monoclonal antibody capture and viral clearance by cation exchange chromatography

ARTICLE

Monoclonal Antibody Capture and Viral Clearanceby Cation Exchange Chromatography

G.R. Miesegaes,1 S. Lute,1 D.M. Strauss,2 E.K. Read,1 A. Venkiteshwaran,2 A. Kreuzman,2

R. Shah,3 P. Shamlou,2 D. Chen,2 K. Brorson1

1Office of Biotechnology Products, CDER/FDA, 10903 New Hampshire Ave., Silver Spring,

Maryland 20903; telephone: 301-796-2095; fax: 301-796-9817;

e-mail: [email protected] Lilly and Company, Indianapolis, Indiana3Office of Testing and Research, CDER/FDA, Silver Spring, Maryland

Received 6 September 2011; revision received 10 February 2012; accepted 16 February 2012

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.24480

ABSTRACT: Traditionally, post-production culture harvestcapture of therapeutic monoclonal antibodies (mAbs) isperformed using Protein A chromatography. We investigat-ed the efficiency and robustness of cation exchange chro-matography (CEX) in an effort to evaluate alternativecapture methodologies. Up to five commercially availableCEX resins were systematically evaluated using an experi-mentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capturea model mAb with a neutral isoelectric point, (b) clear threemodel viruses (porcine parvovirus, CHO type-C particles,and a bacteriophage). This approach identified a narrowoperating space where yield, purity, and viral clearance wereoptimal under a CEX capture platform, and revealed trendsbetween viral clearance of PPV and product purity (but notyield). Our results suggest that after unit operation optimi-zation, CEX can serve as a suitable capture step.

Biotechnol. Bioeng. 2012;xxx: xxx–xxx.

� 2012 Wiley Periodicals, Inc.

KEYWORDS: monoclonal antibodies; viral clearance; cationexchange chromatography

Introduction

Protein A chromatography is the unit operation mostcommonly employed for the initial purification step orcapture of monoclonal antibodies (mAbs) and Fc-fusionproteins from harvested cell culture fluid (HCCF) (Ghoseet al., 2007a). Protein A features high specificity and yield(Duhamel et al., 1979; Forsgren and Sjoquist, 1966; Ghose

et al., 2005, 2007b; Goding, 1978; Hjelm et al., 1975),performance tolerance to high load capacities and relativelycrude HCCF feedstocks (Hahn et al., 2003), and reusabilitywith proper cleaning (O’Leary et al., 2001; Seely et al., 1994).At the same time, Protein A can serve as a virus removal step,clearing 2–4 log10 retroviruses and 1–3 log10 parvoviruses(Miesegaes et al., 2010a) although this modest level is notlikely to improve until a better mechanistic understanding ofhow viruses are cleared has been obtained (Miesegaes et al.,2010a). Protein A chromatography has other drawbacks,which include a relatively high material cost, the need tomonitor or clear leached Protein A, and obviously theinability to capture protein products without an Fc region.As a result, there is a constant interest in identifying suitablealternatives for Protein A chromatography as a productcapture step.

Ion exchange chromatography is a frequently discussedalternative mAb capture strategy (Arunakumari et al., 2007;Follman and Fahrner, 2004; Lain et al., 2009; Necina et al.,1998) that also can demonstrate viral clearance (Miesegaeset al., 2010a). Attractive aspects include lower resin costsrelative to Protein A and resistance to highly concentratedalkaline cleaning buffers such as NaOH. Our meta-analysisof a CDER regulatory documents database (Miesegaes et al.,2010b) revealed that approximately 6% of submissions inwhich a capture step was claimed for viral clearance hadcited cation exchange chromatography (CEX) as the capturestep, making it second only to Protein A chromatography(Fig. 1a). The analysis also revealed that compared to othermajor unit operations, viral clearance by CEX is somewhatmore variable (our database found a standard error of themean for CEX retroviral LRV records¼ 0.20 vs. 0.16 forProtein A; Fig. 1b), and has LRV distributions for retro- andparvoviruses that shift to lower ranges, relative to Protein A(Fig. 1c and compare d with e and f with g). We also foundthat somewhat better virus partitioning seems to occur when

Conclusions in this manuscript are those of the authors and do not necessarily

represent official policy of the Food and Drug Administration. The FDA does not

recommend or endorse specific chromatography resins.

Correspondence to: K. Brorson

Additional supporting information may be found in the online version of this article.

