Global Adventitious Agent Regulation of Raw Materials Used in

Biopharmaceutical ManufacturingBarbara J. Potts, Ph.D. Biologics Consulting Groups, Inc. USA

T.W. Tanaka Biologics Consulting Group, Inc. Japan

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Bio Process International ConferenceSeptember 20-24th 2010

Rhode Island Convention CenterProvidence, RI

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USDA 9 CFR 113.53 – Requirements for Ingredients of Animal Origin Used for Production of Biologics and 113.47 – Detection of Extraneous Viruses by the Fluorescent Antibody Technique

ICH Q 5A CBER Guidance – Cell Substrates and Biomaterials USP <1046> Cell and Gene Therapy Products USP <1043> Ancillary Materials for Cell, Gene and Tissue-

Engineered Products

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Global Regulations that apply to the Control of Adventitious Agents In Raw Materials

European Medicines Agency – Note for Guidance on Minimizing the Risk of Transmitting Animal Spongiform Encephalopathy Agents via Human and Veterinary Medicinal Products (EMEA/410/01 Rev.2, 2004). Rev.4 has been released for consultation 2008.

WHO Recommendations on the Evaluation of Cell Substrates Evaluation. Released for public consultation 2010.

Japanese Pharmacopeia 210 European Pharmacopeia 2.6.7

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Global Regulations that apply to the Control of Adventitious Agents In Raw Materials

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Risk Assessment for Adventitious Agent Control Identify and Assess

Agent Likelihood of Contamination

Consequences Impact

Prions Very Low Recall of productsLong term shut down of manufacturingReduced inventory of drugs for patients Reduced public trust

Very high

Viruses Medium Short term shutdown of manufacturing

High

Mycoplasma Low - Medium Rejection of impacted lots Medium – low (depends on frequency)

Bacteria Medium - high Depending on level - may lead to rejection of impacted lots

Medium – low (depends on frequency)

Mold-Yeast Low Depending on level – may lead to rejection of impacted lots

Medium

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Diagram from Scientific American, July 2004, p.88 in article by Stanley Prusiner

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Disease Causing Prion (PrPsc)

TSE

Builds up, and if not removedby cell’s machinery, illness can result.

(Transmissible Spongiform Encephalopathy)

Deer, Elk Cows Cats Sheep, Goats Humans

Chronic WastingDisease

BSE Feline SE Scrapie Kuru, Creutzfeldt-Jakob Disease (CJD)vCJD, Fatal Familial Insomnia

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Viruses

Parasites at the genetic level Virus reproduction is strictly dependent on host cell’s biochemical machinery. This parasitism separates viruses from free – living parasites such as some Mycoplasma Viruses are made up of:

• Genome (RNA or DNA)• Few proteins• Some have a lipid envelope acquired from host cells

A successful viral infection requires a “team” of virions for “infectious unit” to infect a host cell. >Like a touchdown in Football.< Cannot be seen using light microscopy

• Must use transmission electron microscopy to see virions.

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Major Groups of Viruses

Bacterial Viruses(Bacteriophage)

Host range does notcut across taxonomic boundaries between plants or animals

Animal Viruses

Replicate only in bacteria

Replicate in animal cells

Plant Viruses

Replicate in plant cells

Some viruses may replicate in plants and animals.

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Mycoplasma

Smallest free – living microorganisms Pleomorphic

• Vary in size from 0.12 to 0.25 µm• Pliability allows Mycoplasma to pass through

small pore filtration devices. Derived from ancestral anaerobic bacteria (clostridia)

by gene deletion. Contains no cell wall hence, they do not take up Gram

stain Assume various shapes from round to filamentous. Can be seen by dark – field and phase – contrast light microscopy. One Mycoplasma can initiate an infection resulting in 109 CFU/mL within 48 – 72 hours

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Bacteria Prokaryotic

*Absence of a membrane surrounding the nuclear region.*Absence of sub cellular organelles. Much smaller dimensions than most eukaryotic cells, (plants,

animals) which have a membrane bounded nucleus. (1µm compared to 10µm). Thrive in a range of environments that are extreme.*Can use an infinite array of energy sources. Can be seen by using light microscopy Have a cell wall surrounding the cytoplasmic membrane.*Gram Stain of cell wall will identify

> Gram positive bacteria (e.g.. Bacillus)> Gram negative bacteria (e.g.. E.coli) One bacteria can initiate an infection.

