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Retrospective Evaluation of Cycled Resin in Viral Clearance

Studies−−A Multiple Company Collaboration
John Mattila, Sherrie Curtis, Yenny Webb-Vargas, et al.

PDA J Pharm Sci and Tech 2019, 73 470-486

Access the most recent version at doi:10.5731/pdajpst.2018.009605
 Downloaded from on January 21, 2020

RESEARCH

Retrospective Evaluation of Cycled Resin in Viral Clearance

Studies—A Multiple Company Collaboration
JOHN MATTILA1, SHERRIE CURTIS2, YENNY WEBB-VARGAS2, EILEEN WILSON3, OLGA GALPERINA4,
DAVID ROUSH5, SCOTT TOBLER5, BRADFORD STANLEY6, MIKE CLARK7, JUSTIN WEAVER8,
JEREMY PIKE8, DEQIANG YU9, XINFANG LI10#, ANDREAS FLICKER11, JOHANNA KINDERMANN11,
NORBERT SCHUELKE12, RICHARD WHITCOMBE13, and LOUISE BENNETT14,*
1
Regeneron Pharmaceuticals Inc., Tarrytown, NY; 2Genentech, Inc., South San Francisco, CA; 3GlaxoSmithKline plc,
King of Prussia, PA; 4GlaxoSmithKline plc, Rockville, MD; 5Merck & Co., Inc., Kenilworth, NJ, USA; 6Biogen, Research
Triangle Park, NC; 7AbbVie Bioresearch Center, Worcester, MA; 8Alexion Pharmaceuticals, Inc. Boston, MA;
9
Bristol-Myers Squibb, Devens MA; 10ImmunoGen, Inc. Waltham, MA; 11Shire plc, Lexington, MA; 12Takeda
Pharmaceuticals International Co., Cambridge, MA; 13UCB, Brussels, Belgium; and 14BioPhorum Operations Group,
London, U.K. © PDA, Inc. 2019
ABSTRACT: The BioPhorum Development Group Viral Clearance Workstream performed a collaborative retrospective
analysis to evaluate packed bed chromatographic resin performance after repeated cycling for two commonly used
chromatography steps in biopharmaceutical manufacturing: protein A and anion exchange. Key variables evaluated in
the assessment included virus type, resin type, number of reuse cycles, and virus challenge. In this retrospective analy-
sis of viral clearance data on naı̈ve versus cycled resin, powered by the availability of a decade’s worth of accumulated
industry data, clearance capability was not negatively impacted by resin cycling. This finding is consistent with publica-
tions showing that surrogates for viral clearance capabilities could be employed in lieu of testing the viral clearance of
cycled resins for protein A and anion exchange chromatography. The rigorous analysis of the retrospective data sup-
ports the view that viral clearance studies for cycled resins are not necessary provided that appropriate cleaning meth-
ods are applied during repeated use of the chromatography columns.

KEYWORDS: Viral clearance, Packed bed chromatography, Resin reuse, Protein A, Anion exchange.
LAY ABSTRACT: The manufacturing processes for biopharmaceutical products often include reusable chromatographic
resins that remove process- and product-related impurities as well as potential contaminating viruses. Typically, chro-
matography resin is “cycled” through repeated steps of resin conditioning, product purification, and resin cleaning. The
cycling approach has been evaluated in both small- and full-scale studies that show the performance parameters are
maintained. The ability to remove virus is demonstrated separately in a focused small-scale virus-spiking study that is
resource-intensive and costly. This paper is a retrospective review of industry data comparing virus removal by naı̈ve
and repeatedly cycled resins that summarizes the viral clearance impact of re-using protein A and anion exchange chro-
matography resins. The key variables evaluated in the assessment included virus type, resin type, number of cycles, and
virus challenge. In this retrospective analysis, it was found that the viral clearance capability is not negatively impacted
by resin cycling. This finding is consistent with other publications and supports the view that viral clearance studies for
cycled resins are not necessary if appropriate cleaning methods are applied during the repeated use of the chromatogra-
phy columns.

Abbreviations: AAV-2, Adeno-associated virus; A-MuLV, Amphotropic murine leukemia virus; AEX, Anion-
exchange chromatography; B/E, Bind and elute; BVDV, Bovine viral diarrhea virus; C.P.G., Controlled pore
glass; DEAE, Diethylaminoethanol; EMCV, Encephalomyocarditis virus; FT, Flow through; HAV, Hepatitis A vi-
rus; HSV-1, Herpes simplex virus type 1; LOD, Limit of detection; LOQ, Limit of quantification; LRF, Log10

* Corresponding Author: BioPhorum Operations Group, London, U.K.; e-mail: bennettl@biophorum.com

# Present address: MabPlex International Co., Ltd, Yantai, Shandong, China
doi: 10.5731/pdajpst.2018.009605

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reduction factor; mAb, Monoclonal antibody; MVM, Minute virus of mice; NaOH, Sodium hydroxide; PA, Protein
A; PPV, Porcine parvovirus; QA, Quaternary amine; QP, Quaternized polyethyleneimine; qPCR, Quantitative po-
lymerase chain reaction; Reo3, Reovirus type 3; SuHV-1, Suid herpesvirus; SV40, Simian virus 40; X-MuLV,
Xenotropic murine leukemia virus

