Ray_Parker_BMS_Publication

Development of Robust Antibody Purification byOptimizing Protein-A Chromatography inCombination With Precipitation Methodologies

Srinivas Chollangi, Ray Parker, Nripen Singh, Yi Li, Michael Borys, Zhengjian Li

Bristol-Myers Squibb, Biologics Development, Global Manufacturing & Supply,

Hopkinton, Massachusetts; telephone: þ9784 784 6513; fax: 978-784-6639; e-mail: Yi.

[email protected]

ABSTRACT: To be administered to patients, therapeuticmonoclonal antibodies must have very high purity, with processrelated impurities like host-cell proteins (HCPs) and DNA reducedto<100 ppm and<10 ppb, respectively, relative to desired product.Traditionally, Protein-A chromatography as a capture step has beenthe work horse for clearing a large proportion of these impurities.However, remaining levels of process and product related impuritiesstill present significant challenges on the development of polishingsteps further downstream. In this study, we have incorporated highthroughput screening to evaluate three areas of separation: (i)Harvest treatment; (ii) Protein-AChromatography; and (iii) Low pHViral Inactivation. Precipitation with low pH treatment of cellculture harvest resulted in selective removal of impurities whilemanipulating the pH of wash buffers used in Protein-Achromatography and incorporating wash additives that disruptvarious modes of protein–protein interaction resulted in furtherand more pronounced reduction in impurity levels. In addition, ourstudy also demonstrate that optimizing the neutralization pH postProtein-A elution can result in selective removal of impurities.When applied over multiple mAbs, this optimization methodproved to be very robust and the strategy provides a new andimproved purification process that reduces process relatedimpurities like HCPs and DNA to drug substance specificationswith just one chromatography column and open avenues forsignificant decrease in operating costs in monoclonal antibodypurification.Biotechnol. Bioeng. 2015;112: 2292–2304.� 2015 Wiley Periodicals, Inc.KEYWORDS: high-throughput screening; monoclonal antibody;precipitation; protein-a chromatography

Introduction

Monoclonal antibodies and their derivatives are currently a majorsource of revenue generation in global biotechnology market(Lawrence, 2007a,b). However, process economics can be greatlyinfluenced by the choice of expression and purification steps.Recent advances in cell line selection, growth media, and feedingstrategies have led to significant improvement in cell densities andexpression levels are consistently reaching 5–10g/L across theindustry in a 14 day fed-batch process (Huang et al., 2010).Consequently, process bottlenecks have shifted downstream andpurification costs are now outweighing the upstream cell culturecosts (Follman and Fahrner, 2004; Gagnon, 2012; Guiochon andBeaver, 2011). Implementation of platform approach has greatlyreduced downstream operation costs but challenges associated withmeeting final drug substance specifications still remain (Guiochonand Beaver, 2011; Shukla et al., 2007).

For monoclonal antibodies purification platform, Protein-Achromatography is still the most robust step in removing processand product related impurities (Vunnum et al., 2009a). Owing to itsspecific affinity towards Fc region containing antibodies and fusionproteins and its ability to tolerate high conductivities, Protein-Achromatography allows direct loading of harvested cell culture fluid(HCCF) and enables removal of a vast majority of process andproduct related impurities while enriching the antibody pool(Vunnum et al., 2009a). Nevertheless, remaining impurities afterProtein-A still present a significant challenge to the purificationsteps downstream in order to achieve the drug substancespecifications suitable for patient administration (Guiochon andBeaver 2011; Liu et al., 2010). In addition, it has been consistentlyshown that Protein-A chromatography alone constitutes more thana quarter of the total raw materials costs in downstream purificationof the antibodies (Follman and Fahrner, 2004). Thus, optimal andefficient usage of Protein-A affinity resin is critical to achieve highproduct quality and reduce the costs of antibody production.

During Protein-A chromatography, post load wash step is a keymeans to achieve impurity clearance. Manipulating the pH of washbuffer is usually the first choice and conventionally, a wash step witha pH between the load and elution conditions is employed forProtein-A chromatography (Group 2009; Vunnum et al., 2009a).

Correspondence to: Y. Li

Received 18 February 2015; Revision received 23 April 2015; Accepted 28 April 2015

Accepted manuscript online 6 May 2015;

Article first published online 31 July 2015 in Wiley Online Library

(http://onlinelibrary.wiley.com/doi/10.1002/bit.25639/abstract).

