Emerging Viral Risks and Mitigation Strategies in Biologics Manufacturing

Merck KGaA
Darmstadt, Germany
Merck KGaA
Darmstadt, Germany
Reginald Clayton, Ph.D.
Principal Scientist
Global Field Development Services

2
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.

1. Risk assessments
2. Emerging adventitious
viruses & mitigation
▪ Porcine circovirus type 3
▪ Hepatitis E Virus
▪ Schmallenberg virus
▪ Zika virus
▪ Borna disease virus
3. Quality by Design and
holistic screening of raw
materials
3

4
Viral
safety
Lot Release
Testing
Viral clearance or
inactivation
Characterization
of cell lines and
raw materials
Risk Assessment
Recent knowledge, Literature, Experience,
Regulatory

5
 Emerging viruses are potential contaminants of raw materials used in the
manufacture of vaccines and biologicals
 Challenge to the safety testing of products intended for human and
veterinary use.
 Risk mitigation strategies are necessary to preserve the integrity of the
manufacturing process and reduce the likelihood of impact upon animal and
human health
 Risk assessments of emerging viruses with potential to contaminate raw
materials should inform the evaluation of detection platforms that support
and enable risk mitigation.
Emerging viruses – a constant challenge

6
Sources of recent viral contamination

Mitigating the Risk from Two
Porcine Adventitious Viruses:
1. Porcine Circovirus Type 3
2. Hepatitis E Virus

9
An Emerging virus of pigs: Porcine Circovirus type 3
• Porcine circovirus type 3 (PCV3) is a widespread contaminant of the herd.
• Demonstrated to cause disease in pigs: porcine dermatitis and nephropathy
syndrome, and reproductive failure (Palinsky et al 2017, J Virol 91;e01879-
16)
• PCV3 is genetically divergent to PCV1&2, and diverse strains of PCV3 exist
(Fux et al 2018 Virol. J. 15:25-34)
• The plasma burden of PCV3 has been determined at ~4 logs/mL in infected
animals, and in aborted fetuses at ~7 logs per gram of material.
• Cap and Rep genes of PCV3 are genetically divergent from PCV1&2.
• Attempts at in vitro isolation & propagation of PCV3 unfruitful so far.
• PCV3 identified by Next Generation Sequencing of diseased primary material
(skin lesions).

10
PCV3 is distinct from known strains of PCV1/2
Source: Palinsky et al, 2017
• Genetically distal clustering of
PVC3 genomes indicates
divergence of strains over time
• Phenotypes of PCV3 not yet
established, but early data show
PCV3 refractile to culture
• PCR for risk mitigation

Porcine materials and Hepatitis E Virus
Hepatitis E virus (HEV) is a known contaminant of Porcine species and causes
disease in humans:
• 20 million cases worldwide, 70,000 deaths/annum
• Typical presentation; malaise, raised ALTs/GSTs, 6 weeks duration, self limiting
• Often asymptomatic where patient is immunocompetent
• Often fatal for fetus & mother where infection occurs in first trimester
• One serotype of HEV, but four major genotypes; 1&2 associated with humans,
3&4 have zoonotic origin
• HEV culture is difficult: virus isolation is possible by culture, but a sufficiently
sensitive in vitro assay is not yet available.
• Risk mitigation by PCR assay
11

Mitigating the Risk of Schmallenberg
Virus Contamination in Raw
Materials and Cell Lines

14
Regulatory Background
• Appropriate species specific tests should be performed depending on the
passage history of the cell line...(ICH Q5A R1).
• CPMP Note for Guidance on Production and Quality Control of Animal
Immunolgobulins & Immunosera for Human Use (CPMP/BWP/3354/99) –
this lists the viruses of concern and highlights which are classified as
pathogenic for humans.
• In the past two-three years, rigorous regulatory scrutiny has been noted in
regard to the use of antibodies & antisera during cell line development.

15
Schmallenberg virus (SBV): genus Orthobunyavirus. Simbu serogroup.
SBV infects cattle, sheep & goats resulting in severe birth defects and still birth.
Geographical dissemination of SBV
was rapid through Europe during
2011-2013 and is a concern for
agriculture.
An emerging threat - Schmallenberg virus

16
Spherical, enveloped virion, 80-120 nm diameter
Infects livestock via transmission through insect vectors
Can cross placenta and infect fetus
An emerging threat - Schmallenberg virus

17
The risk assessment for Schmallenberg virus
Animal serum, particularly bovine and Ovine/Caprine serum
Bovine serum is known to harbour SBV in infected animals, and is known to cross the
placental barrier, therefore bovine adult and fetal serum have associated risk.
Manufacturing processes using bovine serum (adult, or fetal) should be tested for SBV.
Cell lines exposed to Ovine or Caprine serum (antiserum for clonal selection) should be
tested for SBV.