� 2012 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2012 1

CEX is employed as a polishing step, that is, where processfluids tend to be more pure (mean LRV¼ 2.7� 0.15compared to 1.8� 0.2 for capture, P¼ 0.01) (Miesegaeset al., 2010a). Although this seems to argue that CEX viralclearance may be adversely impacted by feedstock complex-ity, it should be noted that even as a capture step, 46%retrovirus and 29% parvovirus records claimed LRVs greaterthan 2 log10, which in comparison to Protein A is still withinrange (Lute et al., 2009). However, this type of databaseanalysis only provides a broad overview of general trends asit consists of aggregate data across multiple firms andproducts; it cannot definitively reveal mechanisms or criticalprocess parameters for viral clearance efficiency androbustness. One possibility is that when run in a bind-

and-elute mode of operation, buffer conditions areoptimized based on partitioning product from otherimpurities such as host cell DNA and proteins, product-related impurities (like aggregates), rather than for viralclearance.

Previous case studies have provided some insight into thegeneral mechanism of viral clearance by CEX (Miesegaeset al., 2010a). In one case, >4 log10 MuLV clearance wasachieved in CEX columns run at a pH of 5.0. Clearance wassubstantially reduced however when CEX was performedat higher pH, 5.5 or 6.0, and was completely eliminated atpH 6.5. MMV clearance was consistently <2 log10 underall pH conditions tested. It was proposed that MuLV bindsthe CEX resin electrostatically at a greater affinity at

Figure 1. Database assessment of Protein A and CEX chromatography. All panels except (b) represent data from capture records only. a: Frequency of mAb capture methods

reported in submitted regulatory documents; CEX for mAb capture is the second most common, behind Protein A. b: Overall mean LRV was calculated, per unit operation, for all viral

clearance records in our database; calculating the respective standard errors of the mean illustrates that CEX is the most variable unit operation in terms of LRV (y-axis represents

standard error of the mean). c–g: Analysis of mean (c) and distribution of retrovirus (d and e) and parvovirus (f and g) LRV, using infectivity assay, for Protein A capture (left) and CEX

capture (right), respectively. Pro A, Protein A chromatography; CEX, cation exchange chromatography; BE, bind and elute mode; AEX, anion exchange chromatography; other, other

capture methods (e.g., mixed mode, Protein G, etc.); Filter large, large virus retentive filters; Filter small, small virus retentive filters.

2 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2012

lower pH and could be fractionated from the product itself;at higher pH conditions they co-elute. In another case,effective MuLV clearance by CEX at pH 5.0 for a number ofmAb products also noted an unexpected loss of spikerecovery when determining mass balance. It was suggestedthat some degree of virus clearance in these runs may havebeen due to MuLV inactivation rather than separationpower of the column, even though MuLV is fairly stableat pH 5.0. However, the same study reported substantialclearance at similar buffer pH for PRV, another envelopedvirus but with a higher isoelectric point (Williams et al.,1982), and for Reo-3, a non-enveloped virus. In addition,follow-up studies have suggested that inactivation is likelynot an explanation for the observed pH effects on MuLV(Connell-Crowley et al., 2012). Finally, clearance results forHAV, a non-enveloped but more acidic virus (Michen andGraule, 2010) demonstrated an overall reduction similar tothe other studies (1.5 log10) (Adcock et al., 1998), although adirect explanation for this was not addressed.

Here, we systematically evaluated CEX as a potentialcapture operation using a challenging model mAb processfluid (a complex HCCF containing a neutral isoelectricpoint mAb, possessing a fraction of half-antibodies) usingfive commercially available CEX resins and at two loaddensities (25 and 40mg/mL resin). Using an experimentallyoptimized buffer platform we identified a narrow set ofconditions that successfully achieved target yield and purityvalues, as well as adequate viral clearance of PPV, for three ofthe five resins.

Materials and Methods

Equipment

All lab-prep scale CEX chromatography runs wereperformed on a GE Healthcare AKTA Explorer 100(Piscataway, NJ). The following CEX resins were used inthis study: Poros1 50 HS (Applied Biosystems, Foster City,CA), UNOsphereTM S (Bio-Rad Laboratories, Inc.,Hercules, CA), CaptoTM S (GE Healthcare Bio-SciencesAB, Uppsala, Sweden), CM Ceramic HyperD1F (PallBiosepra, Cergy, France), and GigaCap S-650M (TosohCorporation Bioscience Division, Tokyo, Japan). Resinswere packed at a bed height of 20 cm into OmnifitBenchmark 25mm glass columns (Danbury, CT). Afterpacking, HETP measurements were made (N/m rangingfrom 1035 to 1287) from a chromatogram of a 2% acetone(v/v in water, asymmetry values routinely between 0.8 and1.8) pulse to confirm bed quality. The packing quality ofPoros HS 50 resin in our hands could not accurately beassessed by this technique. During the CEX runs, load, flow-through, and eluate samples were collected for (1) yield andpurity analysis using pre-packed, HPLC-grade analyticalPoros A/20 Protein A (Foster City, CA) chromatographycolumns; and (2) relative proportion of monomers assessedby analytical scale size exclusion chromatography (SEC)

across a TSKgel SW guard column and TSKgel G3000SWxlcolumn (Tosoh Bioscience, Grove City, OH), on an Agilent1200 series HPLC system (Santa Clara, CA). For both, PBSwas used as the mobile phase and UV absorbance wasdetected at 280 nm.

