A Novel, Q-PCR Based Approach to Measuring Endogenous Retroviral Clearance by Capture Protein A...

ARTICLE

A Novel, Q-PCR Based Approach to MeasuringEndogenous Retroviral Clearance by CaptureProtein A Chromatography

Min Zhang,1 Scott Lute,2 Lenore Norling,1 Connie Hong,1 Aurelia Safta,1

Deborah O’Connor,1 Lisa J. Bernstein,1 Hua Wang,1 Greg Blank,1 Kurt Brorson,2

Qi Chen1

1Process Research and Development, MS10, Genentech Inc., 1 DNA Way,

South San Francisco, California 94080; telephone: 650-225-1265;

fax: 650-225-7203; e-mail: [email protected] of Monoclonal Antibodies, CDER/FDA, 10903 New Hampshire Ave.,

Silver Spring, Maryland 20993

Received 26 June 2008; revision received 24 September 2008; accepted 8 October 2008

Published online 14 October 2008 in Wiley InterScience (www.interscience.wiley.com
). DOI 10.1002/bit.22172
ABSTRACT: Quantification of virus removal by the purifi-cation process during production is required for clinical useof biopharmaceuticals. The current validation approach forvirus removal by chromatography steps typically involvestime-consuming spiking experiments with expensive modelviruses at bench scale. Here we propose a novel, alternativeapproach that can be applied in at least one instance:evaluating retroviral clearance by protein A chromatogra-phy. Our strategy uses a quantitative PCR (Q-PCR) assaythat quantifies the endogenous type C retrovirus-like par-ticle genomes directly in production Chinese Hamster Ovary(CHO) cell culture harvests and protein A pools. Thiseliminates the need to perform spiking with model viruses,and measures the real virus from the process. Using this newapproach, clearance values were obtained that was compar-able to those from the old model-virus spike/removalapproach. We tested the concept of design space forCHO retrovirus removal using samples from a protein Acharacterization study, where a wide range of chromato-graphic operating conditions were challenged, includingload density, flow rate, wash, pooling, temperature, andresin life cycles. Little impact of these variables on CHOretrovirus clearance was found, arguing for implementationof the design space approach for viral clearance to supportoperational ranges and manufacturing excursions. The viralclearance results from Q-PCR were confirmed by an ortho-gonal quantitative product-enhanced reverse transcriptase(Q-PERT) assay that quantifies CHO retrovirus by theirreverse transcriptase (RT) enzyme activity. Overall, ourresults demonstrate that protein A chromatography is arobust retrovirus removal step and CHO retrovirus removalcan be directly measured at large scale using Q-PCR assays.

Biotechnol. Bioeng. 2009;102: 1438–1447.

� 2008 Wiley Periodicals, Inc.

Correspondence to: Q. Chen

1438 Biotechnology and Bioengineering, Vol. 102, No. 5, April 1, 2009

KEYWORDS: viral clearance; protein A chromatography;production scale; design space; retrovirus-like particles;CHO cells

Introduction

Viral safety is a predominant concern for monoclonalantibodies (mAbs) and other recombinant proteins withpharmaceutical applications. Mammalian cell cultures thatproduce many of these products concomitantly produceendogenous retroviruses (Anderson et al., 1991b; Dinowitzet al., 1992; Lieber et al., 1973; Lubiniecki et al., 1989), andhave on occasion been infected with adventitious viruses(Bartal et al., 1982; Garnick, 1998). Some rodent cells used inproduction culture, such as mouse myeloma and hybridomacells, produce endogenous retroviruses that are infectious inin vitro assays (Adamson, 1998; Moroni and Schumann,1975; Shepherd et al., 2003). Even in the case of defectiveparticles, the potential for recombination events isunpredictable and such events have in the past led toexpression of tumorigenic viruses under highly manipulatedlaboratory conditions (Donahue et al., 1992; Kim et al.,1982).

