5 When
`purified mAb D (IgG2κ) was spiked into mAb A soluble cellular
`components, little reduction (0.9% reduction in % main peak)
`was observed, showing that mAb D reduction is more resistant
`to these conditions. Thus, the product will influence the degree
`of reduction observed in the harvest process. To test the effects of
`cell line and cell culture process independent of product, purified
`mAb A was spiked into mAb B and mAb C soluble cellular com-
`ponents. Although mAb A is susceptible to reduction in its own
`soluble cellular components, little reduction was observed when it
`was incubated in either mAb C or mAb B components (1.5% and
`2.7% reduction in % main peak, respectively). All of the materi-
`als were carefully sparged with nitrogen during processing, and
`the soluble cellular components were prepared and used within
`30 min of cell lysis. In addition, repeat analysis of the (mAb B)
`IgG2λ spike into the mAb A (IgG1κ) yielded identical results, as
`did intermediate time points for the other conditions. The lack of
`reduction for these conditions must be due to differences in the
`reducing power of cellular component samples because the prod-
`uct is identical. Disufide reducing ability of the cellular compo-
`nent sample could arise from differences in the cell line, such as
`differences in expression of thioredoxin or thioredoxin reductase,
`or differences in availability of NADPH due to regulation of the
`pentose phosphate pathway (PPP). Differences in reducing power
`of the soluble cellular component sample could also arise from
`
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`Table1. End of production cell densities, viability and titer for mAbs A, B and C
`Product
`Viable Cell Density (cells/mL)
`Viability (%)
`7.0x106
`IgG2κ (mAb C)
`75.4
`17.2x106
`IgG2λ (mAb B)
`62.4
`27.0x106
`IgG1κ (mAb A)
`78.9
`
`Titer (g/mL)
`1.3
`4.7
`4.4
`
`Thioredoxin Reductase Activity ( mol/min/mL)
`Below detection limit
`0.16
`0.04
`
`©2013 Landes Bioscience. Do not distribute.
`
`authorities. This phenomenon is caused by shearing of cells,
`resulting in the release of intracellular components, and requires
`an anaerobic environment. Different cell lines and processes
`have been demonstrated to have strikingly different reduction
`responses, e.g., mAb B soluble cellular components having less
`than 1/10th the reducing power of those of mAb A. The dif-
`ference in reducing power cannot be attributed to differences in
`thioredoxin and thioredoxin reductase levels, as measurement of
`thioredoxin reductase shows that it is higher in some of the cell
`lysates that show no reduction. Therefore, these differences must
`stem from other redox active components in the media, or more
`likely, from differences in NADPH availability and regulation
`of the PPP.
`The susceptibility of products to reduction by thioredoxin has
`been demonstrated to be dependent on antibody class and light
`chain type, IgG1λ > IgG1κ > IgG2λ > IgG2κ, with potentially
`some sequence dependency within each range. The susceptibility
`of the antibody classes to thioredoxin catalyzed reduction fol-
`lows the same trend as antibody disulfide reduction by DTT.
`Therefore, a general understanding of product reducibility is
`available prior to expression of the product, and a more refined
`understanding of its susceptibility to reduction is possible with
`only micrograms of material in a chemically-defined system.
`With the understanding of the reducing power of the cell line
`and process, screening of cell lines and cell culture conditions is
`possible. Combining process knowledge with the antibody class,
`a good understanding of the overall reduction behavior can be
`obtained early in process development.
`
`Materials and Methods
`
`Materials. Cell culture fluid and purified antibodies were pro-
`duced at Amgen using standard manufacturing procedures.
`Reagents were obtained from Sigma-Aldrich unless otherwise
`specified.
`Cell shearing. Complete cell lysis of end of production (EOP)
`cell culture fluid, which contains both cells and the media con-
`taining product, was achieved by high-pressure homogeniza-
`tion using a Microfluidics M-110Y high shear fluid processor.
`Homogenization was performed with a single pass at 8,000–
`10,000 psi. Complete lysis was verified using the Roche Innovatis
`Cedex AS20 cell counter.
`Small-scale reduction model. A 3 L glass stirred-tank bio-
`reactor (Applikon Corporation) controlled by a customized
`DeltaV distributed control system (DCS) was used to evalu-
`ate harvest conditions. Processed cells were transferred to this
`bioreactor. Agitation was set at 250 rpm. Temperature was
`controlled to 8–10°C by passing chilled water through a ther-
`mal well in the bioreactor. Room temperature conditions were
`
`Table2. Influence of product and cell line/process on reduction
`Soluble Cellular
`Purified mAb
`Difference in NR CE-SDS
`Component
`% Main peak
`IgG1κ (mAb A)
`IgG1κ (mAb A)
`46.3%
`IgG2κ (mAb D)
`IgG1κ (mAb A)
`0.9%
`IgG1κ (mAb A)
`IgG2κ (mAb C)
`1.5%
`IgG1κ (mAb A)
`IgG2λ (mAb B)
`2.7%
`Difference in NR CE-SDS % Main peak, 8 h, relative to initial.