Model Antibody and Feedstock Conditioning

The antibody used in the study was a proprietary CHO cellexpressed Human IgG4 mAb supplied by Eli Lilly. Thisantibody had an isoelectric point (pI) of 7.53 calculated byLilly from the sequence of its amino acids. Before use, themAb harvest was concentrated with a 10 kDa TFFmembrane, conditioned to control pH and conductivityvia buffer exchange, and pH precipitated using depthfiltration, to achieve a concentration of 5.7 g/L, conductivityof 20mS/cm and pH 5.0.

Buffers and Feedstocks

Buffers used in the study were as follows: Equilibrationbuffer: 25mM sodium acetate pH adjusted to theexperimental set point with stock glacial acetic acid diluted1:2 in water and added drop by drop to each respectivebuffer solution while being mixed on a stir plate; final pHvalues were attained with only minimal volume changes.Likewise, conductivity was adjusted to the experimental setpoint with 5M NaCl; feedstocks were pH and conductivityadjusted in a similar manner. Elution buffer: 25mM sodiumacetate, 100mM NaCl; Regeneration buffer: 1M NaCl;Cleaning buffer: 250mM NaOH; post-strip/regenerationpH adjustment buffer: 100mM sodium acetate, pH 4.0. Seetext for specific pH and conductivity values tested.

Run Conditions

The CEX column flow rate was 0.88mL/min (or 7.5 CV/h).Runs were conducted as follows (note for all experiments,one column volume (CV)¼�7.65mL): Cleaning wasfollowed by regeneration (2 CV ea.), pH adjustment (4CV), and equilibration (3 CV). Product loading was targetedat either 25 g/L (initial screening study) or 40 g/L resin(remainder of study). The column was then washed (4 CV)with equilibration buffer, and elution was carried out by a 20CV elution buffer gradient from 0% to 100%. Eluates werecollected in 4mL fractions for all runs with the exception ofPoros HS at 20mg/mL resin, where samples were collectedin 2mL fractions.

Yield and Purity Analyses

CEX run samples (load, flow-through, wash, and elutionfractions) were analyzed using Protein A HPLC to measureyield and purity per fraction and for determination ofnarrow and wide elution pools. Yield and purity values (%)

Miesegaes et al.: Cation Exchange Chromatography as an Alternative Capture Step 3

Biotechnology and Bioengineering

were calculated by integrating the areas under the curve(AUC) of absorbance at 280 nm (A280) from a givensample’s chromatographic profile. Each Protein A HPLCprofile contained a flow-through peak (predominantly non-mAb host cell proteins and DNA, or AUCnon-mAb) and anelution peak (predominantly mAb-containing, or AUCmAb).Percent yield was therefore determined by comparing agiven elution fraction’s AUCmAb to the respective loadsample’s AUCmAb; percent purity was determined bycomparing AUCmAb of a given sample to the total

absorbance (i.e., AUCmAbþAUCnon-mAb). Our rationalefor defining mAb- and non-mAb using AUC 280 readingswere verified qualitatively by examination of reducingSDS–PAGE (Novex 4–12% Bis–Tris 1.0mm, Invitrogen,Carlsbad, CA) (Fig. 2). Protein A-HPLC run conditionswere as follows: Protein A-HPLC was run at 5mL/min usingPBS as equilibration buffer. Samples were diluted 1:10 inPBS, injected (300mL) washed for 3min in PBS, and elutedfor 2min via 150mM acetic acid. For the initial resinscreening study and prior to pooling, target yields for theentire elution step were set to >90%. Subsequent narrowand wide elution pools were generated based on the HPLCresults from individual fractions, with targeted yields of�80and 90% and targeted purities of�70 and 60%, respectively;see the Results Section.

Viral Clearance Evaluation

For phage and viral clearance studies, feedstocks werespiked with 0.1% PR772 and F-X174 bacteriophagepreparations (108 pfu/mL) and/or 0.1% porcine parvovirus(PPV; 106 TCID50/mL). Infectivity was confirmed in loadsamples for each run. Titers of PR772 and F-X174bacteriophage in spiked load and eluate test articles wereassessed by plaque assay (Bolton et al., 2005; Lute et al.,2004). CsCl gradient purified phage stocks were preparedusing standard procedures (Coetzee et al., 1979). Titers ofPPV (produced in-house by Eli Lilly) were calculated byTCID50 assay. Briefly, 96-well plates were seeded with200mL/well of 2.5� 104/mL PK-13 cells (ATCC# CRL-6489) and incubated at 378C overnight. The plates were theninoculated with serial 10-fold dilutions of testing sample(50mL/well) and a total of 8 replicate wells were inoculatedfor each dilution. Following 10-day incubation at 378C5%CO2, plates were examined under a microscope and eachwell was scored for cytopathic effects (CPE). The TCID50

titer was calculated using the Karber formula (Karber, 1931).