The viral safety strategy for biopharmaceuticals includesscreening of cell banks and cell culture harvests foradventitious viruses, quantification of endogenous retro-virus levels in cell culture harvests and virus removalvalidation studies of the drug substance purification process(Dinowitz, 2002; Xu and Brorson, 2003). The validationstudies are typically scaled-down spiking studies using

� 2008 Wiley Periodicals, Inc.

model viruses and production scale in-process intermedi-ates. The virus clearance of individual purification unitoperations, such as chromatography or virus filtration, ismeasured and expressed as a log10 reduction value, or LRV(Brorson et al., 2004; Shi et al., 2004). The LRV oforthogonal steps (modules where clearance mechanismsdiffer) are added together to compute the overall processLRV for comparison to the quantity of endogenousretrovirus in the cell culture harvests. For Good Manu-facturing Practices (EMEA, 1996; ICH Q5A, 1998; FDA,2008) and practical considerations, viral clearance studiesmust be performed in scaled-down laboratory models thatmimic the large scale unit operation. Because ChineseHamster Ovary (CHO) retrovirus particles cannot beassayed in an infectivity assay and are difficult to produce,studies evaluating their clearance are performed with arelated model murine retrovirus, xenotropic murineleukemia virus, or X-MuLV (Shi et al., 2004).

Certain purification modules, including protein Achromatography, have been observed to reliably remove>2–4 log10 of large enveloped viruses, including X-MuLV(Lau et al., 1999; Shi et al., 2004; Valera et al., 2003). Themechanism for protein A resin to remove virus is to bindspecifically to antibodies (Abs), while allowing viruses toflow through the column (Brorson et al., 2003a,b). Thisnon-specific flow-through mechanism for virus suggeststhat small variations in operational parameters (e.g., flowrates, etc.) and absorbent decay during extended cycling areunlikely to substantially impact virus clearance. All availableevidence to date suggests that protein A chromatography isso robust with respect to small changes in operatingconditions and resin reuse (Brorson et al., 2003a), it is nowconceivable that design space concepts (ICH Q8, 2006) canbe applicable if supported by appropriate, prospectivelygenerated data.

Many aspects of the current design of viral clearancestudies leave large areas for improvement. CHO cells areprobably the most widely used cell substrate for productionof biopharmaceutical products. Unlike murine cell sub-strates, the retrovirus particles that these cells release are notable to replicate in vitro because their genomes contain stopcodons and truncations in genes needed for replication(Anderson et al., 1991a,b). It has not been formallydemonstrated whether an infectious, murine virus,X-MuLV, is a truly representative model for use in clearancestudies. Spiking studies using X-MuLV are also expensive,time-consuming, and performed at a scale different fromactual manufacturing. However, at the present time, spikingstudies with X-MuLV are widely considered to be the mostpractical approach for validation (Celis and Silvester, 2004;ICH Q5A, 1998). An ideal approach for process validation,at least for upstream capture stages, would be to track theendogenous particles directly (CBER, 1997). With theadvent of Q-PCR technology, this is now possible. Forexample, Q-PCR can be used to directly measure CHOretrovirus counts in load material and elution pools tomonitor clearance at manufacturing scale. It is also

conceivable to use this assay to generate CHO retrovirusremoval design spaces, that is, small-scale matrices ofprocess parameters and resin age limits that yield compar-able CHO retrovirus removal.

In this report, we analyze levels of CHO retroviralparticles, measured by two orthogonal Q-PCR assays, inHCCF and elution pools from both large-scale manufactureand small-scale matrices. The LRVs obtained by thisapproach are consistent with those obtained using thestandard X-MuLV spiking approach, supporting its feasi-bility for measuring retroviral clearance. In addition, little orno impact was found on clearance over a wide range ofprocess parameters, showing, for the first time, that it isfeasible to define a design space to ensure robust retroviralremoval by protein A chromatography.

Materials and Methods

Model Process Fluids-Harvested CHO CellCulture Fluids

Harvest fluid from commercial CHO cell cultures was takenfrom routine production at Genentech, Inc. (South SanFrancisco, CA). Cells and debris were removed from theharvested cultures by standard bioprocess technology toyield harvested cell culture fluid, or HCCF. HCCFs from sixmAbs, and seven CHO cell culture processes (mAb1 has twoversions), denoted mAb1a, 1b, and 2 through 6, were used asmodel process fluids in this report. The same HCCF wasused in small scale viral clearance studies, some of which wasspiked with X-MuLV and/or other model viruses, such assimian virus type 40 (SV40), murine minute virus (MMV)(Valera et al., 2003). All HCCF samples were eitherprocessed fresh or frozen at �608C within a short timeafter harvest, and were frozen/thawed fewer than threetimes.

