Usp biotherapeutics - biological medicines

Track II, Session I: Biological

Medicines–Biotherapeutics Wednesday, April 17, 2013 (9:00 a.m. to 11:00 a.m.)

IPC–USP Science & Standards Symposium

Partnering Globally for 21st Century Medicines

Moderator: Venkata Ramana, Ph.D. Reliance Life Sciences

Monograph Tests for Monoclonal

Antibody Therapeutics

Venkat Mukku, Ph.D.

Consultant, USP

International non-

proprietary name Trade name Type Indication first approved First EU (US) approval year

Muromonab-CD3 Orthoclone Okt3 Anti-CD3; Murine IgG2a Reversal of kidney transplant rejection 1986* (1986#)

Abciximab Reopro Anti-GPIIb/IIIa; Chimeric IgG1 Fab Prevention of blood clots in angioplasty 1995* (1994)

Rituximab MabThera, Rituxan Anti-CD20; Chimeric IgG1 Non-Hodgkin's lymphoma 1998 (1997)

Basiliximab Simulect Anti-IL2R; Chimeric IgG1 Prevention of kidney transplant rejection 1998 (1998)

Daclizumab Zenapax Anti-IL2R; Humanized IgG1 Prevention of kidney transplant rejection 1999 (1997); #

Palivizumab Synagis Anti-RSV; Humanized IgG1 Prevention of respiratory syncytial virus infection 1999 (1998)

Infliximab Remicade Anti-TNF; Chimeric IgG1 Crohn disease 1999 (1998)

Trastuzumab Herceptin Anti-HER2; Humanized IgG1 Breast cancer 2000 (1998)

Gemtuzumab ozogamicin Mylotarg Anti-CD33; Humanized IgG4 Acute myeloid leukemia NA (2000#)

Alemtuzumab

MabCampath, Campath-

1H Anti-CD52; Humanized IgG1 Chronic myeloid leukemia 2001 (2001)

Adalimumab Humira Anti-TNF; Human IgG1 Rheumatoid arthritis 2003 (2002)

Tositumomab-I131 Bexxar Anti-CD20; Murine IgG2a Non-Hodgkin lymphoma NA (2003)

Efalizumab Raptiva Anti-CD11a; Humanized IgG1 Psoriasis 2004 (2003); #

Cetuximab Erbitux Anti-EGFR; Chimeric IgG1 Colorectal cancer 2004 (2004)

Ibritumomab tiuxetan Zevalin Anti-CD20; Murine IgG1 Non-Hodgkin's lymphoma 2004 (2002)

Omalizumab Xolair Anti-IgE; Humanized IgG1 Asthma 2005 (2003)

Bevacizumab Avastin Anti-VEGF; Humanized IgG1 Colorectal cancer 2005 (2004)

Natalizumab Tysabri Anti-a4 integrin; Humanized IgG4 Multiple sclerosis 2006 (2004)

Ranibizumab Lucentis Anti-VEGF; Humanized IgG1 Fab Macular degeneration 2007 (2006)

Panitumumab Vectibix Anti-EGFR; Human IgG2 Colorectal cancer 2007 (2006)

Eculizumab Soliris Anti-C5; Humanized IgG2/4 Paroxysmal nocturnal hemoglobinuria 2007 (2007)

Certolizumab pegol Cimzia Anti-TNF; Humanized Fab, pegylated Crohn disease 2009 (2008)

Golimumab Simponi Anti-TNF; Human IgG1 Rheumatoid and psoriatic arthritis, ankylosing spondylitis 2009 (2009)

Canakinumab Ilaris Anti-IL1b; Human IgG1 Muckle-Wells syndrome 2009 (2009)

Catumaxomab Removab

Anti-EPCAM/CD3;Rat/mouse bispecific

Mab Malignant ascites 2009 (NA)

Ustekinumab Stelara Anti-IL12/23; Human IgG1 Psoriasis 2009 (2009)

Tocilizumab RoActemra, Actemra Anti-IL6R; Humanized IgG1 Rheumatoid arthritis 2009 (2010)

Ofatumumab Arzerra Anti-CD20; Human IgG1 Chronic lymphocytic leukemia 2010 (2009)

Denosumab Prolia Anti-RANK-L; Human IgG2 Bone Loss 2010 (2010)

Belimumab Benlysta Anti-BLyS; Human IgG1 Systemic lupus erythematosus 2011 (2011)

Ipilimumab Yervoy Anti-CTLA-4; Human IgG1 Metastatic melanoma 2011 (2011)

Brentuximab vedotin Adcetris

Anti-CD30; Chimeric IgG1;

immunoconjugate Hodgkin lymphoma 2012 (2011)

Pertuzumab Perjeta Anti-HER2; humanized IgG1 Breast Cancer 2013 (2012)

Raxibacumab (Pending) Anti-B. anthrasis PA; Human IgG1 Anthrax infection NA (2012)

Trastuzumab emtansine Kadcyla

Anti-HER2; humanized IgG1;

immunoconjugate Breast cancer 2013

Vedolizumab (Pending)

Anti-alpha4beta7 integrin; humanized

IgG1 Ulcerative colitis, Crohn disease In review (NA)

Source: Janice M. Reichert, Editor-in-Chief, Mabs

Recombinant Therapeutic Mabs approved or in review in the EU and US (as of 2-28-2013)

Mabs ranked in the Top Ten Drugs in 2012

• Humira (AbbVie) - Mab

• Advair (Glaxo Smith Kline)

• Rituxan (Roche) - Mab

• Lantus (Sanofi)

• Herceptin (Roche) - Mab

• Crestor (AstraZeneca)

• Remicade (Johnson & Johnson) - Mab

• Avastin (Roche) - Mab

• Cymbalta (Eli Lilly and Co)

• Plavix (Sanofi & Bristol-Myers Squibb)

Developing Standards for Mabs

• Structure is complex, often heterogeneous, with a mix of isoforms,

glycoforms etc.

• Need to understand the importance of each form

• Many types of impurities, requiring different types of analytical

methods. Impurities often are manufacturing process-dependent

• Potency assay (Bioassay) is a key quality attribute. Multiple potency

assays are needed to address different functional domains

Challenges in developing Mab standards

CH1

VL

CL

CH2

CH3

Constant

Region Fc

Variable

Region

• Effector Functions

ADCC (FcgRI, II, III)

CDC (C1q)

• FcRn binding

IgG Structure and Functional Domains

Multiple domains lead to multiple variants requiring a battery

of physicochemical and biological tests

Mab Variants / Isoforms

• N-terminal Pyro-Glu

• Iso-Asp formation

• Deamidation

• Methionine oxidation

• Heavy Chain C-terminal Lysine

• Heavy chain C-terminal Prolinamide variants

• Disulfide Variants

• Free Cys adducts

• Glycosylation variants

– High mannose, G0, G1, G2

– Fucosylation

– Sialic acid capping

CH1

VL

CL

CH2

CH3

Not all variants impact safety and/or efficacy

Characterization Tests for Mabs

• Primary Structure

– LC/MS Peptide Maps, 2nd enzyme

– N-terminal Sequence

– C-terminal Prolinamide formation

– Disulfide Bond Determination

– Intact Mass Determination

– Amino Acid Analysis (AAA)

Others

– DSC

– AUC

– SEC-MALS

– Extinction Coefficient determination

– Excipients methods

• Secondary and

Tertiary Structure

– FTIR

– Far UV CD

– Near UV CD

– Intrinsic fluorescence

Functional characterization tests

– Additional cell-based assays

– Antigen binding

– Effector functions

– Fcg RI, RIII binding

– FcRn binding

Most of these tests are not used in routine testing, but are important for detailed

understanding of the product and Comparability / Biosimilarity

Quality Tests for Mabs

Quality Attribute Test

Identity Bioassay, Peptide map, cIEF

Potency

Function mediated by antigen binding site

Fc-mediated effector functions

Antigen binding

Purity: Glycosylation variants Glycan profiling: Oligosaccharide mapping

Product -Related Impurities

High molecular weight. impurities Size Exclusion Chromatography , CE-SDS (Reduced)

Low molecular weight impurities CE-SDS under non-reducing conditions (CE-SDS-NR)

Charge heterogeneity Cation exchange chromatography (CEX)

Methionine oxidation Protein-A HPLC or Peptide map LC-MS

Strength (protein concentration) A280 nm

Process-Related Impurities Host Cell Proteins, DNA, Protein-A

Compendial tests pH, endotoxin, osmolality, microbial tests, excipients,

particulates, visual tests

Potency Assays for Mabs

Depend upon the mechanism of action (MoA)

• Anti-Proliferation, cytostatic effects

• Antibody-toxin Mediated Cytotoxicity

• Complement dependent cytotoxicity (CDC)

• Antibody Dependent Cell-mediated Cytotoxicity assay (ADCC)

• Apoptosis

• Inhibit inflammatory cytokine activity or secretion

• Etc.