Figure 2. Representative overview of chromatogram interpretation, Protein

A-HPLC, reducing SDS gel electrophoresis, and SEC–HPLC to assess CEX perfor-

mance. a–c: Poros HS 50 control condition shown (25mM sodium acetate pH 5.7,

conductivity 4.0 mS/cm), depicting purification capability within the determined oper-

ating space. a: Curves represent A260 (red), A280 (blue), and conductivity readings

(beige) over time (see arrows in panel). b: Load, flow-through, wash, and elution

fraction samples (representative to CEX run shown in (a)) were assessed for ‘‘yield’’

and ‘‘purity’’ by Protein A-HPLC. For each CEX run sample shown, the first peak

contains non-mAb host protein, that is, the Protein A-HPLC flow-through, whereas the

second peak contains the mAb intermediate, that is, the Protein A-HPLC elution

fraction. See the Materials and Methods Section for further detail. c: CEX run samples

were also were loaded onto an SDS–PAGE gel, coomassie-stained, and compared

with HPLC results. Band intensities correlate with Protein A-HPLC data, thus support-

ing our methodology. Concentration and purity values in (c) were calculated from the

Protein A-HPLC results and are shown for ease of reference. d: SEC–HPLC to assess

for percentages of HMW species such as dimers (A) and to determine the amount of

monomeric (B) and half-antibody (C) forms of CEX purified IgG4 product intermediate

(see inset; %A, %B, %C, respectively). Note chromatogram data represent multiple

runs at various dilutions and should not be directly compared. Ld, load); FT, flow-

through; W, wash; E1–E7, elution fraction numbers 1–7; HC, heavy chain; LC, light

chain; VV, void volume; CV, column volume.


CHO type C particle removal was assessed by Q-PCR on asubset of the original design-of-experiment (DoE) samplesas described (Zhang et al., 2008).

Results

Setting the Initial CEX Operating Space

We first needed to set an initial operating space wherecapture of our model antibody by typical commerciallyavailable CEX resins could be expected. Our initial scoutingwas guided by general industry knowledge, experience withother antibodies at Eli Lilly, and an observation from ourdatabase that greater LRV claims correlated with lowerpH and conductivity values during loading and a highsalt differential between loading and elution conditions(Miesegaes et al., 2010b). Accordingly, sodium acetatebuffers (pKa 4.75) with pH ranging from 4.5 to 5.7 andconductivity in the range 4.0 to 8.5mS/cm were used in theinitial screening studies. NaCl was used to adjust the ionicstrength of the antibody solution and buffers for columnoperation.

Using data generated from all five resins tested (see theMaterials and Methods Section and Table I), our prelimi-nary scouting studies identified an acceptable center-pointcondition of pH 5.3 and 5.0mS/cm conductivity (data notshown). These studies also revealed that the acceptableoperating range for our particular model antibody waslimited to pH 5.0–5.7 and conductivity 3.0–6.0mS/cm. Thisis however somewhat wider than what others have reportedfor a different model antibody (Lain et al., 2009). We foundthat pH conditions below 5.0 caused precipitation ofundesired host cell proteins in addition had denatured theantibody prior to running on the column (SupplementaryFig. S1). Compromises in mAb integrity at lower pH valuesare a known complication for CEX (Ghose et al., 2007a).However, this phenomenon was not observed with a loadpH of 5.0. Thus, pH <5.0 was considered to be outsidethe acceptable operating range for this particular mAbfeedstock.

Conversely, conductivity conditions above 6.0mS/cmalso fell outside of an effective operating range(Supplementary Fig. S2). Here, the majority of mAb didnot bind tightly to the resin; rather, a large portion remainedin the flow-through and wash fractions, leading to minimal

yield (% bound ranging from 0.2% to 6.0%). Loweringthe conductivity to below 6.0mS/cm improved both purityand yield values. Thus, a conductivity of >6.0mS/cm wasdefined as outside the range of an effective DoE for thisparticular mAb product. Lastly, we found that anaggressive pH neutralization strategy was required post-cleaning for most resin types. After screening various buffersystems we found that effective neutralization was accom-plished with 100mM sodium acetate, pH 4 after the 1MNaCl regeneration. This was probably needed due to a slowrelease of OH� ions from the column resin after it hadequilibrated to the high pH conditions of the cleaningbuffer.

