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Quality by Design in Raw Materials Testing: Considerations,

Strategies and Experience of a Testing Laboratory

Reginald Clayton1, Donna McMutrie1, Anne Ilchmann1, David Onions2, Audrey Chang2, Colette Cote2, John Kolman2, Alison Armstrong1

1BioReliance, West of Scotland Science Park, Todd Campus, Glasgow G20 OXA, 2BioReliance,14920 Broschart Road, Rockville MD 20850, USA

Abstract

Testing of raw materials is an essential step in the production cycle of

biological therapeutics and vaccines. The implementation of Quality by Design

(QbD) in manufacturing processes is required across the pharmaceutical

industry to ensure the consistent production of a product to the required level

of quality. The holistic mapping of extraneous agents in raw materials is an

essential step in the QbD process, and requires molecular techniques capable

of detection of known and novel contaminants.

Results from the evaluation of testing technologies are presented, highlighting

appropriate technologies for evaluation of raw materials, and the drawbacks of

comparable technologies, specifically for the identification of extraneous

viruses.

Case studies regarding the discovery of several viruses in animal sera and cell

lines will be presented, and will be considered in the context of current

regulatory recommendation and guidelines for testing. Strategies for routine

testing to mitigate risk of extraneous agents in raw materials will also be

presented.

Conclusions

In evaluating the viral risk of biological products several factors have to be

considered. These include the risks associated with individual viruses, the

probability of the viruses being present in the material and the procedures

used to inactivate or clear contaminating viruses.

New methods of virus detection based on massively parallel sequencing

(MP-SeqTM) or molecular based technologies i.e., degenerate PCR have

initiated a new era of virus discovery. Recently, the number of human

polyomaviruses has increased from 2 to 5. Scientists at BioReliance have

identified a new bovine parvovirus in bovine serum using MP-SeqTM (Onions

and Kolman 2010) and new porcine viruses in the Bocavirus and Hokovirus

genera have been reported (Lau et al. 2008; Cheung et al. 2010).

Testing regimes have often lagged behind this recent phase of virus discovery.

(see adjacent tables). For instance assays for porcine parvovirus 1 are

always required, but until recently the other porcine parvoviruses were rarely

considered. This is now changing with tests for porcine hokovirus being

requested by regulatory authorities evaluating the safety of porcine pancreatin,

and assays for anellovirus being required for veterinary vaccines.

Introduction

• There are numerous routes through which adventitious viruses may be introduced into the Biologicals manufacturing process. Most are associated with the use of animal-derived raw materials.

• Materials such as serum, trypsin, insulin, plasma proteins, attachment factors, tissue extracts and established cell lines, plant peptones and fish products such as cod liver oil and protein extracts can all potentially harbor viral contaminants.

• Risks can be minimized by testing raw materials by MP-SeqTM to enable identification of the contaminants present in the batches of raw materials.

• Once identified, the use of screening processes using PCR directed methods or Molecular based methods for detection of specific contaminants are necessary.

• Massively parallel sequencing enables the holistic and detailed analysis of raw

materials, the detection of viruses specific to raw materials, and the implementation of

screening programmes for the assessment of raw material safety.

Table 1. Regulatory update; bovine and porcine contaminants

Global regulatory requirements

New enabling technologies:

Massively parallel sequencing: MP-SeqTM

• Screening of raw materials using molecular technologies has

highlighted the detection of novel viruses (see case studies).

• MP-Seq™ is performed using the Roche/454 GS-FLX "Next

Generation" sequencer coupled with FLX Titanium™

chemistry.

• This system can generate as many as 1,000,000 sequences

of about 800-1000 bases each, per run. The "depth" or

"coverage" of the sequencing run, which is the average

number of times that a nucleotide is actually sequenced, is

often several thousands (see case studies for MVS).

• If a plasmid, MVS or PCR product is analyzed, the depth can

be large, possibly 1000-10,000 times.