DOI 10.1002/bit.25639

ARTICLE

2292 Biotechnology and Bioengineering, Vol. 112, No. 11, November, 2015 � 2015 Wiley Periodicals, Inc.

The pH of this intermediate wash is lowered as low as possiblewithout initiating premature elution of the product and thisrequires molecule specific effort. With this approach, HCP levelspost Protein-A are usually in the range of 3,000 to 12,000 ppm asreported in a case-study conducted by a consortium of biotechindustry specialists (Group, 2009). Typically, this requires anadditional of at least two chromatography steps to further purifyand polish the product to achieve drug substance specifications(Group, 2009). In 2007, Shukla and Hinckley (2008) reported one ofthe first successful attempts to enhance the performance ofProtein-A chromatography by evaluating a number of washadditives like sodium chloride, urea, propylene glycol, ethanol,isopropanol, tween-80, spermine, and sodium sulfate duringProtein-A chromatography. Based on the results, they haveidentified urea as the best wash additive to achieve effectiveremoval of HCPs. The levels of HCPs reported upon incorporatingurea wash were in the range of 1,000–3,500 ppm where recovery ofthe product varied between 70 and 100%. However, it is also to benoted that at high concentrations, agents like urea, ethanol, andisopropanol can destabilize the structure of proteins throughformation of hydrogen bonds with peptide groups and exposinghydrophobic residues (Herskovits and Jaillet 1969; Herskovits et al.,1970a, b; Lim et al., 2009). On the other hand, presence of smallamounts of sodium chloride, sodium sulfate, propylene glycol, andtween-80 are shown to help stabilize the protein structure byinteracting with the polar residues on the surface of the molecule(Agarkhed et al., 2013; Damodaran and Kinsella 1981; Kerwin et al.,1998; Thurow and Geisen 1984). In addition, like other agentsmentioned above, L-Arginine is known to help stabilize monoclonalantibodies by breaking non-specific interactions between proteinsand preventing aggregation (Lange and Rudolph 2009; Schneideret al., 2011; Shukla and Trout 2010). Studies by Yumioka et al.(2010) and Sun (2013) have shown that L-Arginine can also be usedas a wash reagent to reduce impurity levels during Protein-Achromatography (Sun, 2013; Yumioka et al., 2010). HCP levels werereported to be reduced by about 90–95% in Protein-A product poolusing this strategy. However, in both of these cases, the HCP levelsare still high compared to final drug substance specifications andrequire two more steps for further polishing. Also, with the lack ofhigh-throughput automation technologies at the time, all of theseexperiments were carried out in a single case by case manner andlimited the scope of identifying the most optimal concentration andcombinations of the additives that can yield better results.Following the above works, significant efforts were focused on

identifying the HCPs that are binding to Protein-A column andco-eluting with the product during elution (Hogwood et al., 2013b;Jin et al., 2010; Levy et al., 2014; Sisodiya et al., 2012; Tait et al.,2012; Tarrant et al., 2012). These studies demonstrated that thetype of HCPs co-eluting with product varied from molecule tomolecule and from one expression system to the other, making thedevelopment of effective platform purification process for Protein-Achromatography highly challenging (Sisodiya et al., 2012). Morerecently, Aboulaich et al, (2014) have covalently cross-linkedmonoclonal antibodies to NHS-activated sepharose resin to studytheir interaction with various host-cell proteins. Again, thesestudies confirmed that the type of HCPs associating with theproduct during Protein-A chromatography varies from molecule to

molecule. In addition, they showed that wash additives like argininecan help reduce the HCP levels in product pool. However, the studyis limited by the fact that the full extent of interactions betweenprocess related impurities and the antibody is not captured because,unlike in cell culture broth, the complete surface of antibody isinaccessible to the HCPs after its prior immobilization to thesepharose resin. This limits the number of sites on the monoclonalantibody available for HCP interaction and thereby not all HCPpopulations that normally interact with the antibody are accountedfor. Also, the HCP interaction with Protein-A ligand and co-elutionwith product during low pH treatment is not captured in suchsetting. In addition, in all the above studies, the focus has beenprimarily on understanding HCP interaction and removal duringProtein-A chromatography and did not evaluate the effects of HCPclearance during Protein-A chromatography in context with othersteps (e.g., harvest clarification and low pH viral inactivation)involved in typical purification platform.Recent advancements in high-throughput automation technol-

ogies have enabled rapid acquisition of large datasets under severaldifferent operating conditions (Coffman et al., 2008; Kelley et al.,2008; Kramarczyk et al., 2008). This enables a practical strategy toestablish a framework where customized wash steps for a particularmAb is optimized together with harvest clarification and viralinactivation. Exploiting this, in this study we incorporatedhigh-throughput screening to investigate the effects of pH, buffercomposition, and a range of wash additives (caprylic acid,propylene glycol, triton x-100, arginine, urea, isopropanol, EDTA,and sodium chloride) to identify the most optimal conditions foreffective HCP removal and further improve the performance ofProtein-A chromatography. The wash additives employed and thehigh-throughput design of the study was aimed at not onlyidentifying an effective wash condition, but also understand thenature of interactions between the HCPs and the antibody duringProtein-A chromatography. This method of screening can serve as atemplate for all therapeutic antibodies coming in the pipeline torapidly identify an effective wash strategy during Protein-Achromatography. In addition, combining this strategy withadditional screening on separation techniques like precipitationduring cell culture harvest and optimizing the neutralization pHpost Protein-A chromatography yielded highly improved impurityclearance essentially meeting drug substance specifications for HCPand DNA clearance with just one column process. When testedacross multiple molecules, this method proved very robust and canbe effectively used to significantly improve the product quality whileminimizing the load and cost on subsequent operations.

Materials and Methods

Cell Culture and Harvest Treatment

Proprietary CHO cell lines engineered from either DG44 parentalcells or GS parental cells were used to produce the null harvest orharvests containing recombinant monoclonal antibodies (mAb1,mAb2, mAb3, and mAb4). All cells were cultured using a chemicallydefined, animal component-free media. The bioreactors wereoperated in a fed-batch mode with continuous feeds based onmaintaining glucose concentration between 2 and 4 g/L. Titers were

Chollangi et al.: Impurity Clearance During Antibody Purification 2293

Biotechnology and Bioengineering

measured using a Protein-A HPLC system (Agilent 1100, WatersCorporation, Milford, MA) with an established reference standard.During the harvest, cell culture suspensions were collected and thenwere either left untreated or treated using acid titrants (acetic acidor citric acid) to lower the pH to desired range. Treatment wascarried out for 30min under gentle mixing. Following treatment,the harvest material was centrifuged and filtered using a depth filter(60SP05A, 3M, St. Paul, MN) followed by 0.8/0.2mm filter capsule(Pall Corporation, Port Washington, NY). Post filtration, pH of theclarified harvest was adjusted to a range between 7.0 and 7.2 using2M Tris and subjected to analytical analyses to assess impurityreduction.