Model Process Fluids-Protein A Elution Pools

Protein A elution pools from small scale virus clearancestudies may contain X-MuLV and/or SV40 and MMV. Wehave previously demonstrated that the three viruses are non-interfering in clearance studies (Valera et al., 2003). Smallscale elution pools were adjusted to pH 6–8 before freezing.Protein A elution pools from the process characterizationstudies and resin reuse studies were pH adjusted to aroundfive before freezing.

At manufacturing scale, a protein A column may be usedup to 11 cycles to process one batch of HCCF. Protein Aelution pools were taken after all cycles were completed andthe combined pool was held for lipid-enveloped virusinactivation, and the pH was adjusted to around 5. Allprotein A pool samples were frozen/thawed fewer than threetimes.

Zhang et al.: Endogenous Retroviral Clearance by Protein A 1439

Biotechnology and Bioengineering

Protein A Chromatography—Small Scale

Prosep vA media was purchased from Millipore (Billerica,MA), and was packed into a 0.66 cm diameter glasschromatography housing unit (Omnifit, Cambridge UK).Bed heights were approximately the same as those used atmanufacturing scale purification processes. The resin reuseexperiments used a cycled resin. The columns wereintegrated into an AKTApurifier or AKTAexplorer chro-matography system run by UNICORN version 4.0 software(GE Healthcare, Uppsala, Sweden).

The load in the non-virus spiked runs was spiked withvirus re-suspension buffer, at the same volume ratio as thevirus being spiked during a virus-spiked run. The load invirus-spiked runs was spiked with X-MuLV alone ortogether with other model viruses, at 1/100th the volume ofload for each virus. X-MuLV, SV40 and MMV werepurchased from BioReliance, Inc. (Rockville, MD).

Protein A Chromatography—Process CharacterizationStudy

As part of process characterization, a series of protein Achromatography runs are performed at small scale withvarying process parameters. With the exception of testingparameters, all other parameters were run at target oracceptable manufacturing ranges.

Four HCCF lots containing mAb1b were used asfeedstocks for the process characterization study. Out of atotal of 61 runs, 12 were performed at target conditions.Process parameters were selected to represent the fullpotential range of process variables, with an emphasis onparameters that may affect column performance (PDA,2005). Wide ranges were set to study normal operatingranges, acceptable upper and lower limits and processextremes. Variables that were studied included:

� M

14

Ab load density.
� L oad/elution flow rate. � W ash flow rate and phase duration. � P rotein A elution pool collection criteria. � H CCF temperature.
All feedstocks and protein A pools were analyzed byQ-PCR assay and an RT activity Q-PERT assay.

Protein A Chromatography Resin Reuse Study

The resin reuse experiments were performed using operatingparameters and operational set points equivalent to thoseused in the manufacturing scale. Because of the extendednature of these experiments, six lots of mAb1b were used.The column was cycled up to 250 times. Protein A elutionpools from every 10 cycles were analyzed by genome Q-PCR


and Q-PERT. To maintain acceptable chromatographicperformance and provide coverage for manufacturingconditions, the column was repacked at cycle #205 as astandard practice during a reuse study.

Real-Time Quantitative (Q)-PCR and Q-PERT Assays

Viral RNA was extracted from samples using a QIAampViral RNA kit (Qiagen, Valencia, CA), according to vendorinstructions as described (de Wit et al., 2000). Sample sizeswere 70–100 mL (undiluted and 1:10 diluted HCCF,undiluted protein A pool). To increase signal in someprotein A pool samples, sample volumes were increased witha scaled-up extraction procedure consistent with the kitvendor instructions (i.e., lysis buffer volumes were increasedand the spin columns supplied with the Viral RNA kits wereloaded several times). Extraction efficiency is confirmed byincluding reference standard mAb1a HCCF sample withknown CHO retrovirus particle titer.