Background are often performed early in product development to

support clinical trial applications, whereas studies on
Viral safety is one of several measures in place to cycled resin are often performed months or years later
ensure the safety of biotechnology products derived to support marketing applications. Resin-cycling stud-
from mammalian cell lines and intended for therapeutic ies often do not include a contemporaneous comparison
use. Rodent cell lines may contain endogenous retrovi- of naı̈ve and cycled resin because that requires costly
ruses or retrovirus-like particles as measured by trans- reevaluation of naı̈ve resin in parallel with the evalua-
mission electron microscopy (1). Furthermore, cell tion of cycled resin. Within this analysis, evaluations
cultures may become contaminated by adventitious of naı̈ve and cycled resins that occur at different times
viruses introduced through raw materials or handling (that is, not in parallel) have been identified as “non-
(2, 3). To contribute to the safety of biopharmaceuti- concurrent,” as this study design may add variability
cals, purification processes include multiple orthogonal
(e.g., virus stock purity, virus stock titer, virus assay
virus reduction steps, and there are regulatory require-
conditions) that can confound the comparison of naı̈ve
ments to demonstrate the ability of the purification pro-
and cycled resin.
cess to remove or inactivate a wide variety of potential
viral contaminants in investigational and commercial
Extensive industry data regarding the impact of chro-
products (4–7).
matography resin cycling on viral clearance exists but
Chromatography steps contribute to the cumulative vi- not in an accessible, collated format. Individual firms
ral clearance claimed for many biopharmaceutical puri- often have experience with fewer products with limited
fication processes. Chromatography resins are routinely variability in process conditions. Regulatory agencies
reused for many discrete batches produced over months have access to cycled resin viral clearance data submit-
or even years while performance and product quality ted with marketing applications but often with limited
are maintained with an appropriate resin cleaning re- virus study information. Contract laboratories conduct-
gime. For chromatography steps that are included in vi- ing viral clearance studies have data from a variety of
ral clearance claims, the impact of “repeated use” must clients’ studies but often have limited process informa-
be understood (7). The naming convention for chroma- tion. Some published reports have indicated that chro-
tography resin reuse differs across firms, but this study matography resins that have not shown performance
uses “cycled resin” to denote chromatography resin that decline for other attributes with cycling also do not
has been exposed to repeated cycles of use with expo- show a decline in viral clearance. This has been reported
sure to product and to process solutions including clean- for protein A chromatography and anion-exchange chro-
ing solutions. The age of the resin, with respect to the matography (AEX) resins, and additional experience
resin manufacturing date, is not considered in this retro- with multiple chromatography resins has been reported
spective evaluation. Naı̈ve resin refers to resin that has by individual firms (8–14).
not been exposed to virus and has been exposed to few
or no product cycles. The retrospective analyses presented here result from a
rigorous statistical analysis of a large data set compiled
Firms have addressed the regulatory guidance by com- by multiple individual firms. Data from studies repre-
paring the viral clearance for model viruses on naı̈ve senting real-world variation in product type, molecule
and cycled resin for individual products and processes. isoelectric point, load impurity profile, resin ligand and
Cycled resin viral clearance studies are resource-inten- back bone, surface chemistry, resin loading, and resin
sive and costly. Generating resin that has been exposed cleaning, as well as virus challenge, have been eval-
to sufficient repeated cycles to simulate a resin lifetime uated. Data were collected over multiple decades. This
may take 1 to 6 months and typically requires hundreds analysis contributes to an evidence-based discussion of
of grams of recombinant protein even at a laboratory whether cycling of resins has a detrimental impact on
bench scale. Viral clearance studies with naı̈ve resin the viral clearance.

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TABLE I
Rules Established to Allow Direct Comparison of Viral Clearance by Naı̈ve and Cycled Resins

Rule Rationale
Rule 1: Result must not use infectivity assay for Viral clearance by protein A chromatography can result from
determination of enveloped virus titer during two components: virus inactivation by acidic elution, and
protein A chromatography virus removal by partitioning. The infectivity assay cannot
differentiate between enveloped virus inactivation and virus
removal, so qPCR should be used.
Rule 2: The difference in load viral challenge The virus amount in the load affects the experimental viral
between the naı̈ve and the cycled resins must be clearance results.
<1.0 log10

Materials and Methods pool, V load is the load volume, and V pool is the pool
volume.
Data Source
The firms indicated whether the naı̈ve and the cycled
A database was constructed using blinded survey resins were tested concurrently or nonconcurrently as
responses from 12 biotechnology companies including described previously. Data were a representative sam-
AbbVie Bioresearch Center (Worcester, MA), Alexion ple of viral clearance studies performed to support
Pharmaceuticals, Inc. (Boston, MA), Biogen (Research product licensure but did not include all historical data
Triangle Park, NC), Bristol-Myers Squibb (Devens MA), generated by the 12 firms. The database did not capture
GlaxoSmithKline plc (King of Prussia, PA), Glaxo- the impact of resin cycling on process performance
SmithKline plc (Rockville, MD), ImmunoGen, Inc. (Wal- except for the ability to remove model viruses. No
tham, MA), Merck & Co., Inc., Kenilworth, NJ, USA, results were excluded owing to the reduced perform-
Regeneron Pharmaceuticals Inc. (Tarrytown, NY), Gen- ance of the cycled resin compared to the naı̈ve resin,
entech, Inc. (South San Francisco, CA), Shire Plc (Lex- and the data are consistent with the published capabil-
ington, MA), Takeda Pharmaceuticals International Co. ities of these manufacturing steps (15).
(Cambridge, MA), and UCB (Brussels, Belgium). Protein
A and anion-exchange chromatography steps were sur- The results were categorized as “mAb” for monoclonal
veyed to provide a large data set of viral clearance for antibody-based processes and “non-mAb” for all other
commonly used manufacturing steps. Conversely, imple- recombinant proteins to assess class effects. Recombi-
mentation of other chromatographic technologies such as nant protein, product, and molecule are all used to refer
cation exchange, hydrophobic interaction, and mixed- to mAb and non-mAb as appropriate. Model viruses
mode chromatography differ widely among firms and were reported, although the strain and preparation
were not included in this analysis. method were not in the scope of this analysis. The
clean-in-place (CIP) solution was reported for the AEX
Firms contributed retrospective GLP-compliant viral resins. Process parameters such as column loading and
clearance data spanning decades. The database was CIP solution contact time were disclosed for some, but
comprised of “paired observations”, where firms not all, paired observations based on the disclosure pol-
reported the clearance for both naı̈ve and cycled resins icy of the firms.
for a given virus. Virus log10 reduction factor (LRF)
Analysis
was calculated and reported by contributors as
described in eq 1.
Data were compiled and blinded by the BioPhorum De-
Cvirus;load  Vload velopment Group Viral Clearance Workstream facilita-
LRF ¼ log10 (1)
Cvirus;pool  Vpool tor and imported into SAS JMP v11.1.1 for statistical
analyses. Results were filtered using two rules to pro-
vide a representative data set (Table I). Rule 1 assured
Where C virus, load is the virus concentration in the that the mechanism of viral clearance on protein A was
load, C virus, pool is the virus concentration in the physical removal rather than inactivation during