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Fungi / Mold Eukaryotic* Membrane bound nucleus* Presence of sub cellular organelles

Develops branching filaments Includes yeast, filamentous fungi, and fungi that produce lichens and slime molds Are saprobes (feeds on dead and putrefying tissues)

orMutualists (association between two different species of organism in which there is

mutual aid and benefit) orParasites (lives in or on another living organism with no benefit to the host)

Distribution of most fungi is restricted to a host, mutualist or special substrate* However, there are common and widespread “weedy” saprobe species associated

with human activities, buildings and soils

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Morphological Examination of Viruses, Mycoplasma, Bacteria and Fungi/Mold

Specimen Size MicroscopicRange

Resolution

Fungi / Mold 100 µm

Red Blood Cells 10 µm

Bacteria 1 µm

Mycoplasma 0.120 – 0.25 µm

Viruses 200 nm - 17 nm

Brightfield

Fluorescence

TEM SEM

Reference: Microbiology and Microbial Infections Vol. 2 Systematic Bacteriology Ed. A. Balows, B.I. Duerden. 1998, Oxford University Press Inc. N.Y.

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Control of Adventitious Agents in Raw Materials

Control sourcing of raw materials of animal origin (Prions)

Testing of bovine serum (fetal, calf) for viruses, Mycoplasma and bacteria

Cleaning and decontamination of raw material process equipment

Filtration Heat treatment pH treatment Gamma irradiation

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Increasing Resistance to Physical and Chemical Inactivation

Least Resistant Lipid or Medium-Size Viruses, Mycoplasma

Vegetative Bacteria

Fungi

Non-Lipid or Small Viruses

Mycobacteria

Most Resistant Bacterial Spores

Enveloped Viruses,

MycoplasmaVegetative Bacteria

Non-Enveloped

Small Viruses

Fungi

Spore Forming Bacteria Prions

70% Alcohol + + +/− − − −

Phenolic based disinfectants + + +/− − − −

10% Bleach + + + +/− +/− −

2.5% Acidified Bleach and Peracetic acid and Hydrogen Peroxide and Acetic Acid

+ + + + + −

0.1N NaOH + + + + + −

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Chemical Inactivation

Pore Size Rating (µm)

Bacteria & Fungi Mycoplasma Viruses Prions

0.1 + +/− − −

0.2 + +/− − −

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Filtration

Enveloped Viruses &

Mycoplasma Vegetative Bacteria

Non Enveloped Small Viruses Fungi

Spore Forming Bacteria Prions

90°C-100°C 5-10 seconds

+ + + + − −

121°C 20 minutes

+ + + + + −

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Heat Inactivation

Enveloped Viruses Mycoplasma

Non-Enveloped

Small Viruses Prion

pH 2.0, Short Time

+/− + − −

pH 3.0*, 30 Hours

+ + + −

pH 12.5Short Time

+ + + −

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pH Treatment

* Will not eliminate viral genome detected by PCR (circovirus)

Dose dependant decline in virus survival Varies with different viruses

◦ e.g. 25 kGy will inactivate below detection Bovine Viral Diarrhea Virus (Flavivirus) Infectious Bovine Rhinotracheitis Virus (Herpes Virus) Porcine Parvovirus (Parvovirus)

◦ But it takes 35 kGy to inactivate below detection Canine Adenovirus (Adenovirus) Bovine Reovirus (Reovirus)

BVDV is generally used to validate gamma irradiation of FBS Dose is generally reported as a target of 25 kGy with an accepted range

between15-30 kGy Gamma irradiation of viruses is not a sterilizing procedure Gamma irradiation of Mycoplasma in plant peptones has been validated at

84 kGy Gamma irradiation may not eliminate detectable viral genome by PCR

Gamma Irradiation Inactivation of Viruses and Mycoplasma

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Examples of Rigorous Processes ThatInactivate Prions

• Cleaning process* Sodium hydroxide or chlorine releasing disinfectants (e.g. 20,000ppm. chlorine for 1 hour) Note: Studies have demonstrated infectious TSE bound to the surface of stainless steel• Tallow derivatives processed by* Trans – esterification or hydrolysis at not less than 200°C for 20 minutes under pressure. (glycerol, fatty acids, fatty acids esters production)* Saponification with NaOH 12M (glycerol and soap production)

> batch process at 95°C for 3 hours> continuous process at 140°C under pressure for 8 minutes