Genomic DNA was removed by one or two sequentialDNase digestions: (1) the samples were treated with 0.6units/mL RNAse-free DNase I (Stratagene, La Jolla, CA) for20 min at 378C prior to capsid destruction and RNAextraction, or (2) the extraction eluate was treated with 0.2units/mL of DNase I at 378C for 30 min, and then the DNasewas heat inactivated at 708C for 15 min. The absence ofretroviral DNA was confirmed by assaying the sampleswithout reverse transcriptase.

Real-time quantitative PCR assays to measure CHOretrovirus genomes were performed as described (de Witet al., 2000). Primers and probe sequences were designed toamplify a fragment in the highly conserved pol region fromthe CHO type C retrovirus genome. Likewise, Q-PCR assaysto measure X-MuLV genomes detect part of the env genesequence (Shi et al., 2004). Each retrovirus particle containstwo genomic RNA molecules. Absence of cross-reactivity ofthe CHO retrovirus with X-MuLV (Shi et al., 2004) wasconfirmed by no amplification of X-MuLV genomes usingCHO retrovirus Q-PCR assay (data not shown). Quanti-tative TaqMan fluorogenic 50 nuclease product-enhancedreverse transcriptase (Q-PERT) assays to measure CHOparticle RT activity were performed as described (Brorsonet al., 2002b). Each retrovirus particle corresponds toapproximately 1,000 picocounts of RT activity. Oligonu-cleotide probes and primers were ordered from AppliedBiosystems (Foster City, CA) and Invitrogen (Carlsbad,CA).

Viral clearance was expressed as log10 reduction value, orLRV, which is the difference of log10 (total virus) in load andin product pool. Total virus is obtained from virus titers(particles/mL or nU/mL) in samples and sample volumes(mL).

LRV ¼ Log10ðvirus titer in load HCCF � load volumeÞ

� Log10ðvirus titer in elution pool � pool volumeÞ

Table I. CHO retrovirus and X-MuLV clearance by small scale protein A

chromatography (mAb1a).

Process conditions Load density

Clearance (LRV)

CHO retrovirus X-MuLV

Naı̈ve resin, target pooling Low 2.6 1.8

High 2.8 2.3

Naı̈ve resin, wider pooling Low 2.4 2.7

High 3.0 2.5

End of use resin, wider Low 3.6 3.0

pooling High 3.5 3.7

Results

Comparable CHO Retrovirus and X-MuLV Clearance atSmall Scale

The conventional approach to virus removal validation ofprotein A columns involves spiking studies, where X-MuLVis spiked into HCCF and clearance of the X-MuLV by thecolumn is measured. In order to test the feasibility ofmeasuring CHO retrovirus clearance by the Q-PCR assay,we used samples from conventional spiking studies. Withthe Q-PCR assay, we measured the particles present in loadHCCF as well as in protein A pools, and compared the LRVwith the X-MuLV clearance from the viral spiking studies.

In our first set of experiments with mAb1a (Table I), X-MuLV clearance by scaled-down protein A columns wasevaluated under different load density and pooling criteria.Clearance of CHO retrovirus was comparable to those of X-MuLV (defined as within 1 log10). For both types of viruses,load density and pooling does not impact LRVs substan-tially. Historically, somewhat higher LRVs are obtained withend-of-use than naı̈ve resin (Brorson et al., 2003a; Chen,unpublished observations; Lute et al., 2008). The sametrend occurred in this study for both viruses (two of twoexperiments).

In the second set of experiments with four modelantibodies (mAb1b, 2, 3, and 4; Table II), X-MuLV andCHO retrovirus clearance were compared in naı̈ve, and end-of-use resins. Duplicate mAb2 studies with different HCCFlots confirmed consistent clearance capacity. The protein Aprocesses for these mAbs differ somewhat from each other interms of wash buffer, bed height and load density, but theCHO retrovirus clearance was comparable to the X-MuLVclearance in each case. In addition, clearance of CHOretrovirus determined in non-virus spiked runs is consistentwith that from virus spiked runs. In the cases of mAb3 andmAb4, only X-MuLV was spiked. In the cases of mAbs 1a(Table I), 1b and 2, other model viruses including SV40 andMMV were spiked together with X-MuLV, a strategy that wehave previously shown that can independently evaluateclearance of the separate viruses (Valera et al., 2003). In all

Table II. CHO retrovirus and X-MuLV clearance by small scale protein A ch

Molecule

Naı̈ve resin

Non-virus spiked run

CHO retrovirus CHO retrovirus

MAb1b 2.8 2.6

MAb2 4.0 4.1

3.7a

MAb3 3.8 3.6

MAb4 1.9 1.7

aSamples were from a duplicated run using a different lot of mAb2 HCCF.bNo CHO retrovirus was detected in product pool. The limit of quantificat

cases, CHO retrovirus particles are present in the HCCF andprotein A pools, which can be measured using the CHOretrovirus Q-PCR assay. The presence of the other viruses inthe samples did not appear to impact the measurement ofCHO retrovirus clearance.