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putative low pH elution (16). Rule 2 assured that the enveloped and nonenveloped viruses. The data set was
virus challenge was within 61.0 log10 for the spiking reduced by the filter rules in Table I to prevent assess-
studies of naı̈ve and cycled resin comprising a paired ment of enveloped virus inactivation and to ensure a
observation, as this was considered a comparable eval- similar virus challenge among paired observations.
uation based on cumulative experimental and assay Figure 1A illustrates the application of Rules 1 and 2.
variability (8). This limitation was necessary because Four paired observations were excluded owing to the
the virus challenge in the load may influence the dem- use of the infectivity assay with enveloped virus,
onstrated virus removal by the step either by overload- whereas 11 paired observations were excluded because
ing of the resin, underloading of the resin, or the the virus challenge in the naı̈ve and the cycled resin
condition in which the LRF is limited by the limit of differed by >1.0 log10. As the virus challenge was not
quantification (LOQ) of the assay. reported for a further 19 paired observations, they
could not be confirmed to conform to Rule 2; therefore,
Change in performance after repeated use was re- they were excluded from the analysis. After the filter
ported as the difference in LRF within a paired obser- rules described in Table I were applied, a total of 97
vation. Zero difference indicates that there is no differ- paired observations remained. The remaining data
ence in virus removal between the cycled resin and the represented protein A processes for sixteen mAbs and
naı̈ve resin. A positive difference indicates that the eight non-mAbs. Figure 2 shows that the model virus
cycled resin removes more virus than the naı̈ve resin. families included in the analysis offer diverse physico-
Alternatively, a negative difference shows that the chemical properties as described in Table II.
cycled resin removes less virus than the naı̈ve resin. A
change <1 log10 was not considered practically mean- The resins were classified by their base matrix and
ingful as implied by the published guidance describing caustic stability, including: caustic stable agarose, non-
a reduction in virus titer on the order of 1 log10 as neg- caustic stable agarose, and noncaustic stable controlled
ligible (7). pore glass (C.P.G.). The source of the protein A ligand
was not considered in this analysis. Most of the obser-
It was hypothesized that effective removal by the naı̈ve vations in this analysis are from agarose resin that is
resin may correlate with robust performance after not stable to caustic solutions.
repeated cycling. As such, the clearance ability for the
naı̈ve resin was categorized as: The selected process parameters included the number
of product cycles and the product loading (gram prod-
1. <1.0 LRF: Step does not provide reliable removal of uct per liter resin). Figure 3 shows the distribution of
the model virus. product cycles and the product loading for the paired
observations. The detailed process conditions were
2. 1.0 LRF  x < 4.0 LRF: Step contributes to virus re- beyond the scope of this study; however, the processes
moval although the mechanism does not provide from the 12 biotechnology firms include a breadth and
effective clearance. diversity of buffer matrices, phase durations, and wash
strategies.
3. 4.0 LRF: Step is considered effective for virus re-
moval, and the mechanism is robust. Viral Clearance by Protein A: The variation observed
in this data set is consistent with previously published
Results literature in which the LRF by Protein A is typically
between 1 LRF and 4 LRF, but on some occasions,
Protein A Chromatography Resin there is <1 LRF or >4 LRF (15–18). Figure 4 shows
that it was most common for the naı̈ve protein A resin
Description of Protein A Paired Observations: The to have between 1 and 4 LRF across the six virus fami-
submitted, blinded, and compiled results included 131 lies and the resin physical properties.
paired observations comparing viral clearance by naı̈ve
and cycled resin for protein A chromatography. The Figure 5 compares the change in LRF for paired obser-
ability of the protein A resin to remove virus was deter- vations by resin type for all virus families. Table III
mined by each company’s standard practices. Six virus summarizes the comparison of the clearance within
families were represented, and the data set included paired observations by resin type within the context of

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Figure 1

Application of filter rules for protein A chromatography and AEX. (A) Protein A chromatography and (B)
AEX. Positive numbers correspond to increased load viral challenge in the cycled resin compared to the naı̈ve
resin. Green (*) denotes paired observations excluded owing to Rule 1. Red denotes paired observations
excluded owing to Rule 2. Black denotes paired observations included in the analysis.