Protein-A Chromatography

GE healthcare’s MabSelect Protein-A resin was used for all capturechromatography experiments. Preparative scale column-basedchromatography experiments were carried out using an €AKTAAvant instrument (GE Healthcare, Uppsala, Sweden) controlled byUnicorn 6.3 software. All columns were packed in the lab accordingto the resin manufacturer’s recommendations. Unless noted,equilibration of the resin was carried out using phosphate-bufferedsaline (PBS) at pH 7.4 followed by loading with harvested cellculture fluid (HCCF). The resin was then subjected to PBS washfollowed by an acidic pH wash (pH 5.0–6.0) before eluting the mAbusing buffers at pH< 4.0.

Viral Inactivation and Filtration

Following elution, mAb pool from Protein-A chromatography wassubjected to viral inactivation by holding at low pH (3.4–4.0) for 1 hat room temperature followed by neutralization to desired pH in arange between 4.0 and 9.0. Titrationwas carried out using 0.1 HCl or2M Tris. Post neutralization, filtration was carried out using0.8/0.2mm filter capsule or a 0.8/0.2mm syringe filter (PallCorporation).

High Throughput Liquid Handling and Chromatography

Batch-mode high-throughput chromatography experiments werecarried out using PreDictor plates packed with 50mL MabSelectresin (GE healthcare). Liquid handling was conducted in a highthroughput manner using Tecan Freedom-Evo system equippedwith a high-precision Liquid Handler arm (LiHa) and RoboticManipulator (RoMa) (Tecan Group, Ltd. Mannedorf, Switzerland).Method creation and execution was carried out using Tecanfreedom-evoware software (Tecan Group Ltd.). Wash bufferscontaining various additives were prepared using respective stocksolutions and diluting them as needed on the tecan. These buffersare dispensed and stored in plate format compatible for tecan liquidhandling. For a typical batch mode Protein-A chromatographyexperiment, the MabSelect resin is equilibrated with PBS followedby loading with HCCF. The resin is then subjected to either PBSwash or wash buffers containing various additives. The targetprotein is then eluted using a low pH buffer (pH< 4.0) andcollected into a 96-well plate. The product pool is then neutralizedusing 2M Tris followed by filtration using 0.2mm filter plate (Pall

Corporation). The filtrate is then subjected various analytical assaysto assess product quality and impurity clearance.

Analytical Characterization

Post capture chromatography and filtration, protein concentrationwas assayed by high-throughput UV-Vis spectroscopy using theDropSense96 polychromaticmicroplate reader (Trinean, Gentbrugge,Belgium). UV-Vis spectra were quantified using DropQuant software(Trinean). Size exclusion chromatography (SEC) was conductedusing Waters’ Acquity H-Class Bio UPLC

1

to measure the monomerand aggregate content in the product pool. Acquity UPLC

1

BEH200SEC 1.7mm column (4.6� 150mm) was utilized to perform thisassay where the mobile phase consisted of phosphate buffered saline(10mM Phoshphate, 137mM Sodium Chloride, 2.7mM PotassiumChloride) at pH 6.8 and was run at a flow rate of 1mL/min.Quantification of monomer, low molecular, and the high molecularweight specieswas performedusing Empower software (Waters Corp.Orlando, FL). In addition, secondary structure, charge profile, andthe binding activity were confirmedusing circular dichroism (Jasco J-715spectropolarimeter), iso-electric focusing (ProteinSimple iCE3),and custom ELISA with a reference standard. For hydrophobicityestimation, primary sequences of the four antibodies in study arealigned using ClustalW2 (Larkin et al., 2007) and identified that thedifferences between the sequences largely lied in complementaritydetermining regions (CDRs). 3D structures of the Fab regions of thesefour antibodies were built using homology modeling tool SWISS-Model (Arnold et al., 2006). Amino acid sequences of CDR loopscontaining all sequence differenceswere identified in these structuresand the hydrophobicity scores of these CDR loops were calculatedusing Kyte-Doolittle hydrophobicity score (Kyte and Doolittle, 1982).ELISA for quantification of residual CHO host cell proteins (CHO-HCP) and residual Protein-A was conducted in a high throughputmanner using the Tecan liquid handling system. Host cell proteinswere quantified using the CHO Host Cell proteins 3rd generation kit(Cat # F550, Cygnus Technologies, Southport, NC) while residualProtein-Awas quantified using Repligen Protein-A ELISA kit (Cat #9000-1, Repligen Corporation, Waltham, MA) according tomanufacturer’s protocol. Absorbance was measured at 450/650 nmusing EnVisionMultilabel reader (PerkinElmer. Waltham,MA). Dataquantification and analysis was carried out using JMP software (SASInstitute Inc. Cary, NC). Residual CHO DNA in the samples weremeasured using real-time quantitative PCR (RT-PCR). RT-PCR wascarried out with the 2� TaqMan Universal PCR Master Mix kit (LifeTechnologies, Carlsbad, CA) and 7900 HT Real Time PCR system(Life Technologies) according the manufacturer’s instructions.