These data support that X-MuLV is a representativemodel for the CHO retrovirus in evaluating virus removalby the capture protein A chromatography steps. Inaddition, it shows that CHO retrovirus removal by proteinA chromatography can be determined using samples fromvirus-spiked or non-spiked experiments.

Comparable CHO Retrovirus Clearance at Large Scaleand Small Scale

Small scale virus removal validation studies of protein Acolumns are designed to be representative of large scaleprocesses. Column load density, flow rate, bed height andphase duration are matched to simulate large scaleperformance to the extent achievable. Routine large scaleprocess parameters such as chromatograms and step yieldare monitored to ensure process consistency between thetwo scales. However, GMPs and common sense precludeintroduction of live viruses into a commercial manufactur-ing site. Thus, spiking virus into large scale runs isprohibited while small scale models of viral clearance havenever been demonstrated to be representative of large scale.

romatography (4 mAbs).

Clearance (LRV)

End of use protein A resin

X-MuLV spiked run

X-MuLV CHO retrovirus X-MuLV

2.2 2.9 2.3

4.1 �4.2b 4.1

4.1a

3.7 — —

2.1 — —

ion of the assay was used to calculate a minimum clearance value.



With the CHO retrovirus assay, demonstration of viralclearance at large scale is now possible.

HCCF and protein A pools from four mAb processes(mAb1b, 4, 5, and 6; Table III) were collected frommanufacturing, with production scales ranging from 100 Lto 12 KL. Clearance of CHO retrovirus at large scale wascomparable to those in model processes that had beenevaluated at small scale (mAb1b and mAb4 processes,Table II), and comparable to X-MuLV clearance in smallscale validation studies (Table III). MAb4 and mAb5 wereeach studied at two process scales, with comparable LRVs,indicating scale of the commercial processes had minimalimpact on CHO retrovirus clearance. Furthermore, sincesamples from two to four production lots were measuredwith consistent results, CHO retrovirus clearance wasconsistent from run to run.

Even though the retroviruses were inactivated during thelow pH hold step when protein A elution pools werecollected from manufacturing, the Q-PCR results were notaffected, at least in the cases of three mAbs (data not shown).Previous studies have also established that low pHincubation does not significantly degrade murine retrovirusgenomes or RT activity when they are protected withinintact capsid structures (Brorson et al., 2003b). Thus, ourstudies have evaluated virus separation by protein A only.

Consistent CHO Retrovirus Clearance in DesignSpace Study

In an attempt to evaluate the robustness of retroviralclearance by protein A step, we evaluated CHO retroviruslevels using samples from a characterization study. Smallscale characterization studies were constructed to be designof experiment (DOE) studies, where operational parametersare varied individually or in combination to study theimpact on unit operation performance (e.g., yield, impurityclearance, etc.). Using the genome Q-PCR assay, we wereable to investigate whether any of these operational

Table III. Clearance by protein A chromatography at production scale

(CHO retrovirus) and small scale (X-MuLV) for 4 mAbs.