Due to high levels of host cell protein and DNA levelsin the crude feedstocks employed in the study, we optedto assess mAb yield and purity using analytical Protein A-HPLC, as opposed to a more host cell protein-specificmethod such as ELISA. To ensure that this methodology wasadequate for intended use, reducing SDS–PAGE and SEC–HPLC on CEX runs within the defined operating space wasperformed (Fig. 2). Data generated by Protein A-HPLCqualitatively corresponded well with our protein gel analysis(e.g., compare % purity values to each corresponding gellane). We also conducted SEC–HPLC (Fig. 4d) on a subsetof CEX runs to determine the amounts of dimeric,monomeric, and half-antibody forms present. We notethat very little (<1%) high molecular weight/dimeric formswere present for samples run within the defined operatingspace (denoted as ‘‘A’’ in panel (d)). We also found thatlevels of naturally occurring half-antibodies (denoted as ‘‘C’’in panel (d)) ranged from �17% to 35% (mean¼ 25.81%);although influenced by various factors such as expressionvector and production cell line employed, half-antibodylevels upwards of 30% is not uncommon (Vasilyeva et al.,2002).

Resin Performance Screening at Center-Point

Having identified adequate initial operating ranges, we nextcompared the performance of five commercially availableCEX resins (Table I) at center-point, as measured byproduct yield and purity, and virus clearance. Thesecommercial resins vary in backbone composition, ligand(i.e., weak vs. strong cation exchanger), binding capacities,and recommended operating flow rates. Each resin wasevaluated at low (25 g/L) and high (40 g/L) loading densities

Table I. General characteristics of CEX resins used in DoE study as determined and reported by the resin manufacturer.

Resin type Backbone composition Ionic strength Binding capacity (mg/mL) Operating flow rates (cm/h)

Poros HS Crosslinked polymer (S-DVB) Strong 55 at 1,000 cm/h1 Up to 1,000

UNOsphere S Hydrophillic polymer Strong 60 at 150 cm/h2 150–600

Capto S Agarose Strong 120 at 600 cm/h1 Up to 700

CM Ceramic HyperD Ceramic, with gel-containing pores Weak 60 at 200 cm/h2 100–300

GigaCap S-650M Methacrylic polymer Strong 145 at 212 cm/h2 10–600

Refer to product brochures for information on backbone composition, ionic strength, binding capacity, and operational flow rate ranges. Note that eitherlysozyme(1) or human IgG(2) was used by the resin manufacturer to claim the referenced dynamic binding capacities shown.



in univariate experiments using the above defined center-point parameters. Experiments at 25 g/L were first con-ducted in order to better match the initial screeningconditions and to validate our chosen center-point, whereasexperiments at 40 g/L were later employed to (a) identifyany resin specific performance differences attributable torunning at increased load densities, that is, when closer tothe dynamic binding capacity; (b) acquire CEX performancedata at load densities closer to that used during large scalemanufacturing; and (c) better compare our results withthe known typical performance capabilities of Protein A,which is also commonly performed close to the dynamicbinding capacity.

Performance target values were also established to helpguide our eluate pooling strategy. We chose a target yield(�90%) that is common for commercial mAb capture andconsistent with sound process economics. Purity targets(�60%) were somewhat less than those typically set forProtein A chromatography, reflecting the expected lowerspecificity of CEX. In a typical manufacturing setting onlythose elution fractions containing substantial amountsof relatively pure antibody are pooled. However, differentprocess goals or quality target product profiles may warranta different balance between yield and purity. We thereforeestablished pooling criteria for generating respective narrow(high purity, lower yield) and wide (high yield, lower purity)elution pools. To do so, the entire elution phase wascollected in 4mL aliquots (per minute) while the CEX yieldand purity data were plotted across elution fraction number,in order to (a) generate elution profiles encompassing the

entire elution step; and (b) decide appropriate poolingcriteria with an emphasis on purity (i.e., narrow pools) andyield (i.e., wide pools) (Fig. 3 and Supplementary Fig. S3).Assessing the overall sharpness of the elution profiles was ofparticular interest from a process efficiency standpoint, as asharper profile corresponds to a more concentrated productand increased resolution with respect to impurities.

These analyses revealed resin-specific differences inimportant performance attributes such as the width ofthe elution profile and subsequent elute volume (Fig. 3), andoverall yield after pooling (Table II). In general, thechromatogram profiles were similar at both load densitiesfor a given brand of resin. However, Poros HS andUNOsphere S possessed broader elution profiles at bothload densities, and when run at 40 g/L failed to reach our

Figure 3. Elution profiles for Poros HS and Capto S CEX resins as tested at center-point parameters. a and b: At both load densities tested, Poros HS (and Unosphere S; data

not shown) consistently presented a wider, less desirable elution profile. c and d: Capto S (as with GigaCap S-650M and CM Ceramic Hyper D; data not shown) presented sharper

profiles. Brackets in (b) and (d) depict ranges of narrow and wide elution pools for Poros HS and Capto S, respectively.

Table II. Yield, purity, and bacteriophage LRV (log10) of the entire eluate,

i.e., before pooling, across CEX resins, at center-point (pH 5.3, 5.0mS/cm).