Results and Discussion

HCPs and DNA Co-Elution With Antibody

Three different load materials: (i) mAb1 expressing CHO cell HCCF;(ii) null-CHO cell HCCF; and (iii) null-CHO cell HCCF spiked withpurified mAb1 were subjected to Protein-A purification process.Figure 1 shows the amount of mAb1, HCPs, and DNA being loadedand eluted from the Protein-A column. Consistent with the resultsreported by Shukla and Hinckley (2008) our results show that there

2294 Biotechnology and Bioengineering, Vol. 112, No. 11, November, 2015

is an increase in the HCP and DNA levels in the elution pools whenthe antibody is present. This phenomenon is attributed to thenon-covalent interactions between the impurities and the antibodywhile the interaction between impurities and Protein-A resin isminimal, particularly in the case of agarose based matrix (Levyet al., 2014; Nogal et al., 2012; Shukla and Hinckley 2008; Sisodiyaet al., 2012; Tarrant et al., 2012). Specifically, Tarrant et al. (2012)have compared the adsorption behavior of host-cell proteins ontoagarose based and glass based Protein-A resin matrices (Tarrantet al., 2012). Using ELISA and SELDI-TOF mass-spectrometricanalysis they have identified that the hydrophilic agarose basedmatrices exhibit low degree of interaction with the CHO host-cellproteins while the glass based ProSep Ultra Plus Affinity (UPA)resin exhibited high degree of non-specific interaction with thehost-cell proteins. This was attributed to the hydrophobic nature ofthe controlled pore glass (CPG) back-bone of the ProSep UPA resin.However, in the presence of antibody, both agarose and glass basedresins started exhibiting adsorptive behavior towards CHO host-cellproteins; indicating non-specific interactions between the antibodyand the host-cell proteins. Previous results have also shown that thedegree of these antibody-HCP interactions is highly variable anddepends on the antibody present in the HCCF (Sisodiya et al., 2012).

HCPs Associate With mAb Through a Complex Mode ofInteractions

While the association of HCPs with antibody during Protein-Achromatography is well established, the mode of this interaction is

not well understood yet. A number of groups have used proteomicbased approaches to investigate the profiles of HCPs present inHCCF and the eluates from Protein-A column leading toidentification of specific populations that exhibit strong interactionwith antibodies (Hogwood et al., 2013a, b; Jin et al., 2010; Levyet al., 2014; Nogal et al., 2012; Shukla and Hinckley 2008; Sisodiyaet al., 2012; Tait et al., 2012; Tarrant et al., 2012). While progress wasmade to disrupt these interactions between HCPs and antibody, theremaining levels of impurities are still high and require at least twoadditional steps to bring the impurity levels down to drug substancespecifications (Group, 2009; Shukla and Hinckley, 2008). To assessimproved ways that can disrupt these interactions, batch modehigh-throughput Protein-A chromatography experiments werecarried out on mAb1 (an IgG4 with a pI between 6.5 and 7.0)using wash buffers at various pH values and incorporating salts andother wash additives into them. Figure 2 shows the effect of washbuffer pH on recovery of antibody and removal of HCPs from theproduct. As shown in panel A, the experiment was carried out bothin the presence and absence of 1M NaCl. In both cases, as expected,results show that there is pronounced loss of antibody when the pHof wash buffer is lower than 5.0 compared to basic wash buffers withpH> 7.0. In addition, the presence of 1M NaCl (high conductivity)made the loss of antibody more pronounced even up to pH 7.0 andstarts to show diminishing effect at pH greater than 7.0. Within thisrange, effect of wash buffer pHon the removal of HCPs fromproductpool is however much more dramatic than the effect on recovery.Traditionally, an acidic buffer (pH in the range of 6.0 to 5.0) is oftenemployed to wash off impurities from the Protein-A column and

Figure 1. CHO host cell proteins (HCP) and host cell DNA associate with the monoclonal antibodies (mAbs) and co-elute as impurities during low pH elution. Top panel shows

the levels of mAb, HCPs, and DNA loaded onto the Protein-A column while bottom panel shows the levels of mAb, HCPs, and DNA present in the Protein-A eluate. The loading

material came from either CHO cell supernatant expressing mAb, null cell supernatant, or null cell supernatant spiked with purified mAb.



also serve as a transition phase buffer to reduce the pH and helpkeep the elution column volumes (CVs) low. However, the resultshere show that a basic buffer (pH� 8.0) is instead much moreeffective in reducing the HCPs from product pool. The presence ofNaCl helps in realizing this benefit even at pH 7.0 or greater.Combined with the recovery data shown in panel A, these resultssuggest that basic wash buffers with pH� 9.0 are effective inkeeping the recoveries high and obtain selective removal of HCPsfrom product pool.

Considering the results shown in Figure 1 and keeping the pI ofmAb1 (6.5–7.0) in view, these results strongly suggest thatelectrostatic interactions do play an important role in antibody-HCPinteractions. Shown in Table I is the list of iso-electric points ofvarious molecular entities found in HCCF and Protein-A elution

pools (Aboulaich et al., 2014; Gottschalk 2009; Levy et al., 2014).Levy et al. (2014) have shown that the diverse population of CHOhost cell proteins has a wide range of iso-electric points but amajority of this population has a pI in the range of 4.5 to 7.0. At pHvalues between 4.5 and 7.0 mAbs are typically neutral or cationicwhile DNA is anionic and a good number of HCPs are either neutralor anionic. This results in a mix of strong electrostatic interactionsand hydrophobic interactions between the mAbs and the impuritiesthus co-purifying during elution. However, when the resinis subjected to washes with buffers at high (pH� 8.0), mAbsbecome either neutral or more anionic and with DNA and a vast ofmajority of HCPs being strongly anionic they start dissociating fromother. With mAbs strongly bound to the Protein-A resin owing totheir specific affinity, HCPs and DNA are washed off the columnduring these high pH washes resulting in product pool enrichedwith pure mAb. However, it is also to be noticed that in the pH rangeof 7.0 to 10.0 there is still some population of HCPs that is chargedor neutral and can bind to the mAbs non-specifically by range offorces like hydrogen bonding, Van der Waals forces or hydrophobicinteractions. To assess whether these interactions can bedissociated using wash additives, a high throughput batch-modechromatography experiment was carried out using wash bufferscontaining a combination of salts and various wash additives.