Activation of

Complement

Cascade

Target B-Cell

Dead cell

subsequent to

CDC

Rituximab

C1q

Complement Dependent Cytotoxicity (CDC)

ADCC Activity

Effector Cell

Target B-Cell

Fc Receptor RI, RIII

Cytotoxic

mediators

Rituximab

Antibody Dependent Cell-Mediated Cytotoxicity

Dead B-Cell

upon ADCC

Potency Assays for Rituximab

CDC assay

Cell line: WIL-2S

Cell viability measured by AlamarBlue

Readout: fluorescence

ADCC assay

Target cells: WIL2-S

Effector cells: Jurkat cell engineered

to express FcgRIII and NFAT-luciferase

reporter gene;

Readout: luminescence

CDC-Based Potency Assay for Rituximab

Acceptable Procedure USP Reference Procedure

Cell line and bioassay: CDC assay using CD20+

cells

CDC assay using WIL-2S cells, human

complement and Resazurin-based

detection reagent

Data Analysis: Relative potency (RP) calculated

according to appropriate data analysis guidelines

Relative potency calculated per USP

<1034> and validated per USP <1033>

Specificity: Unrelated Mab and Formulation buffer

should not show activity.

Unrelated Mab and Formulation buffer

showed no activity.

Precision (Repeatability): RSD ≤ 10% RSD 5%

Precision (Intermediate precision): RSD ≤ 15% RSD 9%

Linearity: r2 > 0.9 measured and expected

potency

r2 1.0

Accuracy: Spike recovery 85%–115% 95%–105%

Range: Minimally cover 80%-125%; wider is better 64% to 156%

Peptide Maps for Identity of Mabs

Peptide Maps for Identity of Mabs

Tryptic peptide maps showing differences between 3 Mabs

Rituximab

Bevacizumab

Trastuzumab

cIEF as an Identity Test for Mab

Role of Fc Glycans in Mab Functions

• Terminal galactose is important for CDC

• N-Acetyl glucosamine essential for ADCC

• Bisecting N-Acetyl glucosamine enhances ADCC

• Core fucose inhibitory to ADCC; afucosylated variants show higher ADCC

• High-mannose glycans reduce half life of IgG

0

20

40

60

80

100

120

% P

ote

nc

y b

y C

DC

as

sa

y

Degalactosylation

abolishes 40% of

CDC activity.

Treatment with Beta-Galactosidase

Glycan Profiling of Mabs

Deglycosylation with PNGase followed by 2-Aminobenzamide labeling and Normal

Phase UPLC. Peaks are characterized using 2AB-Labeled glycan standards

Mannose

N-acetyl glucosamine

Core Fucose

Sialic Acid

Galactose

c

c

EU

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Minutes

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

G0F

G0

G0F

-GN

G0-G

N

Man

5

G1

G1’

G1F

G1F

’

G2F

G2

G2F

S1

G2F

S2

N- & C-terminal Charge Variants of Mabs

S

L

S

L

S

P

G

S

L

S

L

S

P

G

K

Fully Processed Partially Processed

S

L

S

L

S

P

G

K

S

L

S

L

S

P

G

Partially Processed

S

L

S

L

S

P

G

K

S

L

S

L

S

P

G

K

Un-Processed

S

L

S

L

S

P

G

CH1 VL

CL

CH2

CH3

S

L

S

L

S

P

G

VH E E

E pE E

pE pE pE pE

E = N-terminal glutamate; pE = N-terminal pyroglutamate; K= C-terminal lysine

pE

Other charge variants: Deamidation, glycation, terminal sialic acid

Degradation-Related Charge Variants of Mabs

CEX Chromatography of Rituximab

Before Carboxy

Peptidase

treatment

After Carboxy

Peptidase

treatment

Size Exclusion Chromatography

Retuximab

standard

Retuximab

heat treated to

generate HMW

impurities

Capillary Electrophoresis-SDS (CE-EDS)

Non-reducing conditions

(CE-SDS-NR) to quantitate

low molecular weight

impurities

Reducing conditions (CE-

SD-R) to quantitate Non-

Glycosylated Heavy Chain

and

Biosimilar Guideline in Korea and

ASEAN Countries

Mr. Bonjoong Kim,

Celltrion, Inc.

CONTENTS

SECTION I. Overview On Global Biosimilar Guidelines

SECTION III. Biosimilar Guidelines in KOREA

SECTION IV. Biosimilar Guidelines in ASEAN

SECTION V. Executive Summary

SECTION II. KOREA & ASEAN Regulatory Environments

SECTION I

Overview On Global Biosimilar Guidelines

Global Regulation or Guideline on Biosimilar

[SUMMARY]

EU: In fact, world first well-organized biosimilar legislation and guideline were implemented in EU. KOREA: Biosimilar guidelines have been issued since first biosimilar guideline was implemented in 2009. ASEAN: Malaysia and Singapore are leading implementation of biosimilar guidelines in ASEAN.

2011 2012

USA

India

2013

EGY SNG

[SUMMARY]

Biosimilar Pathway: EU, Brazil, Argentina,Turkey, Malaysia, Canada, Mexico, Korea, Japan, India, Singapore, Egypt, Saudi Arabia, Australia, US, Venezuela

Biosimilar Pathway under Development: Peru, Chile, Ecuador, Colombia, Bolivia, Paraguay, Uruguay, Pakistan, South Africa, New Zealand, Philippines, Hong Kong, Vietnam, Indonesia, Thailand

Biosimilar Pathways

Biosimilar Pathways under Development

No Biosimilar Pathway

Global Regulation or Guideline on Biosimilar

SECTION II

KOREA & ASEAN Regulatory Environments

[SUMMARY]

Korea, Singapore and Malaysia has their own biosimilar pathway. Other ASEAN countries does not have official biosimilar pathway yet.

Guideline on Biosimilar in KOREA & ASEAN

Biosimilar Pathways

Biosimilar Pathways under Development

No Biosimilar Pathway

CLASSIFICATION COUNTRY

Biosimilar Pathways (1+2)

Korea, Malaysia, Singapore

Biosimilar Pathways under Development (4)

Philippines, Vietnam, Indonesia, Thailand

No Biosimilar Pathway (4)

Laos, Myanmar, Cambodia, Brunei Darussalam

http://www.google.co.kr/url?sa=i&rct=j&q=asean+countries&source=images&cd=&cad=rja&docid=wng1IW8Qh3keMM&tbnid=VmywB3Fxxy9RNM:&ved=0CAUQjRw&url=http://www.bangkokbusinessbrief.com/2012/04/04/aseans-overall-attractiveness-matters-for-business-investment-decisions/&ei=BA1QUeS_HMj7qwGn54GoAg&bvm=bv.44158598,d.aWM&psig=AFQjCNHnhkzlpBZ8ZkGfOvwX4Lo8wHkyHg&ust=1364287083054035

[SUMMARY]

Regulatory Agency in Indonesia and Thailand has tendency to treat biosimilar filing as new biologics with less requirements

Regulatory Agency in Indonesia and Thailand has tendency to treat biosimilar filing as generic pathway with more requirements

Regulatory Environment in Korea & ASEAN

Regulatory Environment COUNTRY

Biosimilar Pathway Korea, Malaysia, Singapore

New biologics (with less requirement)

Indonesia, Thailand

Generic Pathway Philippines, Vietnam

Unknown Laos, Myanmar, Cambodia, Brunei Darussalam

[SUMMARY]

KOREA: Regulatory Agency is being changed with its name and function. Philippines: Regulatory Agency is being changed with new PFDA commissioner. Thailand and Malaysia: GMP Inspection or Accreditation is time-consuming.