concurrent or nonconcurrent testing. For the agarose stable, the cycled resin LRF is either comparable to or
resin that is caustic stable, the change in the LRF better than the naı̈ve resin for all 28 of the paired obser-
between the cycled and the naı̈ve resin is within the vations. Within this data set, performance changes
generally accepted experimental variability of 1.0 LRF. exceeding 1.0 LRF occurred disproportionately more
For the two observations exceeding the 1.0 LRF differ- often when paired observations were tested non-concur-
ence, one demonstrated increased clearance and one rently rather than concurrently. Among protein A exam-
demonstrated decreased clearance. Both were tested ples where the difference in the clearance between the
nonconcurrently. For the agarose resin that is not caus- naı̈ve and the cycled resin was >1 LRF, 16 of 20 relied
tic stable, the cycled resin LRF is either comparable to upon a qPCR assay. This suggests greater variability ei-
or better than the naı̈ve resin for 47 of the 48 paired ther for enveloped viruses or for the qPCR assay, as the
observations. For the C.P.G. resin that is not caustic enveloped virus quantitation was collinear with qPCR
in this data set. As shown in Figure 5, the LRF for the
cycled resin was generally comparable to or better than
the LRF of the naı̈ve resin, even when the clearance
mechanism was not “effective” (i.e., <4.0 LRF).

The effect of the product cycles and the total product

loading on the change in virus removal was assessed
statistically at 0.05 significance level. Figure 6 shows
the number of product cycles does not have a significant
effect on the change in viral clearance over the resin
Figure 2 lifetime. The lack of effect is illustrated by the shaded
95% confidence region of the fitted line that encom-
Number of paired observations included for each passes the horizontal line representing the mean change
virus family tested with cell-based infectivity assay in the LRF. Not all firms were permitted to disclose the
and qPCR assays for protein A chromatography. gram product per liter resin required to calculate the
Herpesviridae and retroviridae clearances eval- total product loading, so a subset of the data was ana-
uated by the infectivity assay were not included lyzed and demonstrated no significant effect on the dif-
owing to the potential inactivation of the enveloped ference in clearance achieved by the cycled and the
virus by the putative low pH elution. naı̈ve resin (data not shown).

Vol. 73, No. 5, September--October 2019 5

6
TABLE II
Model Viruses Used in Spiking Studies

Family Virus Name Isoeletric Point Size (nm) Envelope Genome Shape Used for PA/AEX
Flaviviridae Bovine viral diarrhea virus Not reported 50–70 Yes RNA Spherical AEX
Herpesviridae Herpes simplex virus 5.5–6.3a 120–200 Yes DNA Spherical PA/AEX
Suid Herpesvirus Not reported 120–200 Yes DNA Spherical PA/AEX
Reoviridae Reovirus type 3 3.8b 60–80 No RNA Spherical PA/AEX
Parvoviridae Adeno-associated virus 6c 18–24 No DNA Icosahedral PA/ AEX
Murine parvovirus 6.2d 18–24 No DNA Icosahedral PA/AEX
Porcine parvovirus 5.1–6.0e 18–24 No DNA Icosahedral PA/AEX
Picornaviridae Encephalomyocarditis virus Strain dependentf 25–30 No RNA Icosahedral PA/AEX
Hepatitis A virus 2.8f 27–32 No RNA Icosahedral PA
Polyomaviridae Simian virus 40 5.4d 40–50 No DNA Icosahedral PA/AEX
Retroviridae Amphotropic murine leukemia virus 6.0g 80–130 Yes RNA Spherical PA/AEX
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Xenotropic murine leukemia virus 5.8d 80–110 Yes RNA Spherical PA/AEX
a
Reference 23.
b
Reference 24.
c
Reference 25.
d
Reference 19.
e
Reference 26.
f
Reference 27.
g
Reference 28.

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Figure 3

Number of product cycles and product loading for paired observations of protein A chromatography. (A) Dis-
tribution of resin cycles and (B) grams of product per liter resin loaded for protein A paired observations.
Note: column loading was not reported for all paired observations.

AEX Resins naı̈ve and cycled resins for AEX. The AEX processes
were described as operating in flow-through (FT)
Description of AEX Paired Observations: The sub- mode or bind/elute (B/E) mode. This description cor-
mitted, blinded, and compiled results included 166 responds to the state of the recombinant protein,
paired observations comparing the viral clearance by which either flows through the packed bed during the

Figure 4

Naı̈ve protein A and AEX resin LRFs by resin type or mode and virus type.

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Figure 5

Change in clearance over protein A resin lifetime as a function of resin type and clearance by naı̈ve resin. Sym-
bol (*) denotes the naı̈ve and the cycled resins tested nonconcurrently. Symbol (1) denotes the naı̈ve and the
cycled resins tested concurrently. Red denotes qPCR-based assay and black denotes infectivity assay.