Figure 3A and B show the contour maps of residual HCPs presentin Protein-A elution pools and the recovery after subjecting the

Figure 2. High pH washes aid in reducing the HCP levels in Protein-A eluate without a loss of recovery. (A) Recovery of mAb in Protein-A eluate is plotted against the pH of

wash buffer used during Protein-A chromatography. (B) HCP (ppm) levels in Protein-A eluate are plotted against the pH of wash buffer used during Protein-A chromatography. All

experiments were conducted either in the presence or absence of 1M NaCl in the wash buffers. Error bars represent standard deviation of the analytical results. (*& **, P< 0.005).

Table I. Iso electric points of various class of molecules commonly

found in CHO cell harvests and Protein-A elution pools (Aboulaich et al.,

2014; Levy et al., 2014; Gottschalk, 2009).

Molecule class pI range

HCPs 2–11DNA 2–3Viruses 4–7.5Protein-A 4.8–5.2Endotoxins 1–4


HCCF loaded resin to washes with various buffers containingadditives: sodium chloride, arginine, EDTA, iso-propyl alcohol(IPA), propylene glycol, sodium octanoate (sodium caprylate/caprylic acid), triton x-100, and urea. In Figure3A, the colorprogression represents the levels of HCPs remaining in the elutionpool while in Figure 3B, the color progression represents recovery of

the monoclonal antibody in elution pool. As expected,Figure 3B shows that the recoveries of the mAb1 are good athigh pH and lower when we use low pH wash buffer in combinationwith high concentrations of NaCl and wash additives particularly,urea. As observed in Figure 2, the control panel shown inFigure 3A demonstrates that high pH buffer is more effective in

Figure 3. High pHwashes and additives like Arginine and Urea help reduce HCP levels in Protein-A eluates. Shown in the figure are contour plots representing (A) HCP levels in

Protein-A eluates and (B) recovery of the antibody in eluate. CHO cell harvest was loaded equally onto a 96-well plate containing Protein-A resin followed by washes with buffers at

3 pH values (pH 5.5, 7.0, and 9.0) containing a range of wash additives in the presence or absence of NaCl. For eachwash additive, the contour plot panel was generated using 12 data

points; four NaCl concentrations in combination with three excipient concentrations. (A) The deep blue color represents low levels of HCPs in the eluate while the deep red color

represents very high amounts of HCPs levels still remaining in the Protein-A eluate. (B) The deep blue color represents high recoveries while the deep red color represents low

recoveries.



removing HCPs from the product pool. In addition, the results alsoshow that supplementing wash buffers with various wash additivescan have a positive effect in reducing the HCPs levels from productpool. Particularly, urea and arginine demonstrate excellentefficiency in disrupting the interactions between mAb1 and theHCPs. In both cases, increasing the amount of the wash additive andincreasing the pH enhanced the ability of the buffers to disrupt theantibody-HCP interactions while maintaining good recoveries(Fig. 3A and B, bottom panels). It has been shown previously thatarginine can stabilize proteins by breaking non-specific protein-protein interactions (Arakawa et al., 2006, 2007; Arakawa and Kita2014; Arakawa and Tsumoto 2003; Borders et al., 1994; Schneideret al., 2011; Shukla and Trout 2010; Tsumoto et al., 2005). Theguanidinium group on arginine was shown to be primarilyresponsible for this ability as it can affect not only the electrostaticand hydrophobic interactions but also hydrogen bonds betweenproteins. On the other hand, urea is shown to interact with proteinsboth directly, by forming hydrogen bonds with the protein andindirectly by altering the solvent environment thereby mitigatingthe hydrophobic effects that lead to protein–protein interactions.However, it is also to be noted that at high concentrations, urea canstart to destabilize the protein structures by forming hydrogenbonds with peptide groups present in the hydrophobic core of themolecule and unraveling the tertiary structure of the protein(Herskovits et al., 1970b; Lim et al., 2009). Thus a careful balanceneeds to be maintained in case of urea to retain the protein structurebut disrupt non-specific protein–protein interactions. Combinedwith the effects of pH described above, both of these wash additivesthus appear to be very effective in disrupting non-specificmAb1-HCP interactions.

A wide variety of metals are often added to cell culture mediato boost cell viability and promote high titers. During the courseof cell culture, these metals can bind to antibodies and Gagnonet.al., have shown that in addition to altering antibody chargeand hydrophobicity, metal ions can cause local conformationalchanges and lead to formation of secondary complexes withcontaminants like HCPs, DNA, and endotoxins (Gagnon, 2010).However, in the case of mAb1, our results show that addition ofEDTA to the wash buffer only has a modest effect in terms ofHCP removal. At pH 7.0, EDTA has slightly improved effect onHCP removal compared to the control while at pH 9.0, it appearsto behave similar or inferior to the control. Albeit better at pH7.0, organic solvent IPA exhibited similar performance to EDTAat pH 9.0. In contrast, propylene glycol and non-ionic surfactantTriton X-100 appear to be much more effective than the controlbuffer in breaking down thenon-specific interactions betweenmAb1 and HCPs at all pH values. Both of these agents arehydrophilic and known to stabilize proteins by altering solventenvironment. Finally, sodium octanoate (also known as sodiumcaprylate/caprylic acid) is an eight-carbon saturated fatty-acidchain that is shown in the literature to selectively precipitateHCPs and other impurities in HCCF (Brodsky et al., 2012).When added to the wash buffers, this agent proved very effectivecompared to the control wash, particularly at pH 9.0, andselectively disrupted the interactions between HCPs and theantibody. Combined together, the effect of high pH, salt, and theeffects of urea, arginine, propylene glycol, and triton x-100 on

strongly disrupting the interactions between HCPs and mAb1suggest that the HCPs associate with antibodies through acomplex mode of interactions which may include electrostaticinteractions, hydrophobic interactions and hydrogen bonds.