• Animal charcoal* Carbonization of bone by using high temperature >800°C• Amino acids from hides or skin* pH 1 to 2 followed by pH of > 11, followed by 140°C for 30 minutes

Reference: Note for guidance on minimizing the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products EMEA/410/01 Rev. 2, July 1, 2004

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Viruses That May Contaminate Biopharmaceutical Processes

Virus Family Genome Envelope Size Resistance a

PoxviridaeRhabdoviridaeParamyxoviridae HerpesviridaeOrthomyxoviridaeRetroviridaeBunyaviridae*CoronaviridaeAdenoviridaeReoviridae*ArteriviridaeTogaviridaeFlaviviridaePapovaviridaeCaliciviridae*Picornaviridae*Parvoviridae*Circoviridae*

DNARNARNADNARNARNARNARNADNARNARNARNARNADNARNARNADNADNA

YesYesYesYesYesYesYesYesNoNoYesYesYesNoNoNoNoNo

250 X 450 nm70 X 175 nm150 – 300 nm120 – 200 nm80 – 120 nm80 – 120 nm80 – 100 nm75 – 160 nm65 – 80 nm65 – 75 nm50 – 65 nm40 – 70 nm50 – 70 nm45 – 55 nm35 – 40 nm25 – 30 nm18 – 24 nm17 nm

Medium – HighLowLowLow – MediumLowLowMediumLowMediumHighLowLowLowMedium – High MediumMediumVery HighVery High

a Resistant to physicochemical treatments

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Viruses That Replicate In CHO CellsVirus Family Virus

(host range)Genome Envelope Size Resistance*

Paramyxoviradae PI 1 (not known) PI 2 (human), PI 3 (human and cattle) Simian virus 5 (monkeys), Mumps virus (humans), Bovine Respiratory Syncytial virus (cattle)

RNA Yes 150-300 nm Low

Orthomyxoviradae Influenza A (human)RNA

Yes80-120 nm Low

Bunyaviridae Cache Valley Fever Virus (mice, mosquitoes, cattle)RNA

Yes80-100 nm Medium

Rhabdoviridae Vesicular Stomatitis Virus (cattle)RNA

Yes70 x 175 nm Low

Reoviridae Reovirus 1,Reovirus 2,Reovirus 3 (human) RNA No 65-75 nm Low

Togaviridae Sindbis (mosquitoes, mice, birds, humans)RNA Yes

40-70 nmHigh

Reoviridae Bluetongue virus(sheep, cattle, bitingmidge) RNA Yes 40 nm Low

Caliciviridae Vesivirus RNA No 35-40 nm Medium

Picornaviridae Encephalomyocarditis virus (mice, chimpanzees pigs, rats, rabbits, guinea pigs, humans)

RNANo 25-30 nm Medium-High

Parvoviridae MVM (mice) DNA No 18-24 nm Very High

* Resistance to physical and chemical inactivation

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Virus Contamination Virus Family Enveloped Size (nm) SourceCache Valley Fever Virus

Bunyaviridae Yes 80-100 Fetal Bovine Serum

Blue Tongue Virus Reoviridae No 65-75 Fetal Bovine Serum

Blue Tongue Virus Reoviridae Pseudo-enveloped 40 Possible insect transmission in testing lab

Bovine Viral Diarrhea Virus

Flaviviridae Yes 40-70 Fetal Bovine Serum

Vesivirus Caliciviridae No 35-40 UnknownPorcine raw material ?

Equine Picorna Virus Picornaviridae No 25-30 Equine Serum

Minute Virus of Mice

Parvoviridae No 18-24 Non-Animal Raw Material

Circoviridae Circovirus Type I No 17 Porcine Trypsin

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Source of Virus Contaminations in Raw Materials

Use raw materials that can withstand heat treatment (e.g. 90-100°C – 10 seconds), gamma irradiation (e.g. 15-30 kGy) or low pH (e.g. pH 3.0 for 30 hours)

If using raw materials of animal origin◦Use only animals designated for human consumption◦A quality assurance system must be in place for monitoring

the production process and for batch delineation of animal raw materials

◦Traceability, self-auditing of supplies Procedures in place to audit supplies of raw/starting materials◦Audit for mouse presence at facility, shipping, storage ◦Detect rodent urine with florescent light

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Minimizing The Risks of Transmission of Viruses

Use no raw materials of animal origin If must use animal raw materials, use porcine or equine sources If bovine raw materials must be used

◦ Source from country with negligible BSE risk (e.g. Australia & New Zealand)◦ Use only animals designated for human consumption◦ Use tissues with no detectable TSE infectivity, e.g.