`
`Figure 4. In(cid:29)uence of Product on Reduction using Thioredoxin System.
`Intact antibody, as measured by % Main peak in the NR CE-SDS analysis
`as a function of time.
`
`was similar between the thioredoxin system and with DTT (Fig.
`4 and 5). This comparison indicates that differences in reduction
`during harvest are the result of overall reducing potential of the
`system and the antibody type, and not any specific interactions
`between thioredoxin and certain antibody types.
`Equipped with the knowledge of the reducibility trend IgG1λ
`> IgG1κ > IgG2λ > IgG2κ, a general understanding of the prod-
`uct contribution to the risk of process-induced reduction can be
`made prior to expression of products. This general understand-
`ing can be further refined by putting a small amount of purified
`product into either the chemically-defined thioredoxin reducing
`system, or by making kinetic measurements of the reducibility of
`the product by DTT.
`
`Discussion
`
`Process-induced partial antibody disulfide reduction is an
`active topic of discussion in the literature and with regulatory
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`Figure 5. In(cid:29)uence of Product on Reduction using DTT. Intact antibody,
`as measured by % Main peak in the NR CE-SDS analysis as a function of
`time.
`
`by incubating mAbs at the concentration of 2 mg/mL in 50
`mM TRIS-HCl, pH 7.5 (Teknova) with 2 mM DTT (Geno
`Technology) at ambient temperature. Aliquots were taken at
`multiple time points and the reduction was quenched by imme-
`diately adding NEM (MP biomedical) to a final concentration of
`25 mM. A non-reducing Caliper CE-SDS assay was performed to
`measure the level of reduction.
`Thioredoxin reductase activity. Thioredoxin reductase activ-
`ity of lysates was assessed using a colorimetric 5,5'-dithiobis-
`(2-nitrobenzoic acid) (DTNB) kit (Cayman Chemical). Briefly,
`lysed CCF was added to a pH 7 sample buffer containing 50
`mM potassium phosphate, 50 mM potassium chloride, 1 mM
`EDTA, and 0.2 mg/mL bovine serum albumin, at a final dilution
`factor of 1:10. For each sample a matrix control was made by add-
`ing 20 M sodium aurothiomalate (final concentration). Excess
`NADPH and 0.5 mM DTNB (final concentration) were added
`to each sample, matrix control, blank, and positive control (rat
`liver thioredoxin reductase). Light absorbance was monitored at
`405 nm for 5 min, and the activity of thioredoxin reductase in
` mol/min/mL was calculated by taking the difference in slopes
`between the sample and the matrix control, dividing by the
`extinction coefficient and path length, and multiplying by the
`dilution factor.
`Non-reduced CE-SDS. Harvested cell culture fluid samples
`were prepared using an automated robotic platform, as previously
`described.16 Briefly, samples were centrifuged at 13,000 rpm for
`1 min and loaded onto a Micro-Extractor Automated Instrument
`(MEA, PhyNexus). PhyTip® 200 L Columns with 20 L pro-
`tein A affinity resin protein A tips were used to remove host cell
`proteins. Non-reducing sample buffer with a final concentration
`of 7 mM NEM, 57 mM sodium phosphate, 1.9% SDS, pH 6.5
`was added to the purified samples. Incubation was set for 5 min
`at 60°C and samples were injected onto a 30 cm bare fused silica
`capillary with a 20 cm effective length and 50 m inner diameter
`using electrokinetic injection. Separation was performed using
`CE-SDS gel (Beckman Coulter) and 15 kV effective voltage, and
`detection was by UV light absorbance at 220 nm.
`
`unregulated, at ~22°C. Dissolved oxygen (DO) was measured
`using a Mettler Toledo DO probe connected to a Rosemont
`Transmitter. DO was lowered by sparging nitrogen gas through
`a drilled tube sparger with a flow sufficient to achieve a zero
`response for dissolved oxygen. To achieve oxygen at the 100%
`level, air was passed through the drilled tube sparger at 100
`to 200 mL/min. DO, temperature, airflow and agitation data
`were collected by the DeltaV DCS and archived into a PI data
`historian (OSIsoft).