load (FT) or binds and is subsequently eluted (B/E). (i.e., quaternary amine [QA]) ligand on varying back-
The ability of AEX to remove virus was determined bones. Non-mAb AEX was generally operated in B/E
by each company’s standard practices, and the data set mode, using a variety of combinations of ligands and
was reduced by a filter rule to ensure similar virus backbones.
challenge among paired observations. Figure 1B
shows application of Rule 2 for AEX. Fifteen paired The selected process parameters included the number
observations were excluded because the virus chal- of product cycles and the product loading (gram prod-
lenge in the naı̈ve and the cycled resin differed by uct per liter packed resin). Figure 9 shows the column
>1.0 log10, whereas seven paired observations were loading and the number of product cycles for both the
excluded because the virus challenge was not re- B/E and the FT AEX. When comparing the two modes,
ported. After applying the filter rules described in the B/E AEX features 2- to 10-fold lower product load-
Table I, a total of 144 paired observations remained. ing than the FT, although both are routinely cycled 50–
The remaining data represented AEX processes for 150 times. The AEX operating conditions are typically
17 mAbs and 11 non-mAbs. Figure 7 shows the dis- dependent on the recombinant protein isoelectric point,
tribution of the model virus families included for and the AEX data set included products with a broad
the B/E and the FT modes. Flaviviridae and polyo- distribution of isoelectric points ranging from 4.2 to
maviridae were evaluated exclusively with the B/E 9.5. The detailed process conditions were beyond the
processes. A relatively similar number of paired scope of this study; however, the processes from the 12
observations for the B/E mode (75) and the FT mode biotechnology firms include a breadth and diversity of
(69) were reported. buffer matrices, pH, conductivity, phase durations, and
product collection strategies.
The physical properties of the resin included the combi-
nation of the resin backbone (i.e., agarose, methacry- Viral Clearance by AEX: The performance by the na-
late, or polystyrene divinylbenzene) and the functional ı̈ve resin in this data set is shown in Figure 4. Viral
group (i.e., diethylaminoethyl or quaternary amine) are clearance was consistent with previously published
shown in Figure 8. For mAbs, the AEX resin was gener- data in which the LRF is typically >4 log10 for the FT
ally operated in FT mode using a strong anion-exchange mode whereas the B/E mode was more dependent on

8 PDA Journal of Pharmaceutical Science and Technology

 TABLE III
Summary of the Viral Clearance in the Paired Observations Using Concurrent or Non-Concurrent Testing Approaches

Protein A (Number of Paired Observations) AEX (Number of Paired Observations)

Agarose, Not Caustic C.P.G., Not Caustic
Comparison of Agarose, Caustic Stable Stable Stable AEX B/E AEX FT
the Cycled and Non- Non- Non- Non- Non-
the Naı̈ve Resins Concurrent Concurrent Concurrent Concurrent Concurrent Concurrent Concurrent Concurrent Concurrent Concurrent

Vol. 73, No. 5, September--October 2019

The Cycled 1 0 7 1 4 5 1 0 6 1
Resin
Clearance
Exceeds the
Naı̈ve Resin
Clearance by
>1 LRF
The Cycled 12 7 21 18 4 15 56 18 34 26
Resin
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Clearance Is
Within |1|
LRF of the
Naı̈ve Resin
The Cycled 1 0 1 0 0 0 0 0 2 0
Resin
Clearance Is
Less Than the
Naı̈ve Resin
by >1 LRF

9
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Figure 6

Change in clearance over resin lifetime as a function of the number of cycles for protein A. Shaded gray is the
95% confidence region for the linear fit, whereas the red line is the mean. Symbol (*) denotes the naı̈ve and
the cycled resins tested nonconcurrently. Symbol (1) denotes the naı̈ve and the cycled resins tested concur-
rently. Red denotes qPCR-based assay and black denotes infectivity assay.
the virus type (15). Figure 10 compares the change in So far contributing firms have not seen product contact-
the LRF for the paired observations for AEX in the ing cycles or total mass loading impact the ability of
B/E and the FT modes according to the previously the resin to remove virus over the resin lifetime
described categories. The dashed lines represent a whether AEX is operated in the FT or the B/E mode.
change of 1.0 log10, which is the generally accepted The team evaluated the hypothesis that the number of
experimental variability. Table III summarizes the cycles has no impact on the change in clearance over
comparison of the paired observations within the con- the resin lifetime at 0.05 significance level, as shown in
text of concurrent or nonconcurrent testing. Regard- Figure 11. For B/E, there is no significant change in the
less of the performance of the naı̈ve resin and the clearance as a function of product cycles. There is a
mode of operation for AEX, the paired observations statistically significant increase in the clearance as a
typically provided clearance within 1 LRF (134 of 144 function of product cycles for the FT AEX, although
cases). For AEX resin operated in the B/E mode, the the apparent increase relies on instances where the vi-
only instance where the difference exceeded one LRF rus was reduced below the assay detection limit in the
was an increase in clearance capability. For AEX oper- cycled resin. The increased clearance in 5 of these 6
ated in the FT mode, the variation in LRF was within cases may reflect a change in the spiking study design
1.0 log10 in 60 of 69 of the paired observations, and (e.g., assay detection limit or load virus titer) associ-
there are two cases where a decrease in clearance ated with the nonconcurrent testing of the cycled and
exceeded 1.0 log10. In these two cases, the reduction in the naı̈ve resin rather than a change in the resin per-
the clearance exceeding 1.0 LRF correlates with non- formance. As was the case for protein A, it was not per-
concurrent testing. When the cycled AEX resin clear- missible for all firms to contribute detailed process
ance was not within 1 LRF of the naı̈ve resin, 90% (9 information such as column loading. The available data
of 10 cases) were tested nonconcurrently. were evaluated, and the total product loading had no

Figure 7

Number of paired observations reported for each virus family tested with B/E or FT AEX.

