Vaccine clinical trial -Quality, include control of cell substrate Ywan-Feng Li Center for Drug Evaluation 4-7-2011 新醫藥品法規人才培訓課程（ I ） The views expressed

Vaccine clinical trial -Quality, include control of cell substrate Ywan-Feng Li Center for Drug Evaluation 4-7-2011 I The views expressed in this presentation are not necessary those of Center for Drug Evaluation-Taiwan

4-7-20112 The views offered here do not necessarily reflect official positions of TFDA

4-7-20113 Heterogenicity of the biological/biotechnological products Peptides (20-30 a,a,) rDNA proteins (Mab, fusion protein) Allergens Traditional vaccines Blood/plasma products Gene/cell therapy P. Tissue engineering P. rDNA-derived vaccines

4-7-20114 Scope Vaccine Vaccine Information from research stage Information from research stage Clinical trial-IND Clinical trial-IND Cell substrate-Testing for adventitious agents Cell substrate-Testing for adventitious agents Quality, starts from phase 1 Quality, starts from phase 1 Quality, (almost) finalizes at phase 3 and continues through product life span Quality, (almost) finalizes at phase 3 and continues through product life span Collaboration from all parties makes a trial going Collaboration from all parties makes a trial going

Type of vaccine Vaccines represent the most diverse type of products Attenuated or killed pathogens (bacteria, virus, parasites) (~traditional vaccine) Purified and recombinant protein Synthetic peptides Polysaccharide (free or conjugate to carrier) DNA, viral vectors (Cell-based product) 4-7-20115

Preventive versus therapeutic vaccines 4-7-20116 CharacteristicPreventiveTherapeutic PopulationHealthy subjectUsually patient Clinical outcomeDecrease microbial infection and/or transmission Cure or postpone disease progression (usually as a 2 nd line strategy) RegimenLow dose, episodicUsually high dose, continual (more like a drug) Evaluations in early trial Safety, immunogenicity In general:

Preventive versus therapeutic vaccines 4-7-20117 CharacteristicPreventiveTherapeutic Regulatory evaluation Emphasis on safety Efficacy Benefit/risk assessment Efficacy Public expectation Highly concern and sensitive to the potential risks Less concern regarding the potential risks However, quality and assessment of the vaccine (Ag and adjuvant) is the same for either type of vaccine

First licensed cancer therapeutic vaccine-Provenge (FDA) Autologous dendritic cells, activated by prostatic acid phosphatase (plus GM-CSF) Autologous dendritic cells, activated by prostatic acid phosphatase (plus GM-CSF) asymptomatic or minimally symptomatic metastatic hormone-refractory prostate cancer 2010, approved by FDA to treat asymptomatic or minimally symptomatic metastatic hormone-refractory prostate cancer 2011, FDA approved Dendreon's request to increase production capacity FDA approved "36 additional workstations at the company's New Jersey facility, adding to the 12 already approved" 2011 (Mar.), US Medicare proposed to cover the cost of $93,000/patient prostate cancer vaccine 4-7-20118

Type of vaccine IND New vaccines New vaccines Include addition or change of adjuvant Include addition or change of adjuvant Modification of original product Modification of original product Formulation (e.g., lyophilized vs. liquid) Formulation (e.g., lyophilized vs. liquid) Strength Strength Route of administration Route of administration Change in indication, age group, schedule, etc. Change in indication, age group, schedule, etc. Concomitant administration with other vaccine Concomitant administration with other vaccine 4-7-20119

10 Vaccines in development/trial, Taiwan, 2011 Ag type Ag type Virus vaccine Virus vaccine Polysaccharide conjugate vaccine Polysaccharide conjugate vaccine rDNA protein vaccine rDNA protein vaccine Indication Indication Infectious disease Infectious disease Cancer Cancer