Resin

25 g/L 40 g/L LRV (40 g/L)

% Yield % Purity % Yield % Purity PR772 F-X174

Capto 98.2 62.5 98.6 54.0 5.0 4.9

Giga 100 57.6 98.6 60.3 5.3 3.6

HyperD 100 60.0 98.0 61.8 4.7 4.9

Poros 90.8 67.2 85.8 59.1 3.9 4.5

Unos 98.2 68.9 73.1 54.8 4.2 4.3

Samples consisted of the entire elution profile (3 CV for all runsperformed) from each corresponding run. Two load densities were assessed,revealing a slight loss in % yield for Poros HS and Unosphere S. Note phageclearance studies were performed only at the 40 g/L load capacity condition.


90% target yield (Fig. 3, compare a and b; Poros HS, withc and d; Capto S, Table II, and Supplementary Fig. S3).Comparing narrow- and wide pooled fractions we foundthat Poros HS and UNOsphere S again failed to meet targetyield (Table III). Here, values ranged from >45 to 74%,

whereas elution pools for the remaining resins ranged from>80% to 98%. Purity counts for elution pools across allresins were not impacted, indicating that our poolingstrategy had been optimized to reduce unwanted host cellprotein impurities.

As a first step toward assessing viral clearance, phages witheither acidic or neutral pI were spiked into 40 g/L loadcondition feedstocks. CEX run LRVs were measured bycomparing volume-adjusted load titers with titers in pooledelution samples (narrow and wide elution pools were nottested separately, but as part of the five-point DoE; seebelow). The center-point studies initially used phage insteadof mammalian viruses as they are considered safe andadequate surrogates under certain circumstances (mamma-lian parvovirus PPVwas later assessed, with results similar tothat shown in Table IV under the center point condition; seebelow). The phages PR772, a model for mid-large viruses,and F-X174, a surrogate for smaller virus, were chosen

Figure 4. Design strategy and representative chromatograms for a Resolution III DoE that was implemented in this study. a: Factors and respective levels, which were

defined based on an experimentally determined operating space. Three resins were assessed, for a total of 18 experiments (center-point condition performed in duplicate).

b: Representative chromatograms for Capto S across all five conditions. Chromatograms from CEX runs performed using GigaCap and HyperD resins were similar with the exception

of (þ/þ). See also Supplementary Figures S4 and S5.

Table III. Yield and purity results for center-point runs, using narrow and

wide elution pooling strategies.

Resin

% Yield (40 g/L) % Purity (40 g/L)

Narrow Wide Narrow Wide

Capto 93 98 72 70

Giga 94 97 69 68

HyperD 80 91 67 64

Poros 60 74 72 68

Unos 45 57 65 63

While % yield for Poros HS and Unosphere S drop substantially at thehigher load value, % purity remained relatively unchanged for all resins.



based on size, resistance to pH extremes (data not shown)and previous use in filtration and chromatography studies(Lute et al., 2007; PDA, 2008). We found that viruspartitioned into many fractions, but all resins appeared toclear both phages with >3–4 log10 LRV (Table II). Overall,phage clearance was not a factor in eliminating resins fromour study.

Based on the above data and with an emphasis on productyield, we ruled out further study of Poros HS andUNOsphere S. The other three resins seemed to be adequatecandidates for further assessment in a screening DoE. CaptoS and GigaCap had sharp elution peaks at both loaddensities. HyperD presented a unique double peak patternwhen run at the higher load (Supplementary Fig. S3, panelc); a similar double peak has been noted in the past when runat high load densities (P. Shamlou, personal communica-tion). Only the latter of the two peaks contained significantlevels of mAb (% purity of >90% compared to <0.06%for the first peak), indicating that this peak consists ofextraneous material that adequate purification could beachieved if it was discarded.

CEX Design of Experiment

Successful design of an ion exchange unit operation includescareful optimization of load and buffer pH and conductivi-ty. With the remaining three resins, we next implemented ascreening design strategy to assess the independent effectsof pH, conductivity, or resin type on product yield andpurity, and viral clearance of PPV and type C endogenousretrovirus particles (Fig. 4; Table IV). Most reported viralclearance studies traditionally assess retrovirus clearance,and do so by using MuLV spike preparations (Miesegaeset al., 2010b). The large number of reported studies onretrovirus is likely because retrovirus particles are producedin many of the commonly used mAb production cell lines,and consequently, retroviral clearance validation studies are

sought by regulatory authorities as early as possible, that is,by IND stage. Here, we chose to study PPV since (a)parvoviruses are generally more hardy than retroviruses andthus could serve as worst-case in terms of clearancecapability by CEX, and (b) there currently exists noavailable data on PPV clearance by CEX. CHO type Cparticle removal was assessed by Q-PCR on a subset of theoriginal DoE samples; this allowed us to track anendogenous retrovirus of concern (Zhang et al., 2008).Finally, since the overall goal was to better understandindependent effects and not necessarily have a fullyoptimized design space, we used a Resolution III screeningstudy, which by design does not confound individual effectsamongst each other. As outlined in Figure 4a, conductivitywas varied between 3.0 and 6.0mS/cm while pH was variedbetween 5.0 and 5.7. Center-point runs were repeatedindependently to those described above. Figure 4 presents arepresentative set of chromatograms from CEX runs forCapto S (see Supplementary Figs. S4 and S5 for GigaCap andHyperD, respectively).