HCPs can be Selectively Precipitated by Optimizing theNeutralization pH Post Protein-A

The ICH Q5A guidance document requires the use of at least twoorthogonal steps for viral clearance to ensure the safety ofproducts produced using mammalian cell culture processes (FDA,1998). In addition to nanofiltration, incubating product pools inlow pH environment (pH 3.0–4.0) is often used as a robustmethod to inactivate enveloped viruses (Brorson et al., 2003;Gagnon 2012; Omar et al., 1996). Given the requirement of low pHbuffers to elute antibodies from Protein-A column, this capturestep is often utilized as both a separation as well as transition forviral inactivation step. However, Protein-A elution pools are oftenreported to be associated with turbidity and the extent of thisturbidity was shown to be highly variable from antibody toantibody (Tobler et al., 2006; Yigzaw et al., 2006). Depending uponthe operating pH of the second chromatography step downstreamof Protein-A, the low pH viral inactivated pool needs to beneutralized to a pH suitable for loading onto the next column.During this process, a significant increase in turbidity is oftenreported (Tobler et al., 2006; Yigzaw et al., 2006). Thisprecipitation and turbidity was viewed as a risk for downstreamprocess as it poses problems of clogging inline sterile filters. Therisk is usually mitigated by using depth filters to remove theprecipitants and higher order aggregates (Yigzaw et al., 2006). Inour studies with mAb1, we observed similar precipitationbehavior when we used the control washes without any additives.To further characterize the phenomenon, we have titrated the viralinactivated pool to various pH values between 4.0 and 8.5 inincrements of 0.5 and then filtered the pools using a 0.2mm filter.Figure 4A shows the visual representation of turbidity in thesamples pools after titration while panel 4B shows thatthe turbidity can be removed by filtration. Panel 4C shows thequantitative measurement of absorbance reading at 410 nm beforeand after filtration. As it can be noticed, the turbidity peakedbetween the pH range of 5.0 and 7.5 and as the pool was titratedto pH values further high, the turbidity went down. When thefiltered pools were subjected to analysis for antibody and HCPcontents, results showed that the precipitation strongly correlatedwith selective removal of HCPs (Fig. 5). In contrast to what onemight expect for an affinity chromatography step, the HCP levelsin the elution pool from Protein-A chromatography have beenreported to be as high as 2,000–50,000 ppm (Group, 2009;Sisodiya et al., 2012; Yigzaw et al., 2006). As described above insection 3.2, a vast majority of CHO host cell proteins have a pI inthis pH range of 4.5–7.5 and can become less soluble during theneutralization process leading to aggregation and precipitation.However, as the pH moves further away from this range i.e.,pH> 7.5 or pH< 4.0, the HCPs become polar and stay solubilizedin the solution leading to a decrease in turbidity. Thus, insolubleaggregate formation during neutralization post viral inactivation


is not necessarily an undesirable phenomenon and one can designa process where the neutralization pH is optimal to get selectiveprecipitation of HCPs while minimizing any product loss. Thisoptimal pH would vary from one expression system to the otherand from one antibody to another depending up on its surfaceproperties and requires empirical characterization.

Enhancing Impurity Clearance by Optimizing PurificationTrain

The most common purification schemes for monoclonal antibodiesutilize Protein A affinity chromatography as a capture step followed by2 to 3 chromatographic steps for polishing (Kelley et al., 2009; Vunnumet al., 2009b). Even though these chromatography steps are able tomeet the stringent purification requirements, they are expensive andoften the downstream purification train contributes to about 50–80%

of the total costs involved in antibody purification (Guiochon andBeaver, 2011). With rapidly rising demand for therapeutic antibodies,significant attention is thus being focused on reducing manufacturingcosts and improving process efficiency for industrial scale production.Based on the findings described so far, we have tested variouspurification schemes to evaluate the robustness of early separationsteps and the most effective and economical purification train.As described in sections 3.2 and 3.3, majority of CHO host cell

proteins are unstable in acidic pH range between 4.5 and 7.0 and canbe precipitated out by optimal adjustments to the pH. This can beperformed post Protein-A as well as before Protein-A during cellculture harvest. Lydersen et al. (1994) have previously shown thatacidification of fermentation broths can successfully induceprecipitation of host cell proteins and cellular debris (Lydersenet al., 1994). In case ofmAb1, we have observed similar behavior (SeeFig. 6). Acidification of the cell culture broth below pH 5.0 usingeither citric acid or acetic acid, lead to precipitation and removal ofhost cell proteins while maintaining antibody recovery above 95%.mAb1 was then subjected to preparative scale purification using

various trains (see Table II) where acid precipitationwas used eitherwith control Protein-A process or with optimized Protein-A washesidentified in section 3.2 and optimized neutralization pH identifiedin section 3.3. Table III shows the results achieved using thesepurification trains. As noted in train 1, purification of the antibodywithout harvest treatment and control washes inProtein-A resultedin high levels of host cell proteins while the DNAwas reduced to 10–26 ppb. In contrast, using either of the enrichment techniques i.e.,harvest treatment or enhancedProtein-A washes, lead to significantdecrease in both HCP and DNA levels. However, among the two,enhanced Protein-A washes lead to a much greater decrease in theimpurity levels compared to acid precipitation (Train 2 vs. Train 3).When combined with optimization of neutralization pH post viral

Figure 4. Neutralization to optimal pH after low pH hold for viral inactivation leads to increase in turbidity of the pool. (A) Pool turbidity of samples as a function of pH. VI hold is

at pH 3.5. (B) Pool turbidity of samples shown in panel A after filtration with 0.2 mm filter. (C) Absorbance at 410 of the samples shown in panels A and B.