Reference In general Guideline on the requirements for quality documentation concerning biological IMP in clinical trials, draft, EMA, 2010 Guideline on the requirements for quality documentation concerning biological IMP in clinical trials, draft, EMA, 2010 Guideline on strategies to identify and mitigate risks for FIH clinical trials with investigational medicinal products, EMA, 2007 Guideline on strategies to identify and mitigate risks for FIH clinical trials with investigational medicinal products, EMA, 2007Vaccines WHO: Biologicals TRS WHO: Biologicals TRS Japan NIID: Minimum requirements for biological products Japan NIID: Minimum requirements for biological products Pharmacopeia: Ph. Eur, USP Pharmacopeia: Ph. Eur, USP 4-7-201111

Quality of a product (EMA) NDA, to ensure a consistent, state-of-the art quality of a product NDA, to ensure a consistent, state-of-the art quality of a product IMP, quality attributes related to safety aspects IMP, quality attributes related to safety aspects Nature of product, clinical phase, patient population, nature/severity of illness, duration of trial. Nature of product, clinical phase, patient population, nature/severity of illness, duration of trial. IMP documentation, M3 of CTD IMP documentation, M3 of CTD IMP should be produced in accordance with the principles and the detailed guidelines of GMP.. IMP should be produced in accordance with the principles and the detailed guidelines of GMP.. 124-7-2011

13 A clinical trial Starts with the quality/control of the test drug Quality of a biological/biotechnological product Quality of a biological/biotechnological product Include safety issues, e.g., impurity, adventitious agent (e.g., bacteria/fungi, mycoplasma, virus) Include safety issues, e.g., impurity, adventitious agent (e.g., bacteria/fungi, mycoplasma, virus) Test drug used in animal toxicity studies be representative of the material for human study Test drug used in animal toxicity studies be representative of the material for human study So as to support a phase 1 study with end points of safety and preliminary immunogenicity So as to support a phase 1 study with end points of safety and preliminary immunogenicity

Characterization - Ag vs. therapeutic drug For Ag, immunogenicity is the desired effect, therefore, concept of certain characteristics is different (e.g., product-related impurity, which would otherwise cause undesired immunogenicity for protein drug) For Ag, immunogenicity is the desired effect, therefore, concept of certain characteristics is different (e.g., product-related impurity, which would otherwise cause undesired immunogenicity for protein drug) In general, extent of characterization is less for an Ag (e.g., product-related impurity) In general, extent of characterization is less for an Ag (e.g., product-related impurity) Thus, Process = quality is more likely to be the case for Ag. Therefore, the approach to establish a design space or platform technology is less likely to apply to vaccine product Thus, Process = quality is more likely to be the case for Ag. Therefore, the approach to establish a design space or platform technology is less likely to apply to vaccine product 4-7-201114

4-7-201115 Scope Vaccine Vaccine Information from research stage Information from research stage Clinical trial-IND Clinical trial-IND Collaboration from all parties makes a trial going Collaboration from all parties makes a trial going

4-7-201116 Information from research stage Science Science A vast amount of information has generated from basic research A vast amount of information has generated from basic research However, most information is yet to be interpreted and thus transferable to development However, most information is yet to be interpreted and thus transferable to development Provide rationale based on disease pathogenesis, and identify Ag candidate Provide rationale based on disease pathogenesis, and identify Ag candidate Control of materials Control of materials Raw materials, starting materials, solvents, reagents, catalysts, e.g., Raw materials, starting materials, solvents, reagents, catalysts, e.g., Source, history of the cell substrate Source, history of the cell substrate History of construction of the expression plasmid History of construction of the expression plasmid

4-7-201117 Information from research stage Safety information Safety information Plan to obtain and document relevant safety data from research studies even they are designed to assess biologic effects. Plan to obtain and document relevant safety data from research studies even they are designed to assess biologic effects. This is an effective approach to lunch preclinical safety evaluation This is an effective approach to lunch preclinical safety evaluation Extent and design of toxicity studies could depend on how much prior info exist Extent and design of toxicity studies could depend on how much prior info exist Especially for vaccine product Especially for vaccine product

4-7-201118 Control of materials Documents, starting from research stage Origin, lineage History of passage, testing Media component, e.g., FBS, trypsin All of the documents be transferable to R& D stage Establishment of a cell bank or virus bank ~GMP Storage, inventory, identification, handling, GMP Qualification of cell and virus bank Contract lab GLP/GMP status