Regulatory Environment in Korea & ASEAN

COUNTRY Regulatory Environment

Korea Name and organization of Korea FDA was changed in March 2013. Name: KFDA MDFS (Ministry of Drug and Food Safety) Organization and Function: Being changed to extend its function.

Singapore EU or US CPP is required.

Malaysia Long waiting list for GMP Inspection.

Thailand TFDA is now preparing biosimilar guideline GMP Accreditation Process was implemented.

Philippines Organization of PFDA is being changed with new commissioner.

Indonesia Local Manufacturing is required.

Vietnam Special Quota VISA & Valid VISA are available.

Laos, Myanmar, Cambodia, Brunei Darussalam

Not known.

SECTION III

Biosimilar Guidelines in KOREA

Guideline on Biosimilar in KOREA

<Reference: EMA Guidelines>

mAb

Non-

clinical

Clinical


[SUMMARY]

KOREA has similar structure with EU biosimilar guideline Legislation: Regulations on Review & Authorization of Biological Products (KFDA Notification No. 2009-59) Guideline: 8 relevant guidelines and 1 Q&A are now available.

Definition/Principle

General Guidelines

Quality, Safety, Efficacy

Product Specific Requirement

Overarching Guideline Guidelines on the Evaluation of Biosimilar Products (2009)

SCOPE In principle: Biosimilar concept applicable to any biological medicinal product. In practice: Only for products that can be thoroughly characterised.


[Choice of Reference Product]

Korea EU (Reference Information)

The reference product should be authorized in Korea. However, if the registered reference product is not available in Korea commercially, the same biological product as the one authorized in Korea (including the manufacturing site and the manufacturing process) may be purchased from overseas markets and used as the reference product.

Must be authorised in the EU based on a complete dossier.



[Quality Requirement]

Korea EU (Reference Information)

A complete description of the manufacturing process for the drug substance and drug product should be provided in detail including information on quality control/quality assurance, in-process controls, and process validation. Comparability study along with specifications, analytical procedures and stability study should be conducted.

For recombinant proteins, an extensive comparability exercise is required. Quality aspects of comparability must be considered in relation to implications for safety and efficacy. Purity and impurity profiles of the active substance and medicinal product must be assessed qualitatively and quantitatively for the biosimilar and the reference product.



[Nonclinical Data] Korea EU (Reference Information)

„„▶ In vitro studies: Assays, such as receptor-binding studies or cell-based assays should normally be undertaken in order to establish the comparability of the biological/ pharmacodynamic activity of the biosimilar product and the reference product, should be conducted „„▶ In vivo studies: - Animal studies should be performed in species know to be relevant - At least one repeat-dose toxicity study - Other toxicological studies, including safety pharmacology, reproductive toxicology, mutagenicity and carcinogenicity studies are not required for biosimilar unless warranted by the results from repeated toxicological studies.

Non-clinical studies for biosimilar versions of recombinant proteins should be comparative in nature, designed to detect differences in response between the biosimilar and the reference product. In vitro studies, in vivo studies in relevant animal species, and at least one repeat-dose toxicity study in relevant animal species normally should be conducted.



[Clinical Data] Korea EU (Reference Information)

The clinical comparability studies include pharmacokinetic, pharmacodynamic, and efficacy studies. If the comparability can be demonstrated by confirmatory pharmacokinetic/pharmacodynamic data, an efficacy study may be omitted.

For recombinant proteins, comparative efficacy clinical trials usually are necessary to demonstrate clinical comparability. The clinical requirements depend on the existing knowledge about the reference product and the claimed therapeutic indication(s). For recombinant proteins, a biosimilar’s immunogenicity must always be investigated. Immunogenicity risks in different indications should be considered separately.



[Extrapolation of Indication] Korea EU (Reference Information)

Extrapolation of these data to other indications of the reference product for which post-marketing survey was completed may be possible if all of the following conditions are fulfilled: A sensitive clinical test model has

been used that is able to detect potential differences between the biosimilar product and the reference product;

The clinically relevant mechanism of action and/or involved receptor(s) are the same;

Safety and immunogenicity have been sufficiently characterized.

For recombinant proteins, in certain cases, it may be possible to extrapolate therapeutic similarity to other indications. Justification of extrapolation depends on clinical experience, available literature, whether the same mechanism of action or receptor is involved in both indications, and possible safety issues in different subpopulations.



[PV and RMP] Korea EU (Reference Information)

Safety data obtained from clinical trials are usually sufficient for product authorization, but further close monitoring of clinical safety of the biosimilar product is usually necessary in the post-marketing phase.

By law, a risk management plan or pharmacovigilance plan must be submitted for biosimilars as for other medicines. The plan should take into account risks identified during product development and potential risks and how those risks will be addressed after authorisation For recombinant proteins, clinical safety must be monitored closely after authorisation. The application should include a risk specification and pharmacovigilance plan.

SECTION IV

Biosimilar Guidelines in ASEAN

Guideline on Biosimilar in ASEAN – SINGAPORE & MALAYSIA

[Choice of Reference Product]

SINGAPORE MALAYSIA

GUIDANCE ON MEDICINAL PRODUCT REGISTRATION IN SINGAPORE (APPENDIX 17 GUIDANCE ON REGISTRATION OF SIMILAR BIOLOGICAL PRODUCTS IN SINGAPORE)

GUIDANCE DOCUMENT FOR APPLICANTS : INFORMATION AND SUBMISSION REQUIREMENTS FOR REGISTRATION OF BIOSIMILARS

SINGAPORE MALAYSIA EU (Reference Information)

The reference product must be registered in Singapore.

The reference product must be registered in Malaysia.



SINGAPORE MALAYSIA




Comparability data between the biosimilar product and the reference product (in terms of quality) must be submitted in the quality dossier. The extent of the comparability studies and the assessment criteria depends on the complexity of the product and the capability of the methods used to demonstrate comparability. The comparability exercise should entail evaluation of both drug substance and drug product.

A full quality dossier is always required. In addition, extensive data focused on the similarity, including comprehensive side-by-side physicochemical and biological characterisation of the biosimilar and the reference product should be submitted


[Quality Requirement]


SINGAPORE MALAYSIA




In vitro studies: Assays like receptor-binding studies or cell-based assays should normally be undertaken in order to establish comparability in reactivity and the likely causative factor(s) if comparability cannot be established. Animal studies should be performed to investigate pharmacodynamic effect/ activity relevant to the clinical application, non-clinical toxicity as determined in at least one repeat dose toxicity study, including toxicokinetic measurements, and specific safety concerns. Safety pharmacology, reproduction toxicology, mutagenicity and carcinogenicity studies are not required for biosimilar products, unless indicated by the results of repeat dose studies.

„„▶ In vitro studies: Receptor-binding studies or cell-based assay should be conducted „„▶ In vivo studies: - Animal pharmacodynamic study where approriate, relevant to clinical use - At least one repeat-dose toxicity study Other toxicological studies, including safety pharmacology, reproductive toxicology, mutagenicity and carcinogenicity studies are not required for biosimilar unless warranted by the results from repeated toxicological studies.