Figure 5. Neutralization to optimal pH after low pH hold for viral inactivation leads

to selective precipitation of HCPs. Shown above are the recovery of mAb and HCP

levels in viral inactivated and neutralized bulk samples post filtration. Error bars

represent standard deviation of the analytical results.



inactivation, enhanced Protein-A washes lead to reduction of HCPsand DNA to 10–33 ppm and <1 ppb, respectively, essentiallymeeting the drug substance specifications for HCP and DNAclearance with just one column step. In train 5, addition of the acidprecipitation step prior to Protein-A chromatography lead to a slightimprovement in the product quality, but the gains were at theexpense of antibody recovery. Considering these results train 4 wasidentified as the most optimal for mAb1 purification. In addition,secondary structure analysis using circular dichroism, chargeprofile analysis using iso-electric focusing and binding activityanalysis using ELISA confirmed that the antibody subjected toenhanced washes still retained its structural integrity andcomparable to reference standard (data not shown).

Purification Efficiency Varies by Molecule

Based on the results obtained above, further tests were carried outon additional antibodies to check the effects of wash pH duringProtein-A chromatography and the effect of neutralization pH postviral inactivation on HCP removal. Shown in Table IV is the IgGclassification, iso-electric points and estimated hydrophobicity foreach of the mAbs chosen. For hydrophobicity estimation, 3D modelstructures of the Fab regions of these four antibodies were builtusing homology modeling tool SWISS-Model (Arnold et al., 2006)and the hydrophobicity scores of the CDR loops were calculatedusing Kyte-Doolittle hydrophobicity score (Kyte and Doolittle,1982). Results suggest that mAb3 is the most hydrophobic whilemAb1 is the least hydrophobic. Different surface charge andhydrophobicity from the four mAbs serve the purpose of a broadscreening for common mechanisms.

Consistent with the observations made for mAb1, results inFigure 7A show that high pH washes are more effective in removingthe HCPs from the product pool. However, the extent of removalclearly varies from antibody to antibody. Particularly, in comparisonto IgG4s (mAb1 and mAb2), the IgG1s (mAb3 and mAb4) required awash buffer with a pH of almost 10.0 to achieve similar percentagereduction in HCPs. This is not unexpected because of the differencesin the pI of the molecules and a higher pH is required to make mAb3and mAb4 anionic and dissociate the HCPs. However, it is also to benoted that the pHof buffer solution can have significant impact on thestability of antibodies and alkaline pH, particularly pH> 10 can leadto deamidation of asparagine residues on the antibody (Pace et al.,2013; Patel and Borchardt 1990a, b). Thus, one has to exhibit cautionon choosing the appropriate pH for wash buffers.

Neutralization pH studies on mAb2, mAb3, and mAb4 post viralinactivation yielded results similar to mAb1. In all cases, there wasan increase in turbidity of the pools with an increase in pH and thepeak turbidity started decreasing when the pH was increased above7.5 (data not shown). Strongly correlating with this turbidity,filtration of the samples resulted in removal of HCPs from the pool(Fig. 7B). However, unlike other antibodies in the study, mAb2exhibited significant loss of recovery in the pH range of 6.0–7.0.This pH range coincided with the iso-electric point of the antibodywhich might have resulted in aggregation/precipitation of theproduct and removal by filtration. Similar observation was notmade for mAb1 although the pI was between 6.5 and 7.0. Thissuggests that the stability of molecule is highly variable fromantibody to antibody and needs empirical experiments todetermine the optimum pH for neutralizing the Protein-A pool.

Having identified the optimal Protein-Awash conditions and theneutralization pH post viral inactivation, mAb2, mAb3, and mAb4were subjected to purification using Train 4 (see Table II) andcompared against the control purification process using Train 1. Inall cases, implementation of the enhanced Protein-A washes alongwith optimization of neutralization pH yielded vastly improvedproduct quality with three out of four mAbs meeting the drugsubstance specifications for HCP and DNA removal using just onecolumn process. Even though there was significant reduction inHCPs and DNA compared to the control process, there wasessentially no removal of high molecular weight (HMW) aggregatesin mAb2. For antibodies that have high aggregates in the HCCF,

Figure 6. CHO host cell protein (HCP) levels present in the clarified harvest after

acid precipitation. Acid precipitation was carried out using either Acetic acid or Citric

acid as the titrant.

Table II. Conditions employed in various purification trains tested.

Step Train 1 Train 2 Train 3 Train 4 Train 5

Harvest treatment Control Low pH Control Control Low pHProtein A chromatography Control Control Enhanced washes Enhanced washes Enhanced washesNeutralization to optimal pH No No No Yes Yes

Table III. Recovery and purity levels of the final mAb pool obtained by

purification using various trains shown in Table II.

Train 1 Train 2 Train 3 Train 4 Train 5

Recovery (%) >95 >90 >95 >95 >90HCP (ppm) 7,100–25,500 940–2,400 180–275 10–33 3–12DNA (ppb) 10–26 7–18 <5 <1 <1


further optimization is needed to get efficient removal of HMWspecies. A new generation of Protein-A resins have recently comeinto the market that claim to offer resolution between HMW speciesand the monomer (Eshmuno-A

1

, EMD Millipore). In addition,recent studies have also demonstrated removal of HMWaggregatesby harvest treatments using flocculation (Kang et al., 2013). Theseneed to be evaluated in combination with the above Protein-Awashes to gain further improvements in product quality whereaggregation of the antibody poses a problem.