4-7-201119 History of a virus strain Example ( Example (FDA, Review of Vero cell banks for Rotarix, 2008) The Serotype G1 HRV strain (genotype P[8]) which GSK used to make vaccine product is designated RIX4414. It was derived from strain 89-12, initially developed by Avant Therapeutics, Inc. ---------------. The virus was isolated in ------ ------- from a child in Cincinnati with a natural case of rotavirus with mild diarrhea. This original isolate was passaged 26 times in primary African Green monkey kidney cells (AGMK) by Avant for use as seed material. The P26 virus was ------- passaged by -- --------------- AVANT, -------, which passaged the seed virus an additional 7 passages to P33. This was the material that was clinically tested ---------------. The additional 7 passages were performed in an AGMK cell line that ------- characterized in --------. The Serotype G1 HRV strain (genotype P[8]) which GSK used to make vaccine product is designated RIX4414. It was derived from strain 89-12, initially developed by Avant Therapeutics, Inc. ---------------. The virus was isolated in ------ ------- from a child in Cincinnati with a natural case of rotavirus with mild diarrhea. This original isolate was passaged 26 times in primary African Green monkey kidney cells (AGMK) by Avant for use as seed material. The P26 virus was ------- passaged by -- --------------- AVANT, -------, which passaged the seed virus an additional 7 passages to P33. This was the material that was clinically tested ---------------. The additional 7 passages were performed in an AGMK cell line that ------- characterized in --------.

4-7-201120 Raw material of animal source COA of FBS (partially shown)

4-7-201121 Raw material of animal source COA of porcine trypsin

4-7-201123 Biosafety control Combination of testings (starting material, UPB, intermediates..) and demonstrating production process to remove a wide variety of potential infectious viruses Purified bulk (Calculation of estimated particles/dose) Cell/virus seed & raw material Cell/virus bank and unprocessed bulk Viral clearance steps Contract lab Research stage

4-7-201124 CMC Control of materials (before phase 1) Control of materials (before phase 1) Raw materials, starting materials, solvents, reagents, catalysts Raw materials, starting materials, solvents, reagents, catalysts Biologically-sourced materials, TSE concern Biologically-sourced materials, TSE concern Source, history, and generating of cell substrate Source, history, and generating of cell substrate Expression construct Expression construct Cell banking system, characterization, and testing Cell banking system, characterization, and testing Non-viral agent Non-viral agent Endogenous and adventitious viruses Endogenous and adventitious viruses Tumorigenicity, case dependent Tumorigenicity, case dependent

4-7-201125 A reference - Ancillary materials (AMs) for cell-based product Reagent and materials that are NOT intended to be present in the final product, e.g., FBS, digestion enzymes, GF, cytokines, antibiotics, media Reagent and materials that are NOT intended to be present in the final product, e.g., FBS, digestion enzymes, GF, cytokines, antibiotics, media Vendor qualification Vendor qualification (cGMP), audit/inspection record (cGMP), audit/inspection record Quality control testing program Quality control testing program Documentation Documentation Grade, traceability, or country of origin/source ( animal-derived AMs) Grade, traceability, or country of origin/source ( animal-derived AMs) Batch analytical results Batch analytical results Stability assessment during use Stability assessment during use

4-7-201126 Risk classification of AMs USP Risk classification of AMs USP Risk tier 1 Low-risk, highly qualified materials with intended use as therapeutic drug or biologic, medical device, or implantable material Low-risk, highly qualified materials with intended use as therapeutic drug or biologic, medical device, or implantable material Therapeutic grade Therapeutic grade E.g, HSA, insulin, IL-12, antibiotics E.g, HSA, insulin, IL-12, antibiotics Certificate of analysis (COA) Certificate of analysis (COA) Assess removal from final product Assess removal from final product

4-7-201127 Risk classification of AMs USP Risk classification of AMs USP Risk tier 2 Low-risk, well-characterized materials with intended use as AMs, produced in compliance with GMPs Low-risk, well-characterized materials with intended use as AMs, produced in compliance with GMPs For use in drug, biologic, or medical device manufacture, e.g., growth factor, proteolytic enzymes, density gradient media (Exclude most animal-derived materials) For use in drug, biologic, or medical device manufacture, e.g., growth factor, proteolytic enzymes, density gradient media (Exclude most animal-derived materials) COA COA Assess removal from final product Assess removal from final product Vendor audit Vendor audit