Molecule

Production run

scale and number

Clearance (LRV)

Production scale

CHO retrovirus

Small scale

X-MuLV

MAb1b 12 KL, Run1 2.2 2.2

12 KL, Run2 3.1 2.3

MAb4 400 L, Run1 2.4 2.1

100 L, Run1 2.4

100 L, Run2 2.0

MAb5 12 KL, Run1 2.0 2.0

12 KL, Run2 1.9 2.4

12 KL, Run3 2.1

400 L, Run1 2.7

MAb6 400 L, Run1 2.1 2.5

400 L, Run2 2.3

400 L, Run3 2.3


parameters impact CHO retroviral clearance. By doing so,we were also able to test the concept of design space for viralclearance by this unit operation for the first time. In this setof experiments, four HCCF feedstocks from the sameproduct, mAb1b, were used. Twelve target runs, where allprocess parameters were kept at the operating target setpoints, were performed throughout the study providinginformation about the range of normal variability, includingdifferent feedstock lot usage. In the experimental runs, mAbload density, load/elution/wash flow rates, wash duration,pooling criteria and HCCF temperature were varied fromthe target conditions. To confirm observations obtainedwith the CHO retrovirus Q-PCR assay, a second assay, Q-PERT, was used to quantify reverse transcriptase activity, aseparate component of endogenous retroviruses. The Q-PERT assay has a higher coefficient of variation than the Q-PCR assay, but results from the two orthogonal assaysgenerally correlate with each other (Brorson et al., 2002b).

In the target runs, modest feedstock differences and assayvariability contributed to the variation in LRV (Fig. 1a andb, measured by both assays). Both assays gave LRVs of 2.4–3.9 log10 for the target runs.

Overall, the challenged parameters had little or no impacton LRV that would be significant from a manufacturingstandpoint (Fig. 2). Both the average values and the ranges ofLRV are similar with target runs and runs with variedparameters. All LRVs were above 2.3 log10. Even though thedifference of LRV among the runs was up to 1.5 log10, mostruns had LRV of around 3.1 log10 (Fig. 2).

The set of challenged parameters (varied parameters)included load density, load/elution flow rate, wash flow rateand phase duration, pooling and HCCF temperature(Fig. 3). The DOE was designed to vary these factors inseveral segments. In one of the segments, load density,load/elution flow rate and equilibration phase duration wereinvestigated for potential interaction in a full-factorialdesign. The three factors in this segment were variedsimultaneously using the same feedstock, feedstock A, andsamples from this segment were evaluated in a single assay.Thus, data from this segment allowed a formal regressionanalysis, without feedstock and assay variation. The analysisinvestigated the individual and combined effects of thesethree factors. Analysis detected statistically significant effectsof load density and load/elution flow rate on LRV( P< 0.05), but these effects were too small to be ofpractical importance. The estimated effect of increasing loaddensity from the low level to the high level was 0.4 log10. Theestimated effect of increasing load/elution flow rate from itslow level to its high level was 0.5 log10. Both effects are smallchanges, 13% and 16% respectively, compared to theaverage LRV at target of 3.0 log10 (Fig. 3a and b). Analysisdid not detect either an effect of changing equilibrationphase duration or interactions among the three factors.

None of the other parameters, including wash flowrate and duration, pooling and HCCF temperature wereobserved to produce substantial impacts on LRV, eventhough each parameter was tested under a wide operational

Figure 2. Impact of operating conditions on CHO retrovirus clearance by protein A chromatography. LRV derived from runs at target condition or with varied parameters,

including load density, load/elution/wash flow rate, wash durations, pooling criteria and HCCF temperature. Data are expressed as a variability chart of the LRVs of target protein A

conditions versus all other experiments combined (i.e., a measure of the degree of the variability of LRV of this unit operation), (a) measured by CHO retrovirus assay or (b) measured

by Q-PERT assay.

Figure 1. Effect of mAb1b feedstock lots on CHO retrovirus clearance by protein A chromatography. Data are expressed as a variability chart; results from individual

experiments in the same category are graphed as points, mean and standard deviations are lines and brackets. a: LRV using four feedstock lots, A, B, C, and D. All other conditions

were identical as target conditions. LRV determined by CHO retrovirus Q-PCR assay. b: CHO retrovirus titers in load HCCF and in elution pools. c: Same as in (a), LRV determined by

the Q-PERT assay. d: Reverse transcriptase (RT) activity in load HCCF and in elution pools.



Figure 3. CHO retrovirus clearance by protein A using samples from characterization study. Data are expressed as a variability chart of the LRVs of target protein A

conditions versus runs under wide ranges of specific operating parameters, including: (a) load density (g/L), (b) load and elution flow rate (CV/hr), (c) wash flow rate (CV/hr),

(d) wash phase duration (CV), (e) pooling criteria (CV), and (f) HCCF temperature (degrees).

range (Fig. 3c–f). In summary, the conditions in ourexperimental matrix had at most small impacts on CHOretrovirus LRV. None of the conditions tested led to a failureof CHO retrovirus clearance, even when multiple para-meters were varied simultaneously away from the targetsettings. These results suggested a very large operationalrange for retroviral clearance by protein A chromatography.