18
Detection of SBV:
Detector cells were challenged with 5-1200 TCID50 SBV
and monitored for CPE over 14 days
DC line Inoculum (TCID50) Time to CPE (days)
BHK-21 1200 NT
50 2
5 2
Vero 1200 1-2
50 2
5 2
CHO-K1 1200 1
50 2-3
5 2-3
MRC-5 1200 14
50 NT
5 NT
324-K 1200 5-6
50 6
5 6
FLK 1200 7-11
50 10-12
5 10-12
BPEK 1200 5-11
50 11
5 11
BT 1200 14
50 18-20
5 18-20
MDBK 1200 -
50 NT
5 NT
Vero C1008/SBV 5 TCID50 3d PI
NC
NC
FLK/SBV 1200 TCID50 11d PI
(Ilchmann et al, 2017, Biologicals, 49; 28-32)

Mitigating the Risk of Zika
Virus Contamination in Raw
Materials and Cell Lines

21
Zika virus & safety of substance of human origin
Manufacturers have the responsibility to
perform a risk assessment
• US FDA Guidance for Industry
Donor Screening Recommendations to Reduce the Risk of Transmission of Zika Virus by
Human Cells, Tissues, and Cellular and Tissue-Based Products & Revised Recommendations
for Reducing the Risk of Zika Virus Transmission by Blood and Blood Components Guidance
for Industry (2016)
• European Centre for Disease Prevention and Control, Scientific Advice2016
Zika virus and safety of substances of human origin (2016)
• Committee for Medicinal Products for Human Use (CHMP), EMA
Report on viral safety of plasma-derived and urine-derived medicinal products with respect
to Zika virus. (EMA/CHMP/BWP/596747/2016)

22
Virus characteristics
 Enveloped, (+) ssRNA virus
 Mosquito-borne
 Flaviviridae family, genus Flavivirus
Origin of the recent epidemic
 First identified in rhesus monkeys
(1947)
 Spread through South East Asia to
French Polynesia & Pacific islands
 Early 2015 spread through South &
Central America
 Multiple strains: African & Asian origin
Zika virus: an emerging pathogen of humans
Image: Deutsche Welle

23
• In 80% of cases infection is asymptomatic
(healthy adults)
• Symptomatic infection manifests with rash,
joint pain, conjunctivitis, and fever
• In rare cases infection can cause neurological
complications:
• encephalitis
• meningo-encephalitis
• Guillain-Barré syndrome
• birth defects termed congenital Zika syndrome
(including microcephaly)
Image Source: CDC
Zika virus: Impact on human health

24
Zika virus & safety of Substances of Human Origin
(Zmurko et al 2018, J Gen Virol 99, 2219-229)
• Plasma
• Platelets
• Convalescent serum
• Monocytes
• Heterologous T-cells
• Albumins
• Coagulating factors
• Immunoglobulins
• Human chorionic gonadotropin (hCG)
• Human menopausal gonadotropin or
menotropin (HMG)
• Follicle stimulating hormone (FSH)
• Urokinase
Blood & blood components
Urine-derived medicinal products
Stem cells

25
In vitro adventitious virus testing
Maintain for 14 or 28 days,
examination for CPE
Sample inoculated
onto multiple
detector cells
Negative result Positive result
Examination for CPE
Haemagglutination assay
Haemadsorption assay
Sample
Detector cells: MRC-5,
Vero, others
End point testing

Detection of Zika virus strain MR766 by in vitro assay
End point testing - Cytopathic effect
• retarded cell growth
• Morphological changes in detector cells
• cellular lysis
• visualized under light microscope
Figure Legend: Micrographs show observations made in
detector cells following ZIKV MR766 inoculation (panels
a-f).
NC: mock-infected cells.
Scale bar is 1000 µm.
Zika virus is robustly detectable
by in vitro assay
(Zmurko et al 2018, J Gen Virol 99, 2219-229)26