4-7-201128 Risk tier 3 Moderate-risk materials not intended for use as AMs Moderate-risk materials not intended for use as AMs For in vitro diagnostic use or reagent grade materials, e.g, growth factors, culture media, chemicals For in vitro diagnostic use or reagent grade materials, e.g, growth factors, culture media, chemicals COA COA Confirm critical test result shown in COA Confirm critical test result shown in COA Develop internal specifications, eventually Develop internal specifications, eventually Assess removal from final product Assess removal from final product Vendor audit Vendor audit Risk classification of AMs USP Risk classification of AMs USP

4-7-201129 Risk classification of AMs USP Risk classification of AMs USP Risk tier 4 High-risk materials High-risk materials Toxin, most animal-derived materials Toxin, most animal-derived materials Feeder cells, ascites-derived Ab, cholera toxin, animal-derived additives (e.g., FBS) Feeder cells, ascites-derived Ab, cholera toxin, animal-derived additives (e.g., FBS) COA COA Confirm critical test result shown in COA Confirm critical test result shown in COA Develop internal specifications, eventually Develop internal specifications, eventually Assess removal from final product Assess removal from final product Vendor audit Vendor audit Source animal, country of origin, adventitious agent testing Source animal, country of origin, adventitious agent testing

4-7-201130 Recent guidance - Cell substrate Guidance for industry: Characterization and qualification of cell substrates and other biological starting materials used in the production of viral vaccines for the prevention and treatment of infectious disease, FDA, 2010 Guidance for industry: Characterization and qualification of cell substrates and other biological starting materials used in the production of viral vaccines for the prevention and treatment of infectious disease, FDA, 2010 Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks, draft, WHO, 2010 Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks, draft, WHO, 2010

4-7-201131 Ideal substrate to produce biological/biotechnological products WHO Technical report series, No. 878, 1998 Permanent/continuous cell line Permanent/continuous cell line MCB, WCB MCB, WCB Quality controlled Quality controlled Serum-free and/or protein-free media Serum-free and/or protein-free media Nature Reviews, June 2010, vol.10, p.441- Cell line identification Cell line identification Incidence of misidentification in 1977 was 16%, 1999 was 18% Incidence of misidentification in 1977 was 16%, 1999 was 18% ATCC working group ASN-0002 (BOX), currently developing a standard for human cell line authentication ATCC working group ASN-0002 (BOX), currently developing a standard for human cell line authentication

4-7-201132 Cell substrate From embryonic egg for Flu vaccine Harvest: allantoic fluid, manual or automated systems Inoculation: into allantoic cavity, manually or automated system

4-7-201133 Cell lines for the production of vaccines Licensed vaccine Licensed vaccine Cell substrateVaccine TypeOriginLive attenuatedInactivated Primary tissues or cells Calf lymph, mouse brain, chicken egg, chicken embryo cell Small pox, Influenza, Measles, Mumps JEV, Influenza, Rabies Diploid cellsHuman (MRC-5, WI-38)Rubella, Varicella/Zoster Poliovirus, HAV, Rabies Continuous cells (Non-tumorigenic) Monkey (Vero)Small pox, RotavirusPoliovirus, JEV, Rabies Continuous cells (Tumorigenic ) Canine (MDCK)-Influenza Non-mammalian cells Yeast (S. cerevisiae) Insect (Hi-5) -HBV, HPV (rDNA product)

4-7-201134 Qualification of the cell bank -Biosafety tests Non-viral agent Non-viral agent Sterility, Mycoplasma, (Mycobacteria, Spiroplasma) Sterility, Mycoplasma, (Mycobacteria, Spiroplasma) Adventitious or endogenous viruses Adventitious or endogenous viruses General (in vitro and in vivo test, retrovirus) General (in vitro and in vivo test, retrovirus) Specific (cell line dependent) Specific (cell line dependent) Tumorigenicity, case dependent Tumorigenicity, case dependent

Qualification of the cell bank -Virus tests 35 Specific tests for : Cell lines derived from human, NHP, or other cell lines as appropriate. Culture media using animal-derived components ( e.g., bovine or porcine) USP, Ph. Eur.