Consistent CHO Retrovirus Clearance Using ReusedResin

Samples from a routine small scale resin lifetime study formAb1b were also analyzed by the CHO retrovirus Q-PCRassay. Protein A elution pools were analyzed every 10 cyclesfor up to 250 cycles. Because of the volume of process fluidsinvolved with cycling studies, six HCCF lots were used as


feedstocks. Parameters in this study were held constant foreach cycle. Consistent LRV was observed throughout thelifetime of resin, up to 250 cycles (Fig. 4). The same generaltrend was observed in the data from Q-PCR and Q-PERTassay, although the results from the Q-PERT assay hadhigher variation. The clearance values measured by the Q-PCR assay should be viewed as a more precise measure of theprotein A viral clearance capacity. The overall observation ofthe robustness of viral clearance by protein A chromato-graphy with extended reuse is in agreement with previousstudies (Brorson et al., 2003a).

Discussion

The CHO retrovirus Q-PCR assay was first developed by deWit et al. (2000) to quantify the amount of CHO retrovirus

Figure 4. Impact of resin reuse on LRV by protein A chromatography. CHO particle measured LRV every 10 or 20 cycles from cycle #1 to 250. A total of six feedstocks (E, F, G, H,

I, and A) were needed to complete the experiment. LRV measured using (a) CHO retrovirus assay and (b) Q-PERT assay.

in a dose-equivalent volume of harvest (Brorson et al.,2003b). Even though the assay has been previously shown toaccurately measure CHO retrovirus titers in harvest samplesor HCCF, this is the first report demonstrating that it cansuccessfully be used to evaluate retrovirus clearance by achromatography step in actual large scale manufacturing. Inthis report, we demonstrate the feasibility of this novelapproach, evaluate potential applications, and discuss itsadvantages and potential concerns.

Retrovirus removal by protein A chromatography istypically validated by small scale X-MuLV spiking studies.This model virus is chosen based on phylogenetic similarityto CHO retrovirus, the actual virus particle of concern.However, there have been reports showing that for someviral inactivation/removal steps, a model virus may becleared at a different efficiency than the actual virus ofconcern (Blumel et al., 2002). ICH Q5A cautions thatsimilarities and differences between model and target virusesshould be taken into consideration when spike study resultsare interpreted. In this report we show that removal of CHOretrovirus present in HCCF is comparable to that of X-MuLV in traditional validation studies. These data supportthe use of X-MuLV as an appropriate model virus forCHO retrovirus to evaluate viral removal by the protein Achromatography step. Consistent CHO retrovirus clearancewas obtained in small scale experiments where zero to threemodel viruses was/were spiked in HCCF as load to thecolumn. CHO retrovirus removal is the actual parameter ofinterest in bioprocessing. Thus, measuring CHO retrovirusremoval in small scale experiments, with or without anadventitious virus spike, is a more real assessment ofendogenous retrovirus clearance than a X-MuLV spikestudy.

Small scale validation studies are designed to be relevantto large scale manufacturing. Because of their expense andcomplexity, usually only target conditions are consideredand modeled. It has also never been formally established that

the small scale studies are fully representative of large scale.In this report we show that retroviral clearance measured atlarge scale is comparable to that at small scale. Our dataargue that (1) traditional X-MuLV spike studies, (2) directCHO retrovirus removal measurement at small scale, and(3) direct CHO retrovirus removal measurement duringproduction are all valid approaches to determine retrovirusremoval by capture protein A step.

Direct measurement of CHO retrovirus removal atproduction scale possesses many advantages relative to amodel virus spiking studies. Spiking studies are expensive,time consuming, and need to be performed in specializedvirology labs. This new approach measures the removal ofthe actual virus of concern under real manufacturingconditions instead of a model virus. The Q-PCR assay isinexpensive and provides high throughput evaluation, andcan be incorporated to the in-process testing programduring clinical campaigns or qualification batches forcommercial manufacturing. Thus, we can quickly gatherinformation concerning retroviral removal by protein A stepand overall viral safety during actual manufacturing.