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Figure 8

Number of paired observations reported for AEX backbone, functional group, mode of operation, and product
class.
effect on the difference in the clearance achieved by each company using their standard practices. Protein A
the cycled and the naı̈ve resin at 0.05 significance level resins compatible with sodium hydroxide cleaning
(data not shown). showed no change in performance over the lifetime on
average (Figure 5). Protein A resins that do not tolerate
CIP Procedures caustic cleaning showed increased clearance for cycled
resin exceeding 1.0 log10 for cycled resin in 17 out of
The impact of the harsh resin cleaning regimens after 76 paired observations.
repeated cycles was evaluated for the protein A and
AEX resins based upon the vendor indication that the For the AEX resin, sodium hydroxide was used during
functionality may potentially be lost. This potential CIP for all paired observations, and the concentration
was assessed via the extensive data set contributed by was as high as 1.0 M NaOH. The mean change in

Figure 9

Product loading and the number of resin product cycles for paired observations for AEX. Column loading for
FT (A) and B/E (B) and the number of resin cycles for FT (C) and B/E (D). Note: column loading was not
reported for all paired observations.

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Figure 10

Change in clearance over AEX resin lifetime as a function of the operating mode and clearance by naı̈ve resin.
Symbol (*) denotes the naı̈ve and the cycled resins tested nonconcurrently. Symbol (1) denotes the naı̈ve and
the cycled resins tested concurrently. Red denotes the residual virus detected when testing the cycled resin,
whereas black denotes the virus reduced below assay detection limits when testing the cycled resin.
clearance was determined for the seven categories of 3. Sodium hydroxide combined with sodium chloride
cleaning solutions used for the B/E and the FT mode. with contact time either <1 h, or 1 h (2 categories).
categories included:

1. 0.1–0.5 M sodium hydroxide with contact time either The difference in the mean LRF was calculated for all
non-disclosed, <1 h, or 1 h (3 categories). pairs of cleaning solutions and ranged from 0.00 LRF
to 0.66 LRF for the B/E and the FT. The differences
2. 1.0 M sodium hydroxide with contact time either in means is less than the practical experimental varia-
non-disclosed or 1 h (2 categories). tion of 1 LRF associated with the studies. As such,

Figure 11

Change in clearance over resin lifetime as a function of the number of cycles for the AEX. Light shaded gray
denotes the 95% confidence region of the linear fit, whereas the red line is the mean. Symbol (*) denotes the
naı̈ve and the cycled resins tested nonconcurrently. Symbol (1) denotes the naı̈ve and the cycled resins tested

12 PDA Journal of Pharmaceutical Science and Technology

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Figure 12

Change in viral clearance during concurrent evaluation of the naı̈ve and the cycled chromatography resins.
Variability is reduced for protein A (A) and AEX (B) when paired observations are evaluated concurrently.

comparison of column CIP procedures encompassing a For caustic stable protein A, there is a single case
variety of sodium hydroxide strengths and contact where the clearance is reduced by >1.0 LRF (Figure
times do not support the view that the resin functional- 5). In this case, paired observations were evaluated
ity to remove virus is lost because of harsh cleaning nonconcurrently. Retrovirus clearance was measured
with these solutions. This retrospective review con- by qPCR and was 1.2 LRF lower in the cycled resin
cludes that the cleaning procedures listed above are than in the naı̈ve resin. Parvovirus and reovirus clear-
suitable for the wide variety of anion-exchange func- ances were within 0.5 LRF in the paired observations,
tional groups and resin backbones shown in Figure 8. suggesting that the change in the clearance was not
related to a change in the integrity of the resin.
Timing of the Evaluation for the Naı¨ve and the Cycled
For noncaustic stable agarose protein A, there is a neg-
Resins
ative difference exceeding 1.0 LRF for the cycled resin
in one case out of 48. The decreased LRF was observed
Many sources of variability are not controlled when the
for a single virus type (herpesviridae) measured by
naı̈ve and the cycled resins are tested nonconcurrently,
qPCR, whereas the clearances for four other virus types
with examples including: virus titer, virus lot, virus
tested with the same product and cycled chromatogra-
preparation impurity level, and assay detection limit.
phy resin were either within the accepted assay varia-
Many of these factors tend to be held constant when tion or increased. The clearance of herpesviridae by
the naı̈ve and the cycled resin are tested concurrently. the naı̈ve resin was 5.4 LRF, which exceeds the typi-
For protein A, 51 out of 97 paired observations were cal removal observed by protein A chromatography,
evaluated nonconcurrently. For AEX, 99 out of 144 but there was no justification to invalidate the result
paired observations were evaluated nonconcurrently. (15). In this case, the paired observations were eval-
Figure 12 demonstrates the reduced variability when uated nonconcurrently and there were many factors
the database was limited to concurrent evaluations of that were not held constant.
paired observations. Notably, this limited data set of
concurrent tests includes no cases where the clearance For the FT AEX, the two cases where clearance by
is reduced by >1.0 LRF in the cycled resin. Several the cycled resin was >1.0 LRF lower than the clear-
case studies illustrate the potential variability from the ance by the naı̈ve resin corresponded to nonconcurrent
nonconcurrent testing. testing (Figure 10). One case evaluated herpes virus