• MP-Seq™ detects sequences that are isolable from a sample;

the breadth of viruses or agents detectable is not limited by

oligonucleotide selection, as observed with array hybridization

or PCR, resulting in an holistic analysis of the raw material.

• MP-SeqTM enables us to ask, and answer the question:

what agents and sequences are present in these raw

materials?

MP-SeqTM enables detection of novel viruses

Primers in ID-Plex (Abbott) detect these 2, but

miss most of the 7 new human polyomaviruses

References

Onions D, Kolman J. (2010) Massively parallel sequencing, a new method for detecting

adventitious agents. Biologicals.38:p377-380

Cheung et al., (2010). Identification and molecular cloning of a novel porcine

parvovirus. Arch Virol.155:p801-806.

Lau et al (2008) Identification of novel porcine and bovine parvoviruses closely related

to human parvovirus 4. J Gen Virol.89:p1840-1848

Widdowson et al. (2005 ). Detection of serum antibodies to bovine norovirus ion

veterinarians and the general population in the Netherlands. J Med Virol.76(1):p119-28)

Yamishita et al. (2003). Isolation and characterization of a new species of kobuvirus

associated with cattle. J Gen Virol.84:p3069-77 )

Quality by Design: 3 Complementary Approaches to controlling contamination and assurance of safety

Three complementary approaches are outlined in regulations worldwide

Massively parallel sequencing of raw materials

enables identification of risk, enhanced vigilance by implementation of

appropriate screening regimes, resulting in reduced risk of contamination,

and increased assurance of safety of end product.

Lot Release Testing

Testing the product at appropriate

steps of production

Characterization of materials:

Cell line characterization (CLC)

Testing of raw materials to

regulatory requirements, e.g. BVDV

testing of bovine serum to 9CFR,

EP, CVMP.

QbD in raw materials: reduction to practise

MP-SeqTM Considerations:

Identify viruses

Will viruses report in 9CFR test?

PCR assays to detect virus in

raw materials.

Where positive in PCR, follow up

with in-vitro infectivity assay.

Downstream processes to show

removal/inactivation of specific

viruses.

Raw materials

Master Virus Seeds: Considerations and strategies

• MVS is typically established at the initiation of a new project.

• Wild type isolates/field strains that undergo laboratory adaption can result in quasispecies and

culture-adapted strains in relatively few passages.

• Homogeneity can be established at the MVS stage to show freedom from adventitious viruses

that are otherwise undetectable by classical methods (in-vitro assay, PCR etc).

• Read depth of MVS can be several thousand, enabling detailed profiling of the MVS.

• Discovery of undesired viruses at early stage by holistic analysis of the raw material (MVS)

enables risk reduction in progression of the project.

Case Studies

Sample Reference name Segment Reference

length

Consensus

length

Number

Match

Number

Mismatch

% Match

L1 consensus REO1LAM3P L1 3854 3855 3842 13 99.69%

L2 consensus REO3L2 L2 3916 3917 3912 5 99.90%

L3 consensus AF129822 L3 3901 3901 3899 2 99.95%

M1 consensus AF461684.1 M1 2304 2304 2301 3 99.87%

M2 consensus REO3OCPMUA M2 2203 2203 2196 7 99.68%

M3 consensus AF174384 M3 3901 3901 3899 2 99.95%

S1 consensus REOS1C S1 1416 1415 1412 4 99.72%

S2 consensus REOS2T4A S2 1331 1331 1330 1 99.92%

S3 consensus X01627.1 S3 1198 1198 1193 5 99.58%

S4 consensus REOS4 S4 1196 1199 1191 9 99.58%

Case study: Characterisation of Reo Virus seed

• Bovine kobuvirus, a new genotype of Picornavirus, detected in (2 of 4) 50% of newborn calf sera by MP-Seq™

• Virus first reported as a contaminant of HeLa cells in 2003

(Yamishita et al. J Gen Virol. 2003 Nov;84(Pt 11):3069-77 )