As can be seen from the chromatograms and later verifiedusing Protein A HPLC (Table IV and data not shown),certain buffer conditions did not perform as well as center-point. All conditions except pH 5.7 and conductivity 6.0mS/cm (þ/þ) demonstrated some mAb binding to the CEXresin (i.e., �90% yield in most cases). For þ/þ, Capto Syielded only minimal recovery (9%), and no elutionfractions were generated using GigaCap or HyperD(Supplementary Figs. S4 and S5). For the other fourconditions where recovery was evident, purity was mostnegatively impacted by buffer conditions involving pHvalues of 5.0 (i.e., �/�; �/þ). Also, this reduction in puritycoincided with lower levels of PPV clearance (Table IV). Allof the above observations applied to narrow and wideelution pools. CHO type C particle removal, ranging from0.87 to >2.60 log10, was not particularly efficient andsomewhat variable, probably due to RVLP’s complex andheterogeneous nature.

Table IV. Yield, purity, and PPV viral clearance across CEX resins under defined DoE parameter ranges.

Resin X1/X2 Yield (%) Purity (%) PPV clearance (log10) Type C particle clearance (log10)

Capto S þ/� 90/92 86/84 1.9/1.9 nd/>2.60a

CP 94/>99 88/87 1.6/1.7 nd/1.51

�/� 92 62 1.0 nd

�/þ 87/>99 60/57 0.6/0.0 nd

GigaCap þ/� 89/90 85/83 1.6/1.2 nd/0.87

CP 86/>99 81/77 1.4/1.2 nd/0.99

�/� 91 55 0.7 nd

�/þ 79/96 63/60 1.0/0.8 nd

HyperD þ/� >99/>99 86/83 1.7/1.9 nd/0.72

CP 90/93 77/74 1.2/1.1 nd/1.02

�/� 75 57 0.6 nd

�/þ 95/98 51/54 0.8/0.6 nd

X1: pH (5.0, 5.3, 5.7); X2: conductivity (3.0, 5.0, 6.0). For yield, purity, and viral clearance, values are represented as (narrow elution pool)/(wide elutionpool), where applicable. Note the �/� condition produced yield plots that did not allow meaningful determination of narrow and wide elution pools.

aNo type C particles were detected in the wide elution pool for Capto S under this condition; the value shown is the minimum clearance factor based on theassay limit of quantification. nd signifies that the study was not performed and therefore, no data are available.


Discussion

In order to be considered a viable capture alternative, CEXshould be able to provide acceptable yield, purity, and viralclearance levels while affording improved process economicsrelative to Protein A chromatography. This may be adifficult target, however, as an average Protein A chroma-tography capture step easily provides>90% yield and puritywhile providing 1–3 log10 of retrovirus and parvovirusclearance (Miesegaes et al., 2010a). The high levels of yieldand purity obtained by Protein A mechanistically reflect itshighly specific binding. CEX, on the other hand, which intheory fractionates proteins predominantly by a charge-based mechanism, likely is less specific for mAbs, co-bindingsimilarly charged host cell proteins. Therefore, in order tojustify the use of CEX for primary capture, any reduction inpurity and viral clearance would need to be overcome by anyof the advantages CEX could offer. Examples may includelower costs of goods, a simpler cleaning regimen, or anyopportunities leading to reduced operational complexity.

Since host cell proteins have a wide range of isoelectricpoints, identifying the ideal conditions for partitioning themfrom a mAb product might prove essential for obtainingoptimal yield and purity results. We accomplished thisthrough performing a center-point study to first evaluatedifferent resins; and a five point DoE study determine anoptimal combination of buffer pH and buffer conductivityvalues. As a result of this, a CEX capture step with high yield(>90% for 18 or 21 runs) and in some cases, high purity(>80%) was developed. At the same time, these conditionsresulted in acceptable clearance values for PPV. In lieu ofProtein A, these values for a non-affinity based mAb captureprocess are favorable from a manufacturing standpoint.Since our DoE was a Resolution III screening study, inpractice further optimization in the form of a higherresolution study design would be needed to set the finaldesign space.