4-7-201136 Tumorigenicity Tumorigenicity (when not known, test on EPC) Tumorigenicity (when not known, test on EPC) Cells form tumor in animal (nude mice), Hela as + control, medium/2n cells as control, 12 wks, 4 months Cells form tumor in animal (nude mice), Hela as + control, medium/2n cells as control, 12 wks, 4 months Oncogenicity (when T+ and for product of prophylactic use) Oncogenicity (when T+ and for product of prophylactic use) Agents (e.g., virus, DNA) induce host cell to form tumor (newborn animal), negative control, 4 months Agents (e.g., virus, DNA) induce host cell to form tumor (newborn animal), negative control, 4 months Cell substrate w/ or w/o tumorigenicity (in trial or licensed) Cell substrate w/ or w/o tumorigenicity (in trial or licensed) Traditional vaccine rDNA protein product (drug or vaccine) T- O- Yes (inactivated, attenuated) Yes T+ O- Yes (Inactivated) Yes T+ (O+) e.g., rodent cells NoYes

4-7-201137 PCV Porcine circovirus types 1 and 2 are both small sscDNA viruses and common in pigs. Porcine circovirus types 1 and 2 are both small sscDNA viruses and common in pigs. Neither PCV1 nor PCV2 are known to infect or cause illness in humans, however PCV2 may cause illness in pigs. Neither PCV1 nor PCV2 are known to infect or cause illness in humans, however PCV2 may cause illness in pigs. Detecting PCV1 DNA in Rotarix and PCV1/PCV2 DNA in RotaTeq vaccine products Detecting PCV1 DNA in Rotarix and PCV1/PCV2 DNA in RotaTeq vaccine products Viruses derived from Vero MCB and carried through manufacture process to products Viruses derived from Vero MCB and carried through manufacture process to products PCV1 DNA is present in poliovirus harvests, but not in final bulk or container (due to inactivation step) PCV1 DNA is present in poliovirus harvests, but not in final bulk or container (due to inactivation step) Source: FDA vaccine advisor committee meeting (May 7, 10)

4-7-201138 PCV RotaTeq (Merck) RotaTeq (Merck) A live, oral pentavalent vaccine that contains 5 live reassortant rotaviruses, parent strains were isolated from human and bovine hosts A live, oral pentavalent vaccine that contains 5 live reassortant rotaviruses, parent strains were isolated from human and bovine hosts Package insert (Sep. 2010) Package insert (Sep. 2010) Rotarix Rotarix A live, oral vaccine derived from human 89-12 strain (G1P[8] type) A live, oral vaccine derived from human 89-12 strain (G1P[8] type) Package insert (2010) Package insert (2010)

FDA actions Additional testings are required FDA (Dec., 2010) requested information regarding FDA (Dec., 2010) requested information regarding Plans that the manufacturers may have to implement additional adventitious agent testing methods as part of their manufacturing process as these methods become available including, but not limited to, screening for PCV and PCV DNA, and Plans that the manufacturers may have to implement additional adventitious agent testing methods as part of their manufacturing process as these methods become available including, but not limited to, screening for PCV and PCV DNA, and Any additional in-process testing for adventitious agents that they may have recently added, but not reported to FDA. Any additional in-process testing for adventitious agents that they may have recently added, but not reported to FDA. 4-7-201139

4-7-201140 Validation of viral clearance steps For rDNA vaccine produced from mammalian/insect cell lines, why virus testing alone is not enough? For rDNA vaccine produced from mammalian/insect cell lines, why virus testing alone is not enough? Due to limitations of testing methods Due to limitations of testing methods Sensitivity, susceptibility (indicator cell, animal model) Sensitivity, susceptibility (indicator cell, animal model) Sampling of test material Sampling of test material Reference: Validation of Biopharmaceutical purification processes for virus clearance evaluation , Allan Darling, Mol. Biotec. Vol. 21, 2002 Reference: Validation of Biopharmaceutical purification processes for virus clearance evaluation , Allan Darling, Mol. Biotec. Vol. 21, 2002