Vol. 73, No. 5, September--October 2019 13

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(1.16 LRF decrease in clearance for the cycled resin) not necessarily a demonstration of the chromatography
and the other case evaluated parvovirus (1.30 LRF step’s maximum ability to remove virus nor does it
decrease in clearance for the cycled resin). These reflect that either the naı̈ve or the cycled resin is better
were processes for different mAbs; one process used a or worse at removing virus.
strong ion exchanger on an agarose backbone, whereas
the other used a strong ion exchanger on a polystyrene Protein A chromatography resin is specifically de-
divinylbenzene backbone. signed to primarily bind the Fc portion of mAbs or the
Fc-fusion proteins, while impurities such as virus are
In one example for the FT AEX, the potential variabili- separated. Fc-containing products bind with high speci-
ty associated with the nonconcurrent testing corre- ficity to the protein A chromatography functional group
sponded to an unusual increase in the clearance by the during the load phase and most virus remains unbound.
cycled resin. In this case there appears to be a 3 LRF A portion of the virus associates nonspecifically with
increase in clearance by the cycled FT AEX resin com- the resin backbone, the resin ligand, or the Fc-contain-
pared to the naı̈ve resin. In this single observation, the ing product and may co-elute with product during the
cycled resin demonstrated a 7.5 LRF for minute virus elution phase, which is commonly acidic (18). Physical
of mice (MVM), whereas the naı̈ve resin achieved a removal of virus by protein A chromatography has
3.98 LRF. Evaluation of four other viruses with the been reported to be highly consistent for a given prod-
same product and process resulted in an LRF within uct but varying widely across products, suggesting that
1.0 log10 for the naı̈ve and the cycled resin. both virus–resin and virus–product interactions are re-
sponsible for virus adsorption and co-elution with the
Discussion product (16–18). When considering mechanisms for
potential retention of virus during the load phase, inter-
A virus-spiking study is a model for endogenous retro- actions between the virus and the cycled resin may
virus-like particles and/or a contamination event in decrease as binding sites are occupied or partially
manufacturing and is used to assess the viral clearance denatured. This retrospective analysis showed that in
achieved by the purification process and to assure the real-world applications, protein A chromatography was
virus safety of the product. Viral clearance is assessed robust for viral clearance, in that it removed virus to
by evaluating the amount of “spiked” model virus pres- the same level whether a naı̈ve resin or a cycled resin
ent in the load material and comparing it to the amount was used. For protein A resins that are cleaned with so-
of model virus present in the product pool (eq 1), result- dium hydroxide, the difference in the LRF between
ing in an LRF for that step. Reported LRF for spiking new and used resins was within 1.0 log10 in almost all
studies conducted in this manner can be impacted by observations (19 of 21). For protein A resins that are
the total virus challenge in the load material. For exam- not caustic stable (agarose or C.P.G.), the difference in
ple, a high virus challenge in the protein A chromatog- the clearance between the new and cycled resins was
raphy load may result in increased virus removal within 1 log10 in 58 of 76 cases. When paired observa-
because the majority of the virus flows through the pro- tions of noncaustic stable resin were not within 61
tein A resin, while a small, putatively constant quantity LRF, the clearance by the cycled resin exceeded the
co-purifies with the product (11, 16). Because the AEX clearance by the naı̈ve resin in 17 of 18 cases. A major-
often reduces the virus to nondetectable levels in the ity of these cases measured virus using qPCR (15 of
product pool, increased virus challenge may propor- 18). Overall, there are only 2 of 97 cases where the cy-
tionately increase the demonstrated LRF for this step as cling protein A resin decreases the clearance by >1
shown in eq 1 (15). In these examples, it is not the chro- LRF compared to the naı̈ve resin.
matography that is impacting the LRF, but rather the
experimental variability of the virus-spiking study. A systematic increase in the LRF was observed for pro-
Hence, Rule 2 was incorporated in the analysis of the tein A, especially for resins that cannot be cleaned-in-
data sets (reference Table I). These examples are espe- place with sodium hydroxide. For protein A resins that
cially likely to occur when the replicate runs are not are repeatedly cycled, potential changes to the ligand
conducted at the same time or under the same protocol, function, the ligand density, the ligand accessibility, or
which occurs with some regularity for the naı̈ve and the the base matrix may occur and result in increased virus
cycled resin. The resulting LRF from a virus-spiking flow-through during the load phase, thus increasing the
study may be limited by the total virus challenge and is viral clearance by the cycled resin. One potential

14 PDA Journal of Pharmaceutical Science and Technology

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mechanism to explain the increased clearance by the Conclusion and Recommendations