The highest yield and purity values were achieved for ourmodel (an IgG4 mAb with a calculated pI of 7.5) using abuffer pH of 5.3–5.7 and a conductivity of �5.0mS/cm(0/0 andþ/� conditions of the DoE). Conversely, we foundthat purity had decreased to 60% if run at pH 5.0. This wassimilar to the results of Lain et al. (2009) who performed aDoE study using an IgG1 mAb (pI of 8.1) where optimalload conditions were determined as pH 5.2� 0.2 and4.5� 0.5mS/cm conductivity. Purity counts were found intheir study to be lowest at pH �4.9. Whether the resultingloss of purity obtained at lower pH values can generally bemitigated, for example by increasing buffer conductivity,remains inconclusive based on our data (Table IV; compare�/� with �/þ). Only GigaCap (the resin also studied byLain et al.) but not Capto S or HyperD had demonstratedthis trend.

We also sought to identify CEX capture conditions thatprovided both optimal yield as well as effective viralclearance. Bacteriophages were first used as surrogates anddemonstrated effective removal of �4–5 log10 of the large

phage PR772 and the small phage F-X174. Assessment offlow-through and wash samples demonstrated that phagepartitioned across many CEX fractions overall. This broadfractionation profile has been observed for other viruses inCEX chromatography operations (Miesegaes et al., 2010a).While the pIs of PR772 (3.8–4.2) and F-X174 (6.6–7.2)differ from typical mammalian model viruses such as MuLV(5.8) and MMV (6.2) (Brorson et al., 2008; Dowd et al.,1998; Strauss et al., 2009), they are in the same generalrange, that is, mildly acidic to neutral. Whether phage canbe representative of mammalian viruses when studyingchromatography is controversial. In either case, any datathat furthers the mechanistic understanding of CEX maylead to better process optimization.

Regarding PPV clearance, although values are lower thanthat for phage, the data are within the range commonlyencountered with Protein A for parvoviruses (Miesegaeset al., 2010a). Our regulatory database also revealed thatmost parvovirus clearance records for Protein A and CEXwere distributed within the 1–2 log10 LRV bin (Fig. 1;compare panels f and g). Lastly, we noted in our DoE thatfor conditions where a substantial loss in purity occurred,PPV viral clearance had failed (i.e., LRV <1.0 log10). Thissuggests that purity content may be a rough correlate CEXperformance indicator for viral clearance of PPV. However,further investigation is necessary to definitively address this.Type C endogenous retrovirus clearance was not particularlyefficient and more variable, although with one exception(Hyper D in the þ/� condition), RVLP clearance wasroughly similar to PPV clearance. Capto S resins seemed tobe more effective at clearing Type C particles than the othertwo, especially at the high pH, low conductivity condition(>2.6 log10). Work by Connell-Crowley et al. (2012) foundthat there was no relationship between MuLV LRV andimpurity clearance; we found no relationship in ourendogenous type C particle assessments either. Takentogether, it appears that the level of impurities alone is at-best an imperfect indicator for overall viral clearance.Nevertheless, based on the DoE runs that met target yieldand purity values, viral clearance alone should not be areason to rule out CEX as a capture step.

Whether CEX can serve as a suitable capture method willlikely be dependent on both product characteristics and themedia manufacturer. In our case with a relatively neutral pIantibody, we found experimentally definable yet narrowprocess ranges with respect to buffer pH and conductivity.Though restricted in terms of acceptable operating ranges,these results are acceptable from the collective standpoint ofreaching adequate yield, impurity clearance, and virus LRV.Based on what is currently known about CEX, a higher pIantibody is likely to yield more process flexibility as it willbind tighter to the resin at more neutral buffer pHs and beeasier to partition from neutral pI host cell proteins. Ourdata thus argue that CEX could possibly be developed intoa suitable alternative to Protein A, which itself is notconsidered a robust unit operation in terms of viralclearance either (Miesegaes et al., 2010a). Still, as this is most



likely case (product) dependent, firms would have toperform their own evaluation on the specific product andoperating conditions, and on a case dependent basis.Additional factors to consider include the overall costs ofgoods (whether the decrease in resin, cleaning andvalidation costs compensate for the need to conduct anyadditional up front development work), developmentefforts required such as the type of CEX resin to use for agiven mAb (e.g., two of five resin types assessed failed tomeet our target for purity), feed-stock preparatory work(is there a need to condition the load via TFF in order getsufficient pH and conductivity for CEX to functionoptimally), and the business issues surrounding incorpo-rating a ‘‘new’’ process into existing manufacturing plants.For existing products that already use Protein A as a capturestep, momentum and regulatory requirements may rule outreplacing the capture step with CEX. However, for newproducts, some consideration is warranted, though with thecaveat that very careful optimization will be needed in mostcases.

We thank Laurie Graham and Chikako Torigoe (CDER/FDA) for

careful review of this manuscript. This work was funded in part by a

cooperative research and development agreement (CRADA) between

CDER/FDA and Eli Lilly & Co.

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