4-7-201141 Allan Darling, Molecular Biotechnology, vol. 21, 2002 Besides DL of test method, ability to detect low concentrations of virus is also limited by statistical sampling Besides DL of test method, ability to detect low concentrations of virus is also limited by statistical sampling Probability that a sample v does not contain virus is Probability that a sample v does not contain virus is p(0) = ((V-v)/V)n p(0) = ((V-v)/V)n If V>>v, above equation be simplified by Poisson distribution If V>>v, above equation be simplified by Poisson distribution p(0) = e -cv, c = (In p)/-v p(0) = e -cv, c = (In p)/-v If v=1 mL, c=10-1000 virus particles/L If v=1 mL, c=10-1000 virus particles/L Probability of 1 mL will not contain a virus particle Probability of 1 mL will not contain a virus particle c 10 100 1000 c 10 100 1000 p(0) 0.99 0.90 0.37 p(0) 0.99 0.90 0.37

4-7-201143 A clinical trial -IND dossier Biological/biotechnological products Biological/biotechnological products Poorly characterized, e.g., virus vaccine, cell-based vaccine Poorly characterized, e.g., virus vaccine, cell-based vaccine Well-characterized, e.g., rDNA protein vaccine, DNA vaccine Well-characterized, e.g., rDNA protein vaccine, DNA vaccine Technical related document Technical related document Clinical study proposal Clinical study proposal Investigator brochure Investigator brochure CMC CMC Pharmacology and toxicology Pharmacology and toxicology (PK, when appropriate) (PK, when appropriate) Clinical Clinical

CMC A summary report with supporting documents, e.g., batch analysis, stability data A summary report with supporting documents, e.g., batch analysis, stability data A valid description which reveals all necessary components to demonstrate the quality and control of the test drug A valid description which reveals all necessary components to demonstrate the quality and control of the test drug Present data in tabular form with brief narrative highlighting the main points Present data in tabular form with brief narrative highlighting the main points CTD format is a valid reference to organize the dossier CTD format is a valid reference to organize the dossier After phase 1, any change such as cell line, process, manufacture site, will require comparability After phase 1, any change such as cell line, process, manufacture site, will require comparability 4-7-201144

4-7-201145 CTD M3 (partially shown) 3.2.S DRUG SUBSTANCE ( ) 3.2.S DRUG SUBSTANCE ( ) 3.2.S.1 General Information ( ) 3.2.S.1 General Information ( ) 3.2.S.1.1 Nomenclature ( ) 3.2.S.1.1 Nomenclature ( ) 3.2.S.1.2 Structure ( ) 3.2.S.1.2 Structure ( ) 3.2.S.1.3 General Properties ( ) 3.2.S.1.3 General Properties ( ) 3.2.S.2.Manufacture ( ) 3.2.S.2.Manufacture ( ) 3.2.S.2.1 Manufacturer(s) ( ) 3.2.S.2.1 Manufacturer(s) ( ) 3.2.S.2.2 Description of manufacturing process and process controls ( ) 3.2.S.2.2 Description of manufacturing process and process controls ( ) 3.2.S.2.3 Control of materials ( ) 3.2.S.2.3 Control of materials ( ) 3.2.S.2.4 Controls of critical steps and intermediates ( ) 3.2.S.2.4 Controls of critical steps and intermediates ( ) 3.2.S.2.5 Process validation and/or evaluation ( / ) 3.2.S.2.5 Process validation and/or evaluation ( / ) 3.2.S.2.6 Manufacturing process development ( ) 3.2.S.2.6 Manufacturing process development ( ) 3.2.S.3 Characterization ( ) 3.2.S.3 Characterization ( )

4-7-201146 3.2.S.2.Manufacture 3.2.S.2.2 Description of manufacturing process and process controls ( ) 3.2.S.2.3 Control of materials / Cell bank Virus seed lot identity viability purity ` Unprocessed bulk expression construct 3.2.S.2.4 Controls of critical steps and intermediates DNA/ (conjugation) 3.2.S.2.5 Process validation and/or evaluation /