[Nonclinical Data]


SINGAPORE MALAYSIA




The clinical comparability exercise should begin with pharmacokinetic (PK) and pharmacodynamic (PD) studies followed by clinical efficacy and safety studies. Comparative PK studies designed to demonstrate clinical comparability between the biosimilar product and the reference product with regard to key PK parameters are required. Pharmacodynamic studies to demonstrate therapeutic efficacy of the product is required. The immunogenicity of a biosimilar product must always be investigated. The assessment of immunogenicity requires an optimal antibody testing strategy, characterisation of the observed immune response, as well as evaluation of the correlation between antibodies and pharmacokinetics or pharmacodynamics, relevant for clinical safety and efficacy in all aspects.

Comparative pharmacokinetic studies should be conducted to demonstrate the similarities in pharmacokinetic (PK) characteristics between biosimilar and the reference product. Clinical efficacy trials should also be coundcuted. Other PK studies such as interaction studies or other special populations (e.g children, elderly, patients with renal or hepatic insufficiency) are usually not required. Studies should demonstrate clinical sfaety and immunogenicity of the drug product.


[Clinical Data]


SINGAPORE MALAYSIA




In case the reference medicinal product has more than one indication, the efficacy and safety of the biosimilar product has to be justified or, if necessary, demonstrated separately for each of the claimed indications. In certain cases it may be possible to extrapolate therapeutic similarity shown in one indication to other indications of the reference medicinal product. Justification will depend on e.g., clinical experience, available literature data, whether or not the same mechanisms of action or the same receptor(s) are involved in all indications. Possible safety issues in different subpopulations should also be addressed.

Demonstration of similarity may also allow extrapolation of efficacy and safety data to other indications of the reference product.


[Extrapolation of Indication]


SINGAPORE MALAYSIA




ADR reporting, PSURs, Risk management plan, Educational Materials and Product Sales Data for the biosimilar product is required

The pharmacovigilance, as part of a comprehensive RMP, should include regular testing for consistent manufacturing of the biosimilar. The pharmacovigilance plan must be approved prior to approval of product and the system must be in place to conduct monitoring.

By law, a risk management plan or pharmacovigilance plan must be submitted for biosimilars as for other medicines. The plan should take into account risks identified during product development and potential risks and how those risks will be addressed after authorisation. For recombinant proteins, clinical safety must be monitored closely after authorisation. The application should include a risk specification and pharmacovigilance plan.

[PV&PMP]


SINGAPORE MALAYSIA




A product is interchangeable with another if both products are approved for the same indication, and can be used for the said indication. Two products are substitutable with each other if they can both be used in lieu of the other during the same treatment period. For interchangeable products, one or the other can be used (prescribed) but these products cannot be substituted with one another during a treatment period. Interchangeability does not imply substitutability. Unlike generic chemical drugs, whereby the chemical structure is identical to that of the reference chemical product, a biosimilar product does not usually have an identical structure to the reference biological product. Therefore, even though a biosimilar product may be approved to be similar in terms of quality, safety and efficacy to the reference product, immunogenicity may preclude switching between products.

Given current science, biosimilars cannot be considered inter-changeable with the reference product or products of the same class.

Substitution is determined at the member state level, and therefore this topic is not directly addressed in EMA guidance. EMA guidance states that biosimilars are not generic medicinal products and that the decision to treat a patient with a reference product or a biosimilar should be made following the opinion of a qualified healthcare professional.

[PV&PMP]

SECTION IV

Executive Summary

Executive Summary – Regulation & Guideline

Topic Reference

KOREA ASEAN

EU US JAPAN WHO SINGAPORE MALAYSIA

Law (Regulation)

Directive 2001/83/EC The Biologics Price Competition and Innovation Act of 2009 (sections 7001 to 7003 of the Patient Protection and Affordable Care Act, Pub. L. 111-148 (2010)).

Ministry of Health Labor and Welfare, Pharmaceutical and Food Safety Bureau, Guideline for Ensuring Quality, Safety, and Efficacy for Biosimilar Products, PFSB/ELD Notification No. 0304007.

Regulations on Review & Authorization of Biological Products (KFDA Notification No. 2009-59)

▶ Medicines Act (Chapter 176) ▶ Poisons Act (Chapter 234) ▶ Misuse of Drugs Regulations – subsidiary legislation under the Misuse of Drugs Act (Chapter 185) ▶ Sale of Drugs Act (Chapter 282) ▶ Medicines (Advertisement and Sale) Act (Chapter 177)

Control of Drugs and Cosmetic Regulations 1984 (CDCR 1984)

Guideline

„„▶ Guideline on Similar Biological Medicinal Products EMEA/CHMP/437/04. „„▶ Guideline on Similar Biological Medicinal Products Containing Biotechnology-Derived Proteins as Active Substance: Nonclinical and Clinical Issues EMEA/CHMP/BMWP/42832/2005. „„„▶ Guideline on Similar Biological Medicinal Products Containing Biotechnology-Derived Proteins as Active Substance: Quality Issues EMEA/CHMP/BWP/49348/2005. „„„▶ Guideline on Immunogenicity Assessment of Biotechnology-Derived Therapeutic Proteins EMEA/CHMP/BMWP/14327/06.

▶ Formal Meetings Between the FDA and Biosimilar Biological Product Sponsors or Applicants ▶ Quality Considerations in demonstrating biosimilarity to a reference protein product ▶ Questions and Answers ▶ Scientific Considerations in demonstrating biosimilarity to a reference product

WHO, Guidelines on Evaluation of Similar Biotherapeutic Products (SBPs).

Guidelines on the Evaluation of Biosimilar Products

GUIDANCE ON MEDICINAL PRODUCT REGISTRATION IN SINGAPORE - APPENDIX 17


<Regulation & Guideline>

Executive Summary – Reference Product

Topic Reference

KOREA ASEAN


Reference Product


Must be licensed under a full biologics licence application (under section 351(a) of the Public Health Service Act).

Must be authorized in Japan.

Should be authorised in the country or region in question (or, where the licensing country lacks an authorised reference product, should be authorised and widely marketed in another jurisdiction with a well-established regulatory framework for and experience in evaluation and post-market surveillance of biotherapeutics).

The reference product should be authorized in Korea. However, if the registered reference product is not available in Korea commercially, the same biological product as the one authorized in Korea (including the manufacturing site and the manufacturing process) may be purchased from overseas markets and used as the reference product.

The reference product must be registered in Singapore

The reference product must be registered in Malaysia

<Reference Product>

Executive Summary – Quality

Topic Reference

KOREA ASEAN


Quality Requirement


Unless the Secretary makes a determination that it is unnecessary, an application must contain data from analytical studies demonstrating that the biosimilar is highly similar to the reference product (notwithstanding minor differences in clinically inactive components).

A biosimilar must be fully characterised, including by conducting studies comparing the structure and composition, physicochemical properties, bioactivity, and immunologic properties of the biosimilar against its reference product.

Comprehensive physicochemical and biological characterisation of the biosimilar in head-to-head comparisons with the reference product is required, and all aspects of quality and heterogeneity should be assessed.

A complete description of the manufacturing process for the drug substance and drug product should be provided in detail including information on quality control/quality assurance, in-process controls, and process validation. Comparability study along with specifications, analytical procedures and stability study should be conducted.

Comparability data between the biosimilar product and the reference product (in terms of quality) must be submitted in the quality dossier. The extent of the comparability studies and the assessment criteria depends on the complexity of the product and the capability of the methods used to demonstrate comparability. The comparability exercise should entail evaluation of both drug substance and drug product.

A full quality dossier is always required. In adiition, extensive data focused on the similarity, including comprehensive side-by-side physicochemical and biological characterisation of the biosimilar and the reference product should be submitted.