The design space concept, proposed in ICH Q8, involvesstudying the effects and interactions of process parametersand defining an operational space of these multipleparameters which assures product quality. Changes withinthe space would be considered to have no impact onsubsequent quality attributes of the products and would becandidates for reduced regulatory reporting requirements.Even before ICH Q8, we have conducted conceptuallysimilar modular studies on other operating units, such asflow through anion exchange chromatography (Curtiset al., 2003) and low pH inactivation of retrovirus (Brorsonet al., 2003b). We were able to successfully establish‘‘design spaces’’ for these two steps because of their relativesimplicity.

Protein A chromatography, the most common capturestep in purification of monoclonal antibodies, is somewhat



more complex due to the complex nature of the feedstockand the larger number of process parameters. For example,CHO retrovirus load is cell line dependent and to someextent cell culture process dependent (Brorson et al., 2002a).Thus, the application of a modular approach for protein Achromatography is a larger technical challenge. Defining adesign space for the protein A step for a specific mAb by thetraditional X-MuLV spiking approach could involve morethan 50 small scale column runs—an impractical, costly andtime-consuming study. By instead measuring the CHOretrovirus removal using samples already obtained from aprotein A process characterization study, we were able tobypass these technical hurdles. Process characterizationstudies are normally designed to evaluate the impact ofprocess parameters on yield and impurity removal, but inthis case we leveraged existing samples and a new assay toalso measure CHO retrovirus clearance. Analysis of datafrom this experiment did not detect any important effects onthe LRV of retrovirus removal from parameter variationswithin ranges tested: parameters varied included mAb loaddensity, load/elution/wash flow rate, wash duration, poolingcriteria, and HCCF temperature. Since none of the extremeconditions tested led to a clearance failure, CHO retrovirusremoval by protein A step is robust within wide ranges ofoperational conditions.

When evaluating CHO retrovirus removal during resinreuse, we found that multiply cycled resins also performedequivalently to naı̈ve resins. Historically, viral clearance byend-of-use resins has been investigated for to-be-marketedproducts. This is because the time and cost is not justified forclinical products where the media are product dedicated andhave a limited number of cycles. Using the CHO retrovirusQ-PCR assay, we can easily evaluate the dynamic rangeof retroviral clearance during the actual lifetime of theresin. Normal process events like column repacking, resinregeneration/storage conditions, etc. will then be incorpo-rated into resin evaluation.

The two orthogonal assays, CHO retrovirus Q-PCR assayand Q-PERT assay, measure different components ofretrovirus particles, genomic RNA and RT enzyme activitylevels. The clearance results obtained using both assays, inboth the process characterization and resin reuse studieswere similar. Overall, the combined data provide confirma-tion that protein A is a robust virus removal step (Brorsonet al., 2003a; Valdes et al., 2002).

Thus, this technological advance enables design spacesand resin lifetime limits for this and possibly otherpurification steps, supporting wider operational rangesand providing coverage for unexpected manufacturingexcursions without the need for additional validationstudies. This Q-PCR method can be treated like any otheranalytical method; it can routinely monitor product qualityand impurity levels, and retroviral clearance may be addedto the routine profile of in-process product safety testing forearly recovery processes. This approach is an importantadvance for process development and optimization as wellas product safety. CHO retrovirus removal data can be


quickly generated from protein A process developmentexperiments to screen resins, wash buffers and other processconditions without performing virus spike studies. It canintegrate the viral safety aspects simultaneously with otherimportant process factors to determine the best possibleprocess for production.

In summary, using Q-PCR methods allows a directassessment of retroviral clearance by large scale protein Acolumns. Because of the rapid and high throughput natureof Q-PCR assays, it is now practical to establish designspaces of process parameter ranges that ensure robust viralclearance. These design spaces will likely prove critical tosupport BLA ranges and mitigate manufacturing excursions.

The authors thank Denise Korbe and Janice Chen for X-MuLV

clearance data, Philip Lester, Robert Van Reis for valuable discussions,

and thank Daniel Strauss for manuscript preparation.

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