cycled protein A resin is the blocking of nonspecific
binding sites that may exist in the naı̈ve resin. A retrospective analysis of model viral clearance from
12 biotechnology firms spanning decades has shown
The AEX resin is typically positively charged over a that viral clearance capability of protein A and AEX is
wide range of pH. As such, a recombinant protein will not negatively impacted by resin cycling independent
either bind to the resin or flow through, depending on of the virus type, the resin type, the number of cycles,
protein isoelectric point. In either case, the separation the virus challenge, and the molecule isoelectric point
may be designed such that impurities, including (range 4.2–9.5). The difference in the viral clearance
viruses, are separated from the product based on differ- between the naı̈ve resin and the cycled resin was typi-
ences in electrostatic interactions with the positively cally no greater than the commonly accepted experi-
charged functional group (19). AEX viral clearance has mental variability of 1.0 log10. The variability of the
been reported to be highly consistent across products viral clearance test reagents (for example, virus stock)
when ionic interactions are maintained (i.e., same chro- and associated analytics (e.g., LOQ, precision) needs
matography resin, conductivity and salt composition), to be considered when assigning causality to a given
further supporting the view that separation is driven by parameter as these factors can be significant. The data
interactions between the virus and the functional group show a substantial source of variation is nonconcurrent
rather than by virus–product interactions (20). The evaluation of the naı̈ve and the cycled resins, which
analysis of paired observations in this data set was may confound lifetime effects with effects from the vi-
intended to show if the functional group degrades or rus titer, the virus purity, or the assay. Another source
becomes occupied after repeated cycling, and the anal- of variation for protein A chromatography is virus
ysis suggests that has not occurred. For the AEX resins assay by qPCR, which may be because of less mature
operated in the B/E mode, 74 of 75 paired observations qPCR practices than are the current standard in indus-
were within 1.0 LRF. For the AEX resins operated in try, as this extensive data set represents data collected
the FT mode, 60 of 69 of paired observations were over many years. If firms perform a virus-spiking study
within 1.0 log10. Overall, 142 of 144 paired observa- to determine that virus clearing ability is maintained
tions demonstrated no notable negative impact of cy- throughout the lifetime, the BioPhorum Development
cling on AEX. Repeated use of AEX resins had no Group Viral Clearance Workstream recommends:
negative impact on viral clearance capabilities when
appropriate cleaning techniques were applied, despite 1. The evaluation of the naı̈ve and the cycled resins
the marked differences in the feed streams, the func- with appropriate controls (e.g., virus titer, purity, ra-
tional group, the resin backbone, and the cleaning regi- tio of infectious to noninfectious virus, and assay),
men of the diverse data set. which may be accomplished by testing the naı̈ve and
the cycled resins concurrently for the ability to
Inherent to the spiking study is the laboratory proce- remove model viruses.
dures governing virus stock purity, titer, and assay in-
termediate precision. It was not feasible to account for 2. The use of well-designed qPCR assays to reduce the
this variability in our retrospective analysis. Our analy- variability of the determined LRF versus infectivity
sis identified paired observations in which clearance by assays (21).
the new and the cycled resins differed by >1.0 log10.
The vast majority of these observations were associated The findings of the retrospective review are consistent
with nonconcurrent evaluation of new and cycled res- with publications showing that product yield, product
ins and hence the measured difference in the LRF can- breakthrough, and column pressure provide indicators
not necessarily be attributed to the impact of resin of resin degradation that can be monitored in lieu of
cycling. In contrast, when concurrent evaluation of testing virus removal by cycled protein A chromatogra-
new and cycled resins was employed, the difference in phy resin (9–11). A similar approach has been sug-
the LRF between the new and the cycled resins was <1 gested for AEX, where in-process monitoring of DNA
LRF or not practically significant (reference Figure or host cell protein removal has provided demonstra-
12). Continued efforts to reduce the assay and the virus tion of maintained performance in place of direct
preparation variability are critical to reduce the overall evaluation of viral clearance by cycled resin (13, 14).
variability in spiking studies (21, 22). The BioPhorum Development Group Viral Clearance

Vol. 73, No. 5, September--October 2019 15

 Downloaded from on January 21, 2020

Workstream’s rigorous analysis of the retrospective 4. European Medicines Agency, Guideline on Virus
data supports the view that viral clearance studies for Safety Evaluation of Biotechnology Investigational
cycled resins are not necessary, if appropriate clean- Medicinal Products. EMA: London, 2008.
ing methods are applied during the repeated use of
chromatography columns. 5. European Agency for the Evaluation of Medicinal
Products, Note for Guidance on Virus Validation
Acknowledgements Studies: The Design, Contribution and Interpreta-
tion of Studies Validating the Inactivation and Re-
This article describes a consensus view from the Bio- moval of Viruses; CPMP/BWP/268/95; Committee
Phorum Development Group Viral Clearance Work- for Proprietary Medicinal Products; EMA: Lon-
stream. The authors sincerely thank the members of don, 1996.
the team for their contributions at monthly BPDG
discussions and in the preparation of this manuscript. 6. U.S. Food and Drug Administration, Points to
Consider in the Manufacture and Testing of Mono-
Since its inception in 2004, the BioPhorum has become
clonal Antibodies for Human Use; Center for Bio-
a trusted environment where senior leaders of the
logics Evaluation and Research; U.S. Department
biopharma industry come together to openly share and
of Health and Human Services: Rockville, MD,
discuss the emerging trends and challenges facing their
1997.
industry. BioPhorum currently comprises 71 manu-
facturers and suppliers deploying their top 2000 leaders
7. International Conference for Harmonisation, Qual-
and subject matter experts in seven Phorums: Drug
ity Guideline Q5A: Viral Safety Evaluation of Bio-
Substance, The Development Group, Fill Finish, The
technology Products Derived from Cell Lines of
Technology Roadmap, BioPhorum IT Group, BioPhorum
Human or Animal Origin. ICH: Geneva, Switzer-
Supply Partners, and BioPhorum Cell and Gene Therapy.
land, 1998.
The Viral Clearance Workstream is part of the Bio-
phorum Development Group.
8. Bl€umel, J.; Brorson, K. Session 2: Company-Spe-
This article is a composite view of opinions shared by cific Data on Cycled Resin Testing. PDA J.
the whole of the BPDG Viral Clearance Workstream and Pharm. Sci. Technol. 2016, 70 (5), 428–442.
should not be attributed to the individual positions of the
participating companies. 9. Brorson, K.; Brown, J.; Hamilton, E.; Stein, K. E.
Identification of Protein a Media Performance
Conflict of Interest Declaration Attributes That Can Be Monitored as Surrogates
for Retrovirus Clearance during Extended Re-Use.
The authors declare that they have no competing J. Chromatogr. A 2003, 989 (1), 155–163.
interests.
10. European Medicines Agency, Meeting Report:
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