<Quality>

Executive Summary – Nonclinical Data

Topic Reference

KOREA ASEAN


Nonclinical Data


Unless the Secretary makes a determination that it is unnecessary, an application must include data derived from animal studies (including the assessment of toxicity) to help demonstrate that the product is biosimilar to a reference product. It is important to understand the limitations of such animal studies (e.g., small sample size, intra-species variations) when interpreting results comparing the proposed product and the reference product. A sponsor may be able to provide a scientific justification for a stand-alone toxicology study using only the proposed product instead of a comparative toxicology study. For a more detailed discussion on the design of animal toxicology studies, see ICH S6/S6(R1).

Before performing clinical studies, the biosimilar applicant must conduct non-clinical studies to verify that the product can be safely administered to humans. Before conducting non-clinical studies, a biosimilar must be subjected to a full quality characterisation. The pharmacological action of the biosimilar and its reference product should be compared through nonclinical pharmacological studies, and repeat-dose toxicity and toxicokinetic studies may be useful.

The non-clinical evaluation should include pharmacodynamic, pharmacokinetic, and comparative repeat-dose toxicity studies in a relevant species. The amount of additional non-clinical data required is dependent on product-specific factors. In vitro assays like receptor binding studies or cell-based assays should normally be conducted to establish comparability of pharmacodynamic activity. Animal studies in a relevant species should generally be conducted.

„„▶ In vitro studies: Assays, such as receptor-binding studies or cell-based assays should normally be undertaken in order to establish the comparability of the biological/ pharmacodynamic activity of the biosimilar product and the reference product. should be conducted „„▶ In vivo studies: - Animal studies should be performed in species know to be relevant - At least one repeat-dose toxicity study - Other toxicological studies, including safety pharmacology, reproductive toxicology, mutagenicity and carcinogenicity studies are not required for biosimilar unless warranted by the results from repeated toxicological studies.

In vitro studies: Assays like receptor-binding studies or cell-based assays should normally be undertaken in order to establish comparability in reactivity and the likely causative factor(s) if comparability cannot be established. Animal studies should be performed to investigate pharmacodynamic effect/ activity relevant to the clinical application, non-clinical toxicity as determined in at least one repeat dose toxicity study, including toxicokinetic measurements, and specific safety concerns. Safety pharmacology, reproduction toxicology, mutagenicity and carcinogenicity studies are not required for biosimilar products, unless indicated by the results of repeat dose studies.

„„▶ In vitro studies: Receptor-binding studies or cell-based assay should be conducted „„▶ In vivo studies: - Animal pharmacodynamic study where approriate, relevant to clinical use - At least one repeat-dose toxicity study Other toxicological studies, including safety pharmacology, reproductive toxicology, mutagenicity and carcinogenicity studies are not required for biosimilar unless warranted by the results from repeated toxicological studies.

<Nonclinical Data>

Executive Summary – Clinical Data

Topic Reference

KOREA ASEAN


Clinical Data


Unless the Secretary makes a determination that it is unnecessary, an application must include data derived from a clinical study or studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity, and potency in one or more appropriate conditions of use for which the reference product is licensed and intended to be used. The purpose of these data is to help demonstrate that the product is biosimilar to a reference product.

Clinical studies should generally be required, but they may not be required where non-clinical data are sufficient to assure bioequivalence and quality equivalence (if bioequivalence and quality equivalence have been demonstrated). Pharmacokinetic or pharmacodynamic study results are, however, inconclusive concerning clinical efficacy. Clinical studies should be conducted to verify efficacy for the specific indications for which the biosimilar applicant seeks authorisation.

Clinical studies should be designed to demonstrate comparable safety and efficacy of the biosimilar and reference product. Clinical trials are required to demonstrate similar efficacy. Immunogenicity should always be investigated in humans before authorisation.

The clinical comparability studies include pharmacokinetic, pharmacodynamic, and efficacy studies. If the comparability can be demonstrated by confirmatory pharmacokinetic/pharmacodynamic data, an efficacy study may be omitted.

The clinical comparability exercise should begin with pharmacokinetic (PK) and pharmacodynamic (PD) studies followed by clinical efficacy and safety studies. Comparative PK studies designed to demonstrate clinical comparability between the biosimilar product and the reference product with regard to key PK parameters are required. Pharmacodynamic studies to demonstrate therapeutic efficacy of the product is required. The immunogenicity of a biosimilar product must always be investigated. The assessment of immunogenicity requires an optimal antibody testing strategy, characterisation of the observed immune response, as well as evaluation of the correlation between antibodies and pharmacokinetics or pharmacodynamics, relevant for clinical safety and efficacy in all aspects.

Comparative pharmacokinetic studies should be conducted to demonstrate the similarities in pharmacokinetic (PK) characteristics between biosimilar and the reference product. Clinical efficacy trials should also be coundcuted. Other PK studies such as interaction studies or other special populations (e.g children, elderly, patients with renal or hepatic insufficiency) are usually not required. Studies should demonstrate clinical sfaety and immunogenicity of the drug product.

<Clinical Data>

Executive Summary – Extrapolation of Indication

Topic Reference

KOREA ASEAN


Extrapolation of Indication


The sponsor will need to provide sufficient scientific justification for extrapolating clinical data to support a determination of biosimilarity for each condition of use for which licensure is sought. Such scientific justification should address, for example, the following issues for the tested and extrapolated conditions of use. The MOA(s) in each condition of use for which licensure is sought. The PK and bio-distribution of the product in different patient populations; PD measures may provide important information on the MOA.

Extrapolation may be permitted if the mechanism of action is not unclear, the applicant can show that a similar pharmacological result can be expected for the relevant indications, and the mechanism of action does not differ among indications.

Extrapolation may be possible if a sensitive clinical test model has been used that is able to detect potential differences between the products, that the mechanism of action and/or receptors are the same, and that the safety and immunogenicity of the biosimilar have been characterised and there are no special safety issues expected with the extrapolated indication.

Extrapolation of these data to other indications of the reference product for which post-marketing survey was completed may be possible if all of the following conditions are fulfilled: - A sensitive clinical test model has been used that is able to detect potential differences between the biosimilar product and the reference product; - The clinically relevant mechanism of action and/or involved receptor(s) are the same; - Safety and immunogenicity have been sufficiently characterized.

In case the reference medicinal product has more than one indication, the efficacy and safety of the biosimilar product has to be justified or, if necessary, demonstrated separately for each of the claimed indications. In certain cases it may be possible to extrapolate therapeutic similarity shown in one indication to other indications of the reference medicinal product. Justification will depend on e.g., clinical experience, available literature data, whether or not the same mechanisms of action or the same receptor(s) are involved in all indications. Possible safety issues in different subpopulations should also be addressed.

Demonstration of similarity may also allow extrapolation of efficacy and safety data to other indications of the reference product.

<Extrapolation of Indication>

Executive Summary – Naming

Topic Reference

KOREA ASEAN


Naming

To support pharmacovigilance monitoring, the specific medicinal product given to the patient should be clearly identified. The name, appearance, and packaging of a biosimilar medicine should differ from those of the reference product.

Not addressed. Notification PFSB/EKD No. 0304011 regulates the naming of biosimilars and states that the non-proprietary names and proprietary names of biosimilars should be readily distinguishable from the names of reference products and other biosimilars. For non-proprietary names, the following should be added to the end of the non-proprietary name: Follow-on 1 [2, 3, and so on]. For proprietary names, the letters BS should be added to the end of the name, along with the dosage form, dosage, and name of the manufacturer.

Biosimilars should be clearly identifiable by a unique proprietary name. Where an international nonproprietary name (INN) is defined, that name should be stated and the WHO’s policy on INNs should be followed.

Not Adressed Not Adressed All biosimilars should be distinguishable by name i.e assign a brand name explicitly, using names that are not suggestive towards the originator nor towards other biosimilars.

<Naming>

Executive Summary – Labeling

Topic Reference

KOREA ASEAN


Labeling

Not addressed. Not addressed. Not addressed. The prescribing information for the biosimilar should be as similar as possible to that of the reference product, except for product-specific information, such as different excipient(s). If the biosimilar has fewer indications than the reference product, the information related to those indications may be omitted unless it is considered important to inform doctors and patients about certain risks. In these cases, the prescribing information should clearly state that the biosimilar is not indicated for use in the specific indication(s) and the reasons for it. A national regulatory authority may choose to require prescribing information to mention the product is a biosimilar, discuss the studies performed with the biosimilar, and/or include instructions to the physician on how to use the biosimilar.

Not Adressed Not Adressed It is expected that the labeling of biosimilar meet the following criteria: - A clear indication that the medicine is a biosimilar of a specific reference product - The invented name, common or scientific name and the manufacturer’s name - Clinical data for the biosimilar describing the clinical similarity (i.e safety and efficacy) to the reference product and in which indication(s) - Interchangeability and substitution advice should clearly and prominently state that the biosimilar is not interchangeable or substitutable with the reference product.

<Labeling>

Executive Summary – Pharmacovigilance and risk

Topic Reference

KOREA ASEAN


Pharmaco-vigilance and risk

By law, a risk management plan or pharmacovigilance plan must be submitted for biosimilars as for other medicines. The plan should take into account risks identified during product development and potential risks and how those risks will be addressed after authorisation For recombinant proteins, clinical safety must be monitored closely after authorisation. The application should include a risk specification and pharmacovigilance plan.

The Secretary’s existing authority to require a risk evaluation and mitigation strategy (REMS) for drugs applies to biosimilars (a REMS documents requirements designed to minimise risk associated with a drug). In addition, the Secretary’s existing authority to mandate post-market studies and clinical trials as well as post-market labelling changes applies to biosimilars.

A post-marketing safety surveillance plan and a risk management plan for biosimilars is required and should be submitted with the application.

A pharmacovigilance plan is required when an application is submitted and a risk management plan may be necessary in some cases.

Safety data obtained from clinical trials are usually sufficient for product authorization, but further close monitoring of clinical safety of the biosimilar product is usually necessary in the post-marketing phase.

ADR reporting, PSURs, Risk management plan, Educational Materials and Product Sales Data for the biosimilar product is required.

The pharmacovigilance, as part of a comprehensive RMP, should include regular testing for consistent manufacturing of the biosimilar. The pharmacovigilance plan must be approved prior to approval of product and the system must be in place to conduct monitoring.

<Pharmacovigilance and risk>

Executive Summary – Interchangeability and substitution

Topic Reference

KOREA ASEAN


Inter-changeability and substitution

Substitution is determined at the member state level, and therefore this topic is not directly addressed in EMA guidance. EMA guidance states that biosimilars are not generic medicinal products and that the decision to treat a patient with a reference product or a biosimilar should be made following the opinion of a qualified healthcare professional.

Substitution is determined at state level in accordance with state pharmacy laws. FDA must find a biosimilar to be interchangeable with its reference product if the information submitted by the biosimilar applicant demonstrates that: „„▶ The applicant’s product is biosimilar to the reference product (under the law’s standard for biosimilarity). „„▶ The applicant’s product can be expected to produce the same clinical result as the reference product in any given patient. For products administered more than once to an individual, the applicant must also demonstrate that the risk in terms of safety or diminished efficacy of alternating or switching between use of the biosimilar and use of the reference product is not greater than the risk of using the reference product without such alternation or switch.

Substitution of a biosimilar for, or the combined use of a biosimilar with, a reference product should be avoided during the postmarketing surveillance period.

Not addressed.

Not Addressed

A product is interchangeable with another if both products are approved for the same indication, and can be used for the said indication. Two products are substitutable with each other if they can both be used in lieu of the other during the same treatment period. For interchangeable products, one or the other can be used (prescribed) but these products cannot be substituted with one another during a treatment period. Interchangeability does not imply substitutability. Unlike generic chemical drugs, whereby the chemical structure is identical to that of the reference chemical product, a biosimilar product does not usually have an identical structure to the reference biological product. Therefore, even though a biosimilar product may be approved to be similar in terms of quality, safety and efficacy to the reference product, immunogenicity may preclude switching between products. A warning statement on the risks associated with switching of products during treatment, and against product substitution, is to be included in the package insert of the biosimilar product.

Given current science, biosimilars cannot be considered inter-changeable with the reference product or products of the same class.

<Interchangeability and substitution>

Executive Summary – Data protection for originators

Topic Reference

KOREA ASEAN


Data protection for originators

For a reference product for which the application for approval was submitted in October or November 2005 or later (different rules apply to a reference product for which the application for approval was submitted earlier), a biosimilar may not be marketed until ten years after authorisation of its reference product that is a new active substance. This period may be extended for an additional year, if within eight years after reference product authorisation, the reference product is authorised for a new therapeutic indication that constitutes a significant clinical benefit in comparison with existing therapies.

A biosimilar may not be approved until 12 years after the date on which the reference product was first licensed under section 351(a) of the Public Health Service Act. The date of first licensure does not include the date of approval of a supplement to the reference product application or the date of approval of a subsequent application for either: „„ A change to the reference product, other than a structural change, that results in a new indication, route of administration, dosing schedule, dosage form, delivery system, delivery device, or strength. „„ A structural modification to the reference product that does not result in a change in safety, purity, or potency.

A biosimilar application cannot be approved until the innovative product on which the application relies has completed an eight-year re-examination or post-marketing surveillance period.

Not Addressed Not Addressed Not Addressed Not Addressed

<Data protection for originators>

Biosimilar Guideline in Latin

American Countries

Mr. Daniel Guedes

Bio-Manguinhos / FIOCRUZ / Brazil

Oswaldo Cruz Foundation

Fiocruz / Brazil

Bio-Manguinhos

Institute of Technology in

Immunobiologicals

FIOCRUZ / Bio-Manguinhos

Institute of Technology in Immunobiologicals

Biosimilar Guideline in Latin American Countries

Vaccines Reactives Biopharmaceuticals



Vaccines

Diphtheria, Tetanus, Pertussis and Hib;

Yelow Fever;

Haemophilus influenzae b;

Meningitis A e C;

Poliomyelitis;

Measles, Mumps and Rubella.

Tetravalent viral (SmallPox).



NAT HIV/HCV;

Reactives

Molecular Tests

Rapid Tests

DPP HIV, Syphilis, Canine Leishmaniasis, Leptospirosis;


Reactives

Parasitological Tests Sorological Tests

Imunoenzimatic

Kato-Katz

Ind.

Immunofluorescence C. Leishmaniasis

C. Leishmaniasis

H. Leishmaniasis

Chagas Disease


Biopharmaceuticals

Alphapoetin Alphainterferon

Daniel Guedes, Ph.D.

Quality Control Department

FIOCRUZ / Bio-Manguinhos - Brazil

Biosimilar Guideline in Latin

American Countries

Latin American Countries Pharmacopoeia

Argentina Farmacopoea Argentina

Brazil Farmacopéia Brasileira

Chile Chilean Pharmacopoeia Foundation

México Farmacopea de los Estados

Unidos Mexicanos

NATIONAL HEALTH SURVEILLANCE AGENCY COLLEGIATE

BOARD – ANVISA (BRAZIL)

Integration of Latin American countries Pharmacopeias;

Optimize the use of resources and knowledge;

Mercosur`s Pharmacopoeia


Uruguai

Paraguai Argentina

Brazil


Peru

Venezuela Suriname

Equador

Guidelines from Argentina


Law 16.643 ANMAT

7075/2011

3397/2012

7729/2011

Biologicals

Biotechnologicals

Biosimilars

1490/1992


Law 16.643

“Are subject to this law and regulations, import, export, production,

preparation, processing, commercialization or deposit in national

jurisdiction or interprovincial trade of drugs, chemicals, reagents,

pharmaceutical forms, diagnostic elements, and all other product use

and application in human medicine and people whether natural or

involving in such activities.

All this products shall meet the conditions set in Argentina`s

Pharmacopoeia.”



Rule 7075/2011

Establish the conditions and requirements for registration of medical

specialties with biological origin.



Rule 3397/2012

Authorization Biotechnology products

Monoclonal antibodies;

Recombinant products;



Rule 7729/2011 – Authorization Biosimilars

Medical specialty of biological origin with previous marketing.

Comparability referential product;

Characterization with modern methods;

Similar behavior to the referential product;



NATIONAL HEALTH SURVEILLANCE AGENCY

COLLEGIATE BOARD – ANVISA (BRAZIL)

RDC 55 / 2010

RDC 49 / 2011

RDC 50 / 2011

RDC 61 / 2008

RDC 46 / 2000

RDC 17 / 2010

RDC 233 / 2011


Regulates production processes

and quality control, acquisition

and distribution of medicines for

human use.

RDC 46/ 2000


Provides on Criteria for

Harmonization of Nomenclature of

Vaccines and Serums.

RDC 61/ 2008


Requirements for the Good

Manufacturing Practice in

Medicinal Products for human

RDC 17/ 2010


Provides on the registration of

new biological products

(Biosimilar) and biological

products.

RDC 55 / 2010


RDC 55, Scope

I. Vaccines;

II. Hyper-immune serums;

III. Hemoderivatives;

IV. Biodrugs (Biologicals or by Biotechnology);

V. Monoclonal antibodies;

VI. Drugs (live, attenuated or dead microorganisms).

Provides on post-registration changes and

inclusions, suspension and reactivation of

manufacturing and cancellations of biological

product registration.


RDC 49 / 2011

Stipulates the procedures and conditions for

conducting stability studies for registration or

changes post-registration of biological

products.


RDC 50 / 2011

Technical Regulation of Production and Quality

Control for Registration, Changing Post-

Registration and Revalidation of Allergen Extracts

and Products.


RDC 233 / 2011

The WHO Biosimilar Guidelines influenced Latin America;

The structure is similar to those of USP;

There are new but consistent Guidelines for Biosimilars;

More countries can join the group.


Conclusions


Impact of Product Variants on

Product Functional Activity and

Stability in the Content of

Biosimilars

Anita Krishnan, Ph.D.,

Lupin Limited

Overview of Product and Process inter-relationship

Acceptable ranges for a subset of these QAs are normally established based on a

combination of clinical experience, pre-clinical studies, lab studies and prior knowledge.

The acceptable ranges are used to establish the boundaries for the design spaces in the

Upstream, Downstream and Drug Products sections of the case study.

Critical Quality Attributes

“A CQA is a physical, chemical, biological, or microbiological property or

characteristic that should be within an appropriate limit, range, or distribution to

ensure the desired product quality. Desired product quality includes clinically safe

and efficacious product.” ICH Q8 (R2)

Product-Related Substances

• Glycosylation variants (Galactose; Man5; Sialic Acid; Afucose)

• Acidic Variants (Deamidated/Sialylated Species)

• Oxidation

• Unpaired cysteine residues

• C-terminal Lysine Variants

Product-Related Impurities

• Clips / Truncations

• Aggregates

Process-Related Impurities

• Residual Protein A (Affinity Resin)

• Host Cell Proteins

• DNA

• Endotoxin/Bioburden/Particulates/Viruses

In the case of Biosimilars most of the CQAs are available as prior art.

10

1

5

Glycosylation

Q cyclization Deamidation

Oxidation

Lys variants

Aggregation & Clips

CQAs related to Fab variations

Deamidation

Glycation

CQAs related to Fc effector functions

Gal content and CDC

Afucose and ADCC

FcRn and PK

CDR 1

CDR 3

CDR 1

CDR 2

CDR 3

CDR 2

IgG Sequence analysis for Fab CQAs

Asn 30 to Asp

Asn 55 to Asp

Asp 102 to isoAsp

Fab related

variations

K glycation

Fab glycosylation : N-X-S/T

R. J. Harris et al. / J. Chromatogr. B 752 (2001)

233 –245

isoAsp102 Asp30

CQAs Related to Fab Variations

Reaction of primary amines of lysine residues with

reducing sugars such as glucose/lactose to form

aldimine (Schiff’s base ) and later a ketoamine adduct

Molecular Signatures of Fc effector Functions

HINGE

DP

Fcɣ R I, II, III Fcɣ R I, II, III

FcRn - Met256

C1q C1q

FcRn – His protonation

Deamidation

Mechanistic pathways of IgG

Rituximab

Trastuzumab

Cetuximab

Infliximab

Golimumab

Adalimumab

Omalizumab

Bevacizumab

Role of neonatal Fc receptor (FcRn) in PK

Regulates systemic half-life by

salvaging FcRn bound IgG and

recycling

IgG:FcRn interaction depends on

conserved His in CH2-CH3 interface

Salt bridges between positive

residues of IgG and negative amino

acids on FcRn surface

Minutes

20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52

mA

U

0

50

100

150

200

250

300

mA

U

0

50

100

150

200

250

300

RMP

In-house batch

Minutes

36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80

LU

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225

0.250

0.275

LU

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225

0.250

0.275

RMP

In-house batch

Case Study of In-house Batch

Glycan profile : NP - HPLC

Charge variants : IE - HPLC Potency : CDC activity

0

1000

2000

3000

4000

5000

6000

7000

0.01 0.1 1 10

Ref std B6197 B06

In house Sample

Potency: 67.5%

CI: 91.3% - 109.0%

G2 G0

G0F

G1

+Man5

G1F/G1F’

G2F

Impact of Fc glycosylation heterogeneity on effector functions

Does ADCC and CDC co-relate with the glycan profile?

Structure function relationship

Optimized Process: 10L Bioreactor

Mannosylation Galactosylation

Afucosylation Fucosylation

ADCC: 131% 270%

OPT INIT RMP OPT INIT RMP

OPT INIT RMP

CDC: 96 % 92 %

G1F G0F G2F G1’F

R O I R O I R O I R O I

5.0 10.0 15.0 20.0 25.0 30.0 35.0 min

0

5000

10000

15000

20000

25000

30000uV

mAb-RMP

In-house 1

In-house 2

Oxidized impurities

Oxidation of rHu mAb – reduced binding to Protein A

5% TBHP

3% TBHP

1% TBHP

0.5% TBHP Native Oxidized

Minutes

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

mA

U

0

200

400

600

800

1000

1200

mA

U

0

200

400

600

800

1000

1200

MWD: Signal A, 214 nm/Bw:4 nm

Rituximab RMP Ctrl

Rituximab RMP Ctrl.dat

Resolution (DAB)


rituximab rmp 0.5 a


rituximab rmp 0.25_a


rituximab rmp 0.125_a


rituximab rmp 1_a

Fc/2

LC

Fd

Ox Fc/2

37°C CTRL

1.0% t-BHP

0.5% t-BHP

0.25% t-BHP

0.125% t-BHP

Site Specific Oxidation of rHu mAbs on chemical stress

Minutes

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

mA

U

-200

0

200

400

600

800

1000

1200

mA

U

-200

0

200

400

600

800

1000

1200MWD: Signal A, 214 nm/Bw:4 nm

RMP

RMP.dat

Area Percent


dp5 500 mgper50ml_3m at_a


dp6 100 mgper10ml_3m rt_a

Fc/2

LC

Fd

Ox Fc/2 Ox Fd

Site Specific Oxidation of in-house samples

RMP

Inhouse 1

Inhouse 2

Oxidation of methionines in Fc and Fd

y = -0.7676x + 102.53 R² = 0.9291

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0.0 20.0 40.0 60.0 80.0 100.0 120.0