• A member of Picornavirus with a wide host range

• This may be another ‘vesivirus 2117’ waiting to happen

Bovine Kobuvirus Bovine Norovirus

• Bovine Norwalk-like virus (Norovirus) detected in (2 of 4) 50%

of newborn calf sera by MP-Seq™

• Recent serological data indicated bovine strains are transmitted

to humans

(Widdowson et al. J Med Virol. 2005 May;76(1):119-28)

BPV-2

BPV-3

BAAV-2

Cross

placenta

New Bovine Parvoviruses BioReliance QPCR Data

(number positive number tested. Different sera than

tested by MP-SeqTM)

Serum BPV-2 BPV-3 BAAV-2

Calf 2/5 1/5 0/5*

FBS 2/5 3/5 1/5**

*2 positive below LOQ of

100 copies,

**2 positive below LOQ

BPV-2 up to 6.7 x103 ge/ml

BPV-3 up to 1.1 x104 ge/ml

BAAV-2< 100 ge/ml

• LOQ 10 copies/Automated platform

• Detects BPyV, BPaV1, BPaV2, BPav3, BAAV2

• Validated and commercially available

New contaminants in bovine serum

• BioReliance has identified a new parvovirus in bovine serum (BAAV-2) using MP-SeqTM

• This virus is able to infect human cells and cells of other species.

• BAAV-2 can establish latent infections. Therefore cells that have been exposed to serum in the past need to be

screened.

• BAAV-2 is a dependovirus (AAV) and is likely to be mobilised by adenoviruses & herpesviruses

• Many parvoviruses are capable of autonomous replication, but some are not: the detection of dependoviruses in

classical in-vitro assays is unlikely unless a helper virus is present.

• FDA required 7 fold coverage

• MP-SeqTM gave over 13,000 fold

coverage in some regions:

unequivocal demonstration of

stability!

Case study: contaminants in bovine serum Background • Screening of bovine derived materials including serum, is performed in compliance with 9CFR

and CVMP regulatory guidelines.

• Current culture-based methods will not detect Bovine parvovirus 2 and 3 by classical cytopathic

effect.

• BVDV and BPyV are prevalent in FBS but they are not the most common viruses:

• MP-sequencing of serum lot 1 demonstrated:

BPV3 >10,000 hits

BPV2………high number of hits

And 5 hits against…….

Increasing PCV Copy numbers as a function of passage indicates

the presence of infectious PCV in a test article.

An In-vitro PCV Infectivity Assay

An in-vitro infectivity assay consisting of growth and amplification steps using permissive cells followed by

quantification of PCV nucleic acid by PCR.

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

Day 0 Day 6 Day 15 Day 27

Day post inoculation

PC

V (

gen

om

e c

op

y n

um

be

r /m

l)

PCR Assay for PCV1/2

• LOQ 10 copies/Automated platform

• Detects PCV 1 and 2

• Validated test, commercially available

Lane 1: PCV-1 infected PK-15

Lane 2: PCV-2 infected PK-15

M = MW marker

1 2 M

Case Study: Porcine Circovirus (PCV)

Screening of porcine derived materials, including porcine trypsin, is performed in compliance with

9CFR and CVMP regulatory guidelines. Current culture based methods will not detect PCV.

• PCV: a small (17-22 nm in diameter) non-enveloped ss DNA genome (ambisense)

• Widespread in swine throughout the world.

• Two types: PCV-1, PCV-2

• PCV-1 isolated from PK-15 cells, not associated with disease

• PCV-2 associated with postweaning multisystemic wasting syndrome

• PCV 1 and PCV 2 share 68-76% sequence homology

• In addition to pigs, Circovirus exist in chicken, pigeons….

• PCV is a known contaminant of porcine trypsin

• Discovery of PCV in two vaccine products has escalated regulatory scrutiny

• The presence of DNA does not necessarily indicate the presence of infectious virus

•Hence the following testing strategy is recommended: