Evaluation of antibody drugs quality safety

June 27, 2007June 27, 2007 The 34The 34thth annual meeting of the Japanese Society of Toxicologyannual meeting of the Japanese Society of Toxicology

Evaluation of Antibody DrugsEvaluation of Antibody Drugs-- From the Standpoint of From the Standpoint of

Quality and Safety Quality and Safety --

Toru KAWANISHIToru KAWANISHIDivision of DrugsDivision of Drugs

National Institute of Health SciencesNational Institute of Health Sciences

TopicsTopics

A new era of monoclonal antibody A new era of monoclonal antibody (mAb) drugs(mAb) drugsBasic principles of quality and safety Basic principles of quality and safety evaluation of biotechnological protein evaluation of biotechnological protein drugsdrugsQuality control of mAb drugsQuality control of mAb drugsSafety evaluation of mAb drugsSafety evaluation of mAb drugsNew types of mAb drugsNew types of mAb drugs

Number of new biopharmaceuticals approved Number of new biopharmaceuticals approved from 1999 to 2004 in Japan, EU, and USAfrom 1999 to 2004 in Japan, EU, and USA

-113579

1113151719

Num

ber o

f ND

As

anti bodi es

in terf eron s

in sul insbl ood coagula tion fa ctors

others (e nzymes, hormones )

Classification of engineered monoclonal Classification of engineered monoclonal antibodiesantibodies

Mouse

Mouse

CDR

HumanizedChimeric Fully human

CDR：Complementarity-determining regions

Human

Fusion protein with Fc fragment

Receptor

mAbmAb drugs approved before September of 2006drugs approved before September of 2006Name Type Antigen Indication Company Approval

Orthoclone Mouse CD3 Transplantation J&J 1986(1991)Zevalin Mouse CD20 NHL Schering AG/IDEC 2002Bexxar Mouse CD20 NHL Corixa/GSB 2003Rituxan Chimera CD20 NHL/RA Roche/Genentech/IDEC 1997(1997)ReoPro Chimera GPIIb/IIIa Thrombosis Lilly/Centocor 1994Remicade Chimera TNF Inflammation Schering-Plough/Centocor 1998(2002)Simulect Chimera CD25 Transplantation Novartis 1998(2002)Erbitux Chimera EGF-R CRC/HNC Imclone/BMS/Merck 2003Zenapax Humanized CD25 Transplantation Roche 1997Herceptin Humanized Her2/neu Breast cancer Roche/Genentech 1998(2001)Synagis Humanized RSV Virus infection Abbott 1998(2002)Mylotarg Humanized CD33 AML Celltech/AHP 2000(2006)Campath Humanized CD52 CLL ScheringAG/Millenium/ILEX 2001Xolair Humanized IgE Athma Novartis/Genentech 2003Raptiva Humanized CD11a Psoriasis Xoma/Genentech 2003Avastin Humanized VEGF CRC Genentech/Roche 2004Actemra Humanized IL-6R Castleman's Chugai 2005Humira Human TNF Inflammation Abbott/CAT 2003Tysabri Humanized VLA4 Multiple Sclerosis Elan/Biogen 2006

１．Blue : Products approved in JAPAN. Xolair was submitted in June of 2006.２．Tysabri was approved by FDA in June of 2006, by adding the safety information.

Number of Therapeutic Monoclonal Number of Therapeutic Monoclonal Antibodies entering Clinical StudyAntibodies entering Clinical Study

Nature Biotech. 23, 1073-1078 (2005)

TopicsTopics


ICH Guidelines for Quality and Safety ICH Guidelines for Quality and Safety Evaluation of Biotech productsEvaluation of Biotech products

・・ ANALYSIS OF THE EXPRESSION CONSTRUCT IN CELLS USED FOR PRODUCTION OF R-DNA DERIVED PROTEIN PRODUCTS (Q5B)（厚生省医薬安全局審査管理課長通知（厚生省医薬安全局審査管理課長通知医薬審第医薬審第33号号平成平成1010年年11月月66日）日）

・・ STABILITY TESTING OF BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS (Q5C)（厚生省医薬安全局審査管理課長通知（厚生省医薬安全局審査管理課長通知医薬審第医薬審第66号号平成平成1010年年11月月66日）日）

・・ VIRAL SAFETY EVALUATION OF BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS DERIVED FROM CELL LOINES OF HUMAN OR ANIMAL ORIGIN (Q5A)（厚生省医薬安全局審査管理課長通知（厚生省医薬安全局審査管理課長通知医薬審第医薬審第329329号号平成平成 1212年年22月月2222日）日）

・・ DERIVATION AND CHARACTERISATION OF CELL SUBSTRATES USED FOR PRODUCTION OF BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS (Q5D)（厚生省医薬安全局審査管理課長通知（厚生省医薬安全局審査管理課長通知医薬審第医薬審第 873873号号平成平成1212年年77月月1414日）日）

・・ SPECIFICATIONS : TEST PROCEDURES AND ACCEPTANCE CRITERIA FOR BIOTECHNOLOGICAL /BIOLOGICAL PRODUCTS (Q6B)（厚生労働省医薬安全局審査管理課長通知（厚生労働省医薬安全局審査管理課長通知医薬審第医薬審第571571号号平成平成1313年年 55月月11日）日）

・・ COMPARABILITY OF BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS SUBJECT TOCHANGES IN THEIR MANUFACTURING PROCESS (Q5E)

（厚生労働省医薬食品局審査管理課長（厚生労働省医薬食品局審査管理課長薬食審査発第薬食審査発第04260010426001号号平成平成1717年年44月月2626日日））・・ PRECLINICAL SAFETY EVALUATION OF BIOTECHNOLOGY-DERIVED PHARMACEUTICALS

(S6)（厚生省医薬安全局審査管理課長通知（厚生省医薬安全局審査管理課長通知医薬審第医薬審第326326号号平成平成1212年年22月月2222日）日）

Cell BankCell Bank( MCB,WCB )( MCB,WCB )

Process for Process for cultureculture

Cells at the limit of Cells at the limit of in vitroin vitro cell age for productioncell age for production

Bulk before purificationBulk before purificationProcess for Process for purificationpurification

Drug SubstanceDrug Substance

Process for Process for formulationformulation

Drug ProductDrug Product

Genetic Stability (Q5B)Genetic Stability (Q5B)

Product Stability(Q5C)Product Stability(Q5C)

Cell Substrate (Q5D)Cell Substrate (Q5D)

Viral Safety(Q5A)Viral Safety(Q5A)

Nonclinical Safety(S6)Nonclinical Safety(S6) Specification (Q6B)Specification (Q6B)

Manufacturing Process of BiotechManufacturing Process of Biotech--products and ICHproducts and ICH--GuidelinesGuidelines

Comparability (Q5E)

Nonclinical safety test of Nonclinical safety test of biotechnological protein productsbiotechnological protein products

Almost all of classical biotechnological protein drugs are desigAlmost all of classical biotechnological protein drugs are designed and ned and produced to mimic human proteins produced to mimic human proteins →→ It is possible to predict the It is possible to predict the pharmacological effects and their mechanism without examinationspharmacological effects and their mechanism without examinations ；；However, such proteins administered as drugs could be distributeHowever, such proteins administered as drugs could be distributed in the d in the regions where the natural proteins are physiologically not distrregions where the natural proteins are physiologically not distributed or at ibuted or at higher concentrations than physiological ones.higher concentrations than physiological ones.Often species difference in molecular structureOften species difference in molecular structure →→ Limits of the nonclinical Limits of the nonclinical safety tests using rodents safety tests using rodents （（→→ use of transgenic animalsuse of transgenic animals））

Neutralizing antibodiesNeutralizing antibodies →→ make it difficult to analyze the biological effectsmake it difficult to analyze the biological effectsCarcinogenicity testCarcinogenicity test →→ Standard carcinogenicity tests are generally Standard carcinogenicity tests are generally inappropriate.inappropriate.Reproductive toxicity testReproductive toxicity test →→ Limit in availability of appropriate speciesLimit in availability of appropriate speciesGenotoxicity testGenotoxicity test →→ Conventional mutation tests is usually not appropriate.Conventional mutation tests is usually not appropriate.Immunogenicity testImmunogenicity test →→ Usefulness is low to predict immunogenicity in Usefulness is low to predict immunogenicity in humanhumanPK testPK test →→ Standard pharmacokinetic tests sometimes has a limit.Standard pharmacokinetic tests sometimes has a limit.

Quality control of biotechnological protein drugsQuality control of biotechnological protein drugs-- comparison with small molecule drugs comparison with small molecule drugs --

Product complexity, including molecular heterogeneity (due to post-translational modification) and high order structure. → It is important to keep the consistency of the product (especially in drug substance) during the production by specifications.Difficult to confirm higher order structure because of the limit of analytical methods.→ It is important to measure biological (Potency) assay.→ It is important to set biological assay correlated with clinical safety

or efficacy.It is important to confirm the suitability of manufacturing process by process evaluation/validation (gene expression construct, cell bank, process-related impurities, contaminants) and keep the consistency by appropriate process control. It is important to consider viral contamination, because raw materials derived from animals/humans are used for the production.Unstable at room temperature → It is important to confirm the real-time/real-temperature stability of the products.Strategy for quality control to keep consistency : Both specifications of products and process controls.

TopicsTopics

A new era of monoclonal antibody A new era of monoclonal antibody (mAb) drugs(mAb) drugsBasic principles of quality and safety Basic principles of quality and safety evaluation of biotechnological protein evaluation of biotechnological protein drugsdrugsQuality evaluation of mAb drugsQuality evaluation of mAb drugsSafety evaluation of mAb drugsSafety evaluation of mAb drugsNew types of mAb drugsNew types of mAb drugs

Discussion points in the review of the Discussion points in the review of the submission of submission of mAbmAb drugsdrugs

Quality aspects : Discussion points are Quality aspects : Discussion points are usually similar to other biotechnological usually similar to other biotechnological protein drugsprotein drugs

Management of Cell bank Management of Cell bank Tests for identificationTests for identificationSpecification for sugar chainsSpecification for sugar chainsImpuritiesImpuritiesReference standardsReference standards etc.etc.

Quality: Characterization of Quality: Characterization of mAbmAb drugsdrugsMolecular structure as antibodyMolecular structure as antibody

Class, SubClass, Sub--class, Light chain or Heavy chain, class, Light chain or Heavy chain, etc.etc.

SpecificitySpecificityBinding to targeting moleculesBinding to targeting moleculesQuantitative evaluation of the bindingQuantitative evaluation of the bindingCrossCross--reaction using human cells or tissuesreaction using human cells or tissuesReaction in animalsReaction in animalsAnalysis of epitopeAnalysis of epitope

Biological effectsBiological effectsNeutralizing activityNeutralizing activityMechanisms of the effectsMechanisms of the effects

ADCC, CDC, agonistic, antagonistic, etc.ADCC, CDC, agonistic, antagonistic, etc.Effects Effects in vivoin vivo animalsanimals

Planning of Toxicity, ADME, Clinical tests Evaluation of Toxicity, ADME, Clinical test data

Establishment of Quality Control Tests

Characteristics and functional properties of Characteristics and functional properties of human antibody isotypeshuman antibody isotypes








(From D & MD Reports, 2001)

Mechanisms of Monoclonal Antibody drugsMechanisms of Monoclonal Antibody drugs








Quality: Sugar chains of antibodiesQuality: Sugar chains of antibodies

Sugar chains may influence on antibody Sugar chains may influence on antibody reaction, function, and ADME of drugs.reaction, function, and ADME of drugs.

Specifications: test procedures and acceptance Specifications: test procedures and acceptance criteria of sugar chains should be set.criteria of sugar chains should be set.It is recommended that the consistency of sugar It is recommended that the consistency of sugar chains is monitored after coming onto the chains is monitored after coming onto the market.market.

Recombinant antibody glycosylationRecombinant antibody glycosylation

Recombinant antibody glycosylation depends on Recombinant antibody glycosylation depends on The production system (= cell line)The production system (= cell line)The selected clonal populationThe selected clonal populationThe process (i.e. feeding strategy)The process (i.e. feeding strategy)

Cell culture under low glutamine or glucose concentration lead tCell culture under low glutamine or glucose concentration lead to o increase highincrease high--mannose type oligosaccharides and hybrid typemannose type oligosaccharides and hybrid type

However, in general,However, in general,No direct impact so far demonstrated in clinical studies:No direct impact so far demonstrated in clinical studies:

On halfOn half--life (when compared to human circulating IgG)life (when compared to human circulating IgG)On immunogenicityOn immunogenicity

and no clear link with chimeric, humanized and human.and no clear link with chimeric, humanized and human.

1. Boyd et al. Mol.Immunol.1995:Sialic acid → No effect

2. Kumpel et al. Hum. Antib. Hybrid. 1994: (+)Gal → 2 to 3-fold increase of ADCC

3. Umana et al. Nat. Biotech. 1999: (+)BisecGlcNAc → 10 to 20-fold increase of ADCC

4. Shields et al. JBC 2002: (-)Fuc → increase of ADCC and 50-fold increase

of FcγRIII binding 5. Shinkawa et al. JBC 2003:

(-)Fuc → more effective in ADCC than (+) Gal or (+)BisecGlcNAc

Asn297

:N:N--acetylglucosamineacetylglucosamine

:bisec GlcNAc:bisec GlcNAc

:Mannose:Mannose

:Galactose:Galactose

:Sialic acid:Sialic acid

：：FucoseFucose

In the future glycoproteins whose sugar chains are In the future glycoproteins whose sugar chains are designed to be modified intentionally should be used as designed to be modified intentionally should be used as

pharmaceuticals:pharmaceuticals:

FucoseFucoseFucose

Relationship between ADCC and structure of NRelationship between ADCC and structure of N--linked oligosaccharide linked oligosaccharide of antibody: Sugar residue is a target for enhancing ADCC of IgGof antibody: Sugar residue is a target for enhancing ADCC of IgG

Quality: ProductQuality: Product--related substancesrelated substances

Specifications of every types of antibody Specifications of every types of antibody should be set, if their structure is modified, should be set, if their structure is modified, even their binding to antigen and their even their binding to antigen and their toxicity are almost the same, because toxicity are almost the same, because there may be differences between normal there may be differences between normal antibodies and in their immunological antibodies and in their immunological functions, ADME, induction of antibodies functions, ADME, induction of antibodies (HAMA, HAHA), etc.(HAMA, HAHA), etc.

Quality : aggregatesQuality : aggregates

There is a possibility that aggregates There is a possibility that aggregates causes allergic reaction in human.causes allergic reaction in human.

should use an inshould use an in--line filter.line filter.should set appropriate specifications of should set appropriate specifications of insoluble matter tests, etc.insoluble matter tests, etc.

TopicsTopics


Immunogenicity of mAb drugsImmunogenicity of mAb drugs

Development of antibody (HACA, HAHA) to mAb should be Development of antibody (HACA, HAHA) to mAb should be evaluated timeevaluated time--dependently.dependently.

Antibody concentration should be checked before the first Antibody concentration should be checked before the first administration in repeated administration tests.administration in repeated administration tests.Should examine which region of mAb the antibody raised Should examine which region of mAb the antibody raised against.against.

Should consider the possibility that mAb itself might prevent Should consider the possibility that mAb itself might prevent the detection of the antibody in samples.the detection of the antibody in samples.

Incidence of antibodies Incidence of antibodies （（HACAHACA、、HAHAHAHA）） to mAb drugs and to mAb drugs and their effects on safety are varied.their effects on safety are varied.

Antibody drug Antibody type incidence

Oncoscint® (anti-TAG) murine IgG1 55%OKT®3 (anti-CD3) murine IgG2a ~80%Rituxan® (anti-CD20) chimeric IgG1 < 1%Simulect® (anti-IL2Ra) chimeric IgG1 < 2%ReoPro® (anti-GPIIb/IIIa) chimeric IgG1 Fab 7-19%Remicade® (anti-TNF) chimeric IgG1 10-57%Erbitux® (anti-EGFR) chimeric IgG1 5%Synagis® (anti-RSV) humanized IgG1 < 1%Herceptin® (anti-HER2) humanized IgG1 0.1%Zenapax® (anti-IL2Ra) humanized IgG1 8%Campath® (anti-CD52) humanized IgG1 < 2%Avastin® (anti-VEGF) humanized IgG1 None detectedHumira® (anti-TNF) human IgG1 (phage) > 5%

Incidence of antibodies in humans administered Incidence of antibodies in humans administered mAbmAb drugsdrugs

Current Pharmaceutical Biotechnology 3, 349-360 (2002)

TopicsTopics


Modification of functional properties Modification of functional properties of antibody moleculesof antibody molecules

Examples of Engineered Antibody FragmentsExamples of Engineered Antibody Fragments

Agonist (Signalling) antibodyAgonist (Signalling) antibody

Alternatives of biologically active Alternatives of biologically active substances such as hormones, cell growth substances such as hormones, cell growth factors (factors (antianti--TROP, antiTROP, anti--EPOR, antiEPOR, anti--GHR, GHR, antianti--GG--CSFRCSFR, etc.), etc.)Inducers of apoptosis (Inducers of apoptosis (antianti--FAS, antiFAS, anti--TRAILR, antiTRAILR, anti--HLA, antiHLA, anti--CD20CD20, etc.), etc.)Induction of proteinInduction of protein--protein interactions protein interactions ((Factor VIIIFactor VIII))

(From D & MD Reports, 2001)

Mechanisms of Mechanisms of mAbmAb drugsdrugs

ConclusionConclusion

Quality and safety evaluation methods, which Quality and safety evaluation methods, which have been established for recombinant protein have been established for recombinant protein drugs, are thought to be adequate in the case of drugs, are thought to be adequate in the case of mAb drugs, which neutralize or block mAb drugs, which neutralize or block physiological reaction by their binding to physiological reaction by their binding to bioactive substances as antigen; bioactive substances as antigen; However, further consideration for the safety However, further consideration for the safety should be required, including investigation of should be required, including investigation of mechanism of action in the case of new types of mechanism of action in the case of new types of mAbmAb drugs. drugs.

Thank you for your attention !!Thank you for your attention !!

Health & Medicine

Evaluation of antibody drugs quality safety