Conclusions

The most common purification schemes for monoclonal antibodiesand Fc fusion proteins utilize Protein-A chromatography for capturefollowed by two to three additional steps for intermediatepurification and polishing (Kelley et al., 2009; Vunnum et al.,2009b). While Protein-A removes a vast majority of thoseimpurities, remaining levels of process and product relatedimpurities present significant challenges downstream. Previousstudies have demonstrated that a bulk of those impurities thatco-purify with the antibody do so by associating with the antibodyitself (Levy et al., 2014; Shukla and Hinckley, 2008). Proteomicbased approaches to investigate the profiles of HCPs present inHCCF and the eluates from Protein-A column lead to identificationof specific populations that exhibit strong interaction withantibodies (Hogwood et al., 2013a, b; Jin et al., 2010; Levy et al.,2014; Nogal et al., 2012; Shukla and Hinckley 2008; Sisodiya et al.,2012; Tait et al., 2012; Tarrant et al., 2012). However, commonframework to selectively disrupt these interactions between HCPsand antibody are still lacking.In this study, using batch mode high-throughput experiments,

effect of pH was tested to selectively disrupt interactions betweenthe antibody and the HCPs. In contrast to the conventionaloperation where a wash buffer pH was always chosen to be

Figure 7. (A) Comparison of the effect of Protein-A wash pH on HCP removal from various mAb harvest pools. (B) Comparison of the effect of neutralization pH on HCP removal

from various mAb Protein-A eluate pools. Error bars represent standard deviation of the analytical results.

Table IV. Chemical characteristics of various mAbs used in purification

evaluation.

Molecule Class pI range Hydrophobicitya

mAb1 IgG4 6.5–7.0 �70.0mAb2 IgG4 6.0–7.0 �56.8mAb3 IgG1 8.0–8.5 �26.4mAb4 IgG1 8.0–8.5 �41.4

a3D structures of the Fab regions were built using homology modeling toolSWISS-Model (Arnold et al., 2006) and hydrophobicity scores of the CDR loops werecalculated using Kyte-Doolittle hydrophobicity score (Kyte and Doolittle, 1982) asshown in the last column. According to the surface models, mAb3 is the mosthydrophobic while mAb1 is the least hydrophobic molecule.



between the load pH and elution pH (Vunnum et al., 2009b),results from these studies suggested that a high pH wash buffer ismore effective in removing process related impurities and thiswas demonstrated across multiple antibodies. In addition, thehigh throughput screening technique used in this study alsoprovided an effective template to comprehensively screen avariety of buffers and wash additives in a very short time frame.This has lead to rapid identification of optimal concentrations ofwash additives like arginine, urea, and caprylic acid that result ingetting additional removal of impurities. These results alsosuggested that the HCPs associate with antibodies through acomplex mode of interactions employing a wide variety of forceslike electrostatic interactions, hydrogen bonds, hydrophobicinteractions, and/or van der waal’s forces. Extended studies onproteome and the protein interactions are underway to categorizethe residual HCPs across harvest clarification, Protein-A, andlow pH inactivation respectively.

Further, our studies also demonstrate that turbidity associatedwith Protein-A pools and neutralization thereafter is not necessarilyan undesirable phenomenon. This is usually a consequence of HCPsbecoming less soluble when the product pool pH is at or close to theiriso-electric points and thus provide a way to further reduce theimpurities by filtration. When used in combination with otherseparation techniques like acid precipitation during cell cultureharvest, new wash incorporated Protein-A chromatography andoptimal neutralization of product pool post Protein-A can lead toremoval of HCPs and DNA to drug substance specifications with justone column. This would essentially reduce the need for additionalpolishing and leave opportunities for the subsequent steps to addrobustness and focus on objectives such as viral clearance. Integral topurification process of any therapeuticmolecule is the demonstrationof robust viral clearance along with other impurity clearance (Brayand Brattle, 2004; CPMP/BWP/269/95, 2001; Shi et al., 2004; Zhouand Dehghani, 2007). Initial studies have demonstrated thatcompared to Protein-A chromatography with phosphate bufferwash only, incorporation of the enhancedProtein-Awash led to two tothree additional logs of clearance for retroviruses, and between oneand three additional logs of clearance for parvoviruses (data notshown). Acomprehensive analysis of improved virus clearance acrossmultiple purification steps (i.e., harvest clarification, protein-achromatography,polishing chromatography, and virus filtration) isongoing and will be presented as a part of future work. To beacknowledged is also the fact that current conditions identified herein Protein-A chromatography are not optimal for separating highmolecular weight aggregates or various glycoforms of the proteins.

Those areas are currently being explored together with newgenerationProtein-A resins as well as novel flocculation techniques.

In summary, with high-throughput framework we can rapidlydevelop a robust Protein-A purification strategy and co-optimizewith harvest treatment and viral inactivation. Such screening canemploy common strategies for most mAbs (Figs. 3 and 4, Table II),as well as identify molecule specific strategies (Fig. 7, Table V) toaccomplish the robustness. When combined with recent advance-ments in depth filtration and flocculation techniques, theempowered Protein-A can represent a significant leap in processeconomics while meeting the drug substance specifications andmaintaining critical product attributes.

We would like to acknowledge the contributions from Bristol-Myers SquibbProcess Analytics department for supporting us with various assays and theupstream department for providing us with the harvest material. We alsothank Dr. Yuanli Song for performing molecular simulation.

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