Detection of Zika virus strain PE243 by in vitro assay
(Zmurko et al 2018, J Gen Virol 99, 2219-229)
End point testing - Cytopathic effect
• retarded cell growth
• Morphological changes in detector cells
• cellular lysis
• visualized under light microscope
Figure Legend: Micrographs show observations made in
detector cell lines following ZIKV PE243 inoculation
(panels a-f).
NC: mock-infected cells.
Scale bar is 1000 µm.
Zika virus is robustly detectable
by in vitro assay
27

A Largely Ignored, But Not
Forgotten Adventitious Virus:
Borna Disease Virus (BoDV)

29
BoDV discovered in Borna, Germany, late 1800s
 Responsible for deaths and behavioural changes in horses
 Strong CNS tropism
 No classical symptoms (no raised ALTs, no rash), but
psychological disturbances, and encephalitis in humans
 Documented infections in many species, with distribution
of virus in CNS (Kinnunen et al J Gen Virol 2013, 94, 247-
262)
 Recent cases of transmission and fatalities highlight risks
of human infection
 BoDV is a mononegalovirus
Borna Disease Virus

HEV
BoDV antigens in CNS of squirrels &
Patients by IHC analysis.
Viral antigen in neurons (black
arrows), glial cells (black arrowheads).
Recent cases highlight the BoDV risk
• Several fatalities from zoonotic BDV infection (Hoffman et al 2015 NEJM 373;2 p154)
• Fatalities following donor organ transplantation (European Centre for Disease Prevention and
Control). Acute encephalitis associated with infection with Borna disease virus 1 – Germany,
2018. 26 March 2018. Stockholm: ECDC; 2018)
(Hoffman et al 2015 NEJM 373;2 p154)30

31
Mitigation of risk from BoDV
• Potential sources of BoDV include domestic cats, rodents, and many other
animals, therefore the risk profile may include diverse types of BoDV.
• In vitro isolation and propagation of BoDV is relatively inefficient, and in vitro
phenotypes of variant strains remain unclear; a molecular assay is best suited
to detect BoDV.
• Cell banks of human origin (Stem cells, ATMPs) should be screened by PCR.
• Cell banks & raw materials with exposure to equine serum should be screened.
• Risk profile: Substances of Human Origin must be considered at risk for BoDV
contamination
• Allogeneic stem cell banks, urine-derived products, plasma-derived
products
• ATMPs with substances of human origin (e.g. stem cell banks) must be
screened for BoDV

Quality by Design and
Holistic Screening of Raw
Materials

34
Emerging viruses are a constant. Diligence is required.
Next Generation Sequencing (NGS; Massively Parallel sequencing, Deep sequencing) identifies
all adventitious viruses, mycoplasma and bacteria
NGS has enabled the detection of novel viruses but in biotechnology applications it also provides
reassurance to regulators and sponsors that their product is safe!
Applications for vaccines:
 Virus identification during contamination events
 Raw material testing
 Cell substrate characterization
 Virus seed characterization

35
Next Generation Sequencing
Advantages
 No pre-selection of nucleic acids: agnostic technique
 Can identify contaminants with known genome sequences but also organisms with only
50% homology with known sequences
 Depth of sequencing may allow construction of whole genome of a contaminant
 Will be quicker than many classical infectivity assays
Considerations
 Detects nucleic acids and not infectious particles
 Requires powerful bioinformatics algorithms
 Careful interpretation is required to avoid false positives
 Need experienced virologist to interpret data

36
Raw material qualification:
Holistic assessment enabled by Next Generation Sequencing (NGS)
• Raw materials batch-batch variation
• Novel production substrates
• Bespoke custom media components
• Novel risk profiles
• Viruses not yet discovered.

37
Summary
Risk assessments evolve over time: new knowledge informs risk assessments
Specific viruses may require targeted PCR assays to enable testing
In vitro assays enable sensitive and robust detection of many emerging viruses
Next Generation sequencing enables the identification of novel risks, and
therefore informs the risk assessment of raw materials.
We have not seen the last of emerging viruses, and vigilance must be
constant.

CVR
Medical Research Council
University of Glasgow
Centre for Virus Research
Prof. Alain Kohl
Dr Claire Donald
Alison Armstrong, Ph.D.
Jo Zmurko M. Sc.
Anne Ilchmann Ph.D.
Principal Scientist,
Field Development Services (EMA)
reginald.clayton@sial.com
ACKNOWLEDGEMENTS
REGINALD CLAYTON, PHD
The vibrant M and BioReliance are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners.
Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
Thank you for your attention.
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Emerging Viral Risks and Mitigation Strategies in Biologics Manufacturing

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