4-7-201147 CMC summary -Phase 1 Manufacturer ad manufacturing process Manufacturer ad manufacturing process Flow diagram and description, batch size Flow diagram and description, batch size Controls which relate to product safety Controls which relate to product safety For rDNA products derived from cell lines of human or animal origin, validation of the viral clearance procedure For rDNA products derived from cell lines of human or animal origin, validation of the viral clearance procedure For inactivated vaccines, a validation of the inactivation process For inactivated vaccines, a validation of the inactivation process For live vaccines, a demonstration of the attenuating characteristics For live vaccines, a demonstration of the attenuating characteristics

4-7-201148 CMC summary -Phase 1 Control of materials Control of materials Raw materials, starting materials, solvents, reagents, catalysts Raw materials, starting materials, solvents, reagents, catalysts Biologically-sourced materials, TSE concern Biologically-sourced materials, TSE concern Source, history, and generating of cell substrate and viral/bacterial seed Source, history, and generating of cell substrate and viral/bacterial seed Expression construct Expression construct Cell/virus/bacteria banking system, characterization, and testing Cell/virus/bacteria banking system, characterization, and testing Non-viral agent Non-viral agent Adventitious and endogenous viruses Adventitious and endogenous viruses Tumorigenicity, case dependent Tumorigenicity, case dependent

CMC summary -Phase 1 Analytical method Analytical method Pharmacopeia Pharmacopeia Non pharmacopeia Non pharmacopeia A brief description A brief description Qualification of safety related method Qualification of safety related method E.g., HCP, host cell DNA, residual reagent E.g., HCP, host cell DNA, residual reagent 4-7-201149

4-7-201150 CMC summary -Phase 1 Drug substance (Ag, adjuvant, novel excipient) Drug substance (Ag, adjuvant, novel excipient) Characterization Characterization Specification (preliminary), e.g., identity, strength, potency, and purity (& impurity) Specification (preliminary), e.g., identity, strength, potency, and purity (& impurity) E.g., HCP, DNA, residual reagents E.g., HCP, DNA, residual reagents Drug product Drug product Adjuvant, excipients, diluents Adjuvant, excipients, diluents Dosage form, composition Dosage form, composition Premix, on-site mix (adjuvant, dilution, reconstitution) Premix, on-site mix (adjuvant, dilution, reconstitution) Specification (preliminary) Specification (preliminary)

4-7-201151 CMC summary Phase 1 Stability Stability At least cover the duration of trial At least cover the duration of trial In-use stability information, e.g., after mixing, dilution, reconstitution, multiple withdrawing In-use stability information, e.g., after mixing, dilution, reconstitution, multiple withdrawing Batch analytical result, DS and DP Batch analytical result, DS and DP Batch for animal and clinical studies Batch for animal and clinical studies Same batch/formulation is recommended for vaccine products Same batch/formulation is recommended for vaccine products If not, describe (to compare) If not, describe (to compare) CMC CMC Animal study might be useful Animal study might be useful

4-7-201152 Phase-in of validation Validation of manufacturing processes and analytical methods goes along with the clinical development Validation of manufacturing processes and analytical methods goes along with the clinical development In phase 1, safety related method requires certain extent of validation In phase 1, safety related method requires certain extent of validation E.g., Host cell DNA, protein, viral test (e.g., PCR) E.g., Host cell DNA, protein, viral test (e.g., PCR) Limit - specificity and LOD Limit - specificity and LOD Quantitation- more extensive validation Quantitation- more extensive validation

Potency assay Correlation to in-vivo biological activity should be justified Correlation to in-vivo biological activity should be justified Potency should be in the stability study, even it is not proven to be stability-indicating in the early trial Potency should be in the stability study, even it is not proven to be stability-indicating in the early trial 4-7-201153

4-7-201154 Phase-in of validation Bioassay (potency) No need for LOD, LOQ No need for LOD, LOQ At phase 1/2 At phase 1/2 Specificity Specificity Precision Precision Repeatability, e.g., Repeatability, e.g., Samples from several independent preparations of the same stock Samples from several independent preparations of the same stock Same sample, well to well, %CV