`Reconstituted extract model. Cell culture fluid (CCF)
`depleted in antibody product was generated using a batch bind-
`ing process to remove existing monoclonal antibodies (mAbs).
`CCF was transferred to a 250 mL polycarbonate bottle (Nalgene)
`and homogenized using a Tissue Tearor™ (Biospec Products) for
`one minute of homogenization to ensure complete cell breakage.
`During homogenization, a nitrogen (N2) gas overlay was applied.
`MabSelect SuRe™ Protein A affinity resin (GE Healthcare) was
`washed twice with an equilibration buffer of 100 mM NaCl, 25
`mM Tris, pH 7.4, dried by vacuum over a nylon membrane, and
`applied in excess directly to the bottle. The mixture was placed
`on a rocker for 10 min to facilitate binding. The solution was
`centrifuged for 5 min x 1000 rpm in 50 mL conical tubes to pel-
`let the resin. The supernatant was extracted from each tube and
`sparged with N2 to form the soluble cellular components, and
`used within 30 min of production.
`Sample antibody drug substance was added to a separate 15
`mL polypropylene centrifuge tube and brought to a total vol-
`ume of 7 mL with the soluble cellular component material to
`give a final antibody concentration of 3 mg/mL. An N2 overlay
`was applied to each tube. The tubes were covered with laboratory
`paraffin film and placed in a digitally controlled water bath set at
`10°C. One mL aliquots were pulled at 0, 4 and 8 h and immedi-
`ately frozen at −80°C prior to analysis by NR CE-SDS.
`Reduction by thioredoxin system. The roles of thioredoxin
`and thioredoxin reductase (TR) have previously been described
`as nicotinamide adenine dinucleotide phosphate (NADPH)-
`dependent cellular protein disulfide reductases.14,15 An in vitro
`lab-scale model using this complex was optimized using recom-
`binant human thioredoxin (Sigma), NAPDH (Calbiochem),
`in excess and thioredoxin reductase from rat liver (Sigma). A
`polypropylene 2 mL cryogenic vial (Corning) was sparged for
`1 min with N2 prior to being sealed in a borosilicate septa vial
`(I-Chem). In a separate 1.5 mL microcentrifuge tube, 825 L
`phosphate buffered saline, 14 L NADPH (10 mM), 10 L
`human thioredoxin solution (0.5 mg/mL) and antibody drug
`substance were combined to give a final volume of 1 mL and
`antibody concentration of 4 mg/mL. The reaction was initiated
`with the addition of 18 L TR solution (7 M) and transferred
`immediately into the sealed vial using a syringe. The septa vials
`were placed within a temperature-controlled water bath at 10°C
`with an applied overlay of N2 to exclude oxygen. Aliquots of 100
` L were taken at each time point, quenched immediately with
`8.7 L N-ethylmaleimide (NEM) at 250 mM and frozen at
`−80°C prior to analysis by NR CE-SDS.
`Reduction by DTT. Partially reduced mAbs, with major-
`ity of the interchain disulfide bonds broken, were generated
`
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`Non-reduced Caliper CE-SDS. A LabChip 90 (Caliper Life
`Sciences) was used to separate SDS bound proteins through a
`sieving polymer based on the hydrodynamic size of the SDS-
`protein complex.17 HT Protein Express Sample Buffer (Caliper
`Life Sciences) was combined with iodoacetamide (IAM) to a
`final IAM concentration of approximately 5 mM. A total of 5 L
`antibody sample at approximately 1 mg/mL was mixed with 100
` L of the IAM containing sample buffer. The samples were incu-
`bated at 75°C for 10 min. The denatured proteins were analyzed
`by LabChip 90 with the “HT Protein Express 200” program.
`
`Disclosure of Potential Conflicts of Interest
`No potential conflicts of interest were disclosed.
`
`Acknowledgments
`Heartfelt thanks are due to Tamer Eris, Henry Lin, and the
`manufacturing facility, especially Behrouz Kiamanesh, for the
`EOP materials, to Michael Shearer, for help with removing the
`antibody by Protein A for the reconstitution experiments, and to
`Dayou Liu, for helpful discussions.
`
`References
`1. Maggon K. Monoclonal antibody “gold rush”. Curr
`Med Chem 2007; 14:1978-87; PMID:17691940;
`http://dx.doi.org/10.2174/092986707781368504
`2. Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC.
`Monoclonal antibody successes in the clinic. Nat
`Biotechnol 2005; 23:1073-8; PMID:16151394; http://
`dx.doi.org/10.1038/nbt0905-1073
`3. Kao YH, Hewitt DP, Trexler-Schmidt M, Laird MW.
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