Reproductive tissues Musculo-skeletal tissues Trachea Skin Adipose tissue

Source from young animals (e.g. < 30 months of age) A Quality Assurance system must be in place for monitoring the production process

and for batch delineation of bovine raw materials Traceability, self-auditing of suppliers Procedures in place to audit suppliers of raw/starting materials

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Minimizing the Risks of Transmission of TSE

Use raw materials that can be heat treated (60°C, 10 minutes), gamma irradiated or low pH (< pH 4.0) treated

Assume that plant or fish sourced raw materials have a Mycoplasma risk◦ Plants are a common host for Mycoplasma◦ There are many Mycoplasma that infect fish

Do not assume sterilizing filtration for Mycoplasma with 0.1µm or 0.2µm filters

Remember that Mycoplasma are very stable when in dried state (Mycoplasma stocks are stored in lyophilized state)

Remember that some Mycoplasma are not detected in the traditional culture methods

Implement a rapid detection system to limit impact of a Mycoplasma contamination

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Minimizing the Risks of Transmission of Mycoplasma

Raw Material Mollicutes Genus/Species Detected1 Tryptic Soy Broth

(animal origin)A. laidlawii

2 Tryptic Soy Broth (plant origin)

A. laidlawii

3 Complex Media A. laidlawii4 Bovine Serum A. laidlawii5 Peptones-animal

sourceA. laidlawii

6 Peptone - plant source

A. laidlawii

7 Water A. laidlawii

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Recent Mycoplasma Contamination of Raw Materials

Parental Drug Association Technical Report #50 “Alternative Methods for Mycoplasma Testing”◦To be released in 2010

Special Section in the Journal Biologicals on Mycoplasma March 2010 – Contains nine publication on risks, testing and control of Mycoplasma Vol.38, Number 2, March 2010

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New Reports on Mycoplasma Risks, Testing and Control

Raw Material Documented Vendor Activity

Vendor Risk Documented Consumer Activity

Overall Risk

1. Bovine Serum Validated sterile filtration for bacteria, gamma irradiation for some viruses (BVDV)9 CFR testing for virus, Mycoplasma and bacteria, fungi

High-Medium 9 CFR testing with addition of production cells in virus testing, 0.1µm filtration

Medium-low

2. Recombinant Proteins, e.g. insulin

Virus, Mycoplasma testingViral clearance studies Validated sterile filtration for bacteria

Low Sterile filtration Low

3. Bovine, Porcine and Equine peptones , Plant peptones, Fish peptones

Heat treatments, pH, some filtration, bioburden monitoring, endotoxin test

High Validated heat inactivation, gamma irradiation, pH, 0.1 or 0.2µm filtration

Low

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Risk Assessment: Adventitious Agents in Raw Materials (Bacteria, Fungi, Mycoplasma and Viruses)

Raw Material Documented Vendor Activity

Vendor Risk DocumentedConsumer Activity

Overall Risk

4. Trypsin, Pepsin Validated low pH treatment (pH 3.0 for 30 hours)Porcine virus testing (9 CFR) Post pH treatment Validated sterile filtration

Medium-low Porcine 9 CFR tests with addition of production cells in test

Low

5. Salts, amino acids, sugars, peptones, other raw materials used in large quantities

Rodent and insect control in manufacturing storage and shipping facilities

High if no quality control

Low if there is documented quality control

Routine quality audit of raw material vendors, manufacturing storage and shipping facilities, check for rodent infestation by using fluorescent light

Medium-Low

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Risk Assessment: Adventitious Agents in Raw Materials (Bacteria, Fungi, Mycoplasma and Viruses)

TSE Transfection reagents made from lamb’s wool must come from live lambs Selection antibiotics are often made from bacteria. Make sure bacteria

process does not include bovine raw materialsViruses Make sure trypsin is low pH treated (pH 3.0/30hrs) to inactivate circovirus

and parvovirus. Inactivated viral genome may remain in the raw material and can be detected by PCR

Fetal bovine serum – may be contaminated with a virus that is not inactivated by gamma irradiation and is not detected in 9CFR virus testing.

Decision Making Have senior management review and approve the use of any animal raw

materials used to prepare Master Cell Banks.

Raw Material Watch Outs for Master Cell Bank Preparation

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