% P

ote

ncy

% Oxidation

Sample % Oxidation % Potency RMP 5.0% t-BHP 100.0 18.2

RMP 1.0% t-BHP 60.1 59.9

RMP 0.5% t-BHP 44.2 82.3

RMP 0.25% t-BHP 30.2 81.5

RMP 0.125% t-BHP 17.3 83.7

10% spike of 100% oxidised RMP 10.0 92.8

RMP 37°C CTRL 1.6 97.1

Inhouse 1 3.7 69.1

Inhouse 2 6.1 62.3

Correlation of Oxidation levels and CDC activity of RMP

Risk based approach for prevention of Oxidation

• Enhanced oxidation due to presence of dissolved oxygen

• Metal-catalyzed oxidation is site-specific - Copper induced

catalysis observed (Baker et al 1978)

• Photo-oxidation of Met can form persulfoxide/ Met sulfoxide

• Oxidation due to compromised thermal stability

Nitrogen purging reduces dissolved oxygen

Dis

so

lve

d O

xyg

en

(%

) Dissolved oxygen induces oxidative damage

Before After Storage for 12h DO after 12h

96% 0% Open to air 25%

97% 0% Headspace with

Nitrogen 2%

Tertiary structure – A role in potency (CDC and ADCC)

Hydrogen bonds Van der Waals contacts

Proline sandwich

W87 W110

C1q binding epicentre

Stretch of charged residues

D270 K322 P329

P331

NATURE |VOL 406 | 20 JULY 2000

Structure based sequence alignment of the C1q complex

components

C1q binding

Site

FcɣRIII

binding

Site

FcRn binding

Site

CH2

CH3

Sugar

Chain

IgG1 Fc

Enhanced ADCC (Increased FcɣRIII a binding)

Enhanced CDC (Increased C1q binding)

Improved PK (Increased FcRn binding)

Site directed mutagenesis for enhanced activity

Post-translational Modifications

3 D structure

Protein aggregates

POTENCY

IMMUNOGENICITY

EFFICACY

SAFETY

Critical Attributes to be assessed for Biosimilars

Three properties of therapeutic proteins – in the opinion of FDA

cannot be sufficiently measured but are deemed to be important for

understanding the behaviour of protein drugs: post-translational

modifications, three-dimensional structures and protein aggregation.

Conclusions

• Glycosylation has a direct overbearing effect on potency (ADCC

and CDC)

• Oxidation of Met 256 and 432 have been proved to have a role in

FcRn binding and therefore PK

• Oxidation of Met has a definite role in CDC activity too

• Reasons for reduced potency could be due to multiple factors

namely Oxidation, Tertiary structure differences and Reduced

CD20 binding

• Risk based approach to control oxidation levels and introduction

of stability and release methods for oxidation in mAbs

New USP Bioassay Chapters

Tina S. Morris, Ph.D.,

Vice President, Biologics & Biotechnology, USP

Official since 1950:

<111>: Design and Analysis of Biological Assays

– A major revision will be proposed in Pharmacopeial Forum PF39(4)

The new suite: <1030>: Biological Assay Chapters – Overview and Glossary

– Will be official with the 2nd Supplement of USP36

<1032>: Design and Development of Biological Assays

<1033>: Validation of Biological Assays

<1034>: Analysis of Biological Assays

All of these chapters are focused on relative potency bioassays.

Chapter numbering is important…

USP Bioassay Chapters

Official

From General to Specific – Biological Potency

<1030> Overview of Bioassay

<1034> Analysis of Biological

Assays

<1032> Development of Biological

Assays

<1033> Validation of

Biological Assays

<111> Design and Analysis of

Biological Assays

<121> Insulin Assays

Guidance &

Information

General

Requirement

Product-Specific

Requirement

Insulin Monograph Product Quality

Attributes

Focuses on design and development, rather than analysis

Covers animal assays, cellular assays, enzyme assays and immunoassays

Focuses on design elements, theory and practice

– e.g., describes good and bad plate designs

Addresses activities throughout the product life cycle

Some key ideas

– fitness for use, different applications of assay

– biological and statistical assay fundamentals

– assay from design and development through validation and

performance monitoring

– system and sample suitability

<1032> Design and Development of Biological Assays

Specific points:

Identify and minimize sources of variability in a method

Record cell line information, before banking, including...

– identity

– morphology

– purity

– cryopreservation

– thaw and culture conditions

– thaw viability

– growth characteristics

– functional stability

Prepare two-tiered bank: Master and Working

– the size of the banks depends on the growth characteristics of the cells,

the number of cells required for each assay, and how often the assay will

be performed

<1032> Design and Development of Biological Assays

Where possible, a Standard should be prepared using the same

manufacturing process as the drug substance

Storage conditions may vary from drug substance or product:

– Temperature (e.g., –70 °C or –20 °C instead of 2–8 °C)

– Container (e.g., plastic vials instead of syringes)

– Formulation (e.g., lyophilized formulation or addition of carrier proteins)

Test Standard for stability at appropriate intervals

An initial Standard = Primary Standard

Subsequent Standards = Working Standards

– Separate SOPs usually required

– Trend charts may be useful in identifying the cause of assay drift

Chapter <1032>’s Recommendations for Standards

Describes approaches for validation design (sample selection and replication strategy), validation acceptance criteria, data analysis and interpretation, and bioassay performance monitoring through quality control

Validation parameters discussed include relative accuracy, specificity, intermediate precision, and range

– Laboratories may use dilutional linearity to verify the relative accuracy

and range of the method

– Although robustness is not a requirement for validation, <1033>

recommends that a bioassay’s robustness be assessed either pre-

validation or with additional experiments soon after validation

Some differences from common practice:

– Equivalence testing

– Confidence intervals to demonstrate conformance to acceptance criteria

<1033> Validation of Biological Assays

Intended to be useful for biologists and statisticians alike but heavier on the statistics side

Covers models for different types of bioassays

– linear and nonlinear

– parallel line and slope ratio

– Quantal (e.g., yes/no assays)

Develop and elaborate on appropriate uses of equivalence assessment; e.g. for parallelism/similarity

Have also developed a 4PL Bioassay Simulator that lives on the www.usp.org/usp-nf/compendial-tools website and is freely available to the public. Other simulators are in development

– Please provide feedback on your needs!

<1034> Analysis of Biological Assays

http://www.usp.org/usp-nf/compendial-tools





Include only components that USP intends to be

required or that are commonly needed in

monographs:

– Outliers

– Combination of independent assays

– Confidence Intervals

Revision is on track for publication in PF39(4)

issue, appearing in July-August 2013, with

public comment period closing September 30,

2013

Plan for Revisions to <111>

Product-Specific Potency Assays

Can be called out in a Monograph or General Chapter

Monograph requirements supersede Chapter

Requirements

A potency test or at least bioidentity test based on a

functional assay is required for most biologics and

biotechnology-derived medicines licensed for the US

market. A few smaller proteins/peptides have only

HPLC-based Assays to determine potency (e.g.,

vasopressin, oxytocin, leuprolide, etc.)

Europe and the US Differ in Bioassay Requirements

In Europe, for peptides and small proteins that are

considered “well-characterized” neither a unit-based

quantitative bioassay nor a bioidentity test are

required. This is not the case in the US.

Some examples:

– Insulin(s) - US requires bioidentity based on USP <121>

Insulin Assay: rabbit blood glucose test

– Somatropin - US requires rat weight gain bioidentity test

– Glucagon - US requires primary rat liver cell bioidentity

assay, see USP <123>Glucagon Bioidentity Tests

Compendial Implications

Many bioassays and bioidentity tests for

licensed biologics are still animal-based

Associated concerns:

Assays with generally high variability and poor

precision

Challenges in unit assignments and maintenance,

especially in relationship to the International Unit

where it exists

Ethical issues regarding animal use

USP Initiative: Replacement of Animal Assays

Current: <126> Somatropin Bioidentity Tests – USP is

evaluating a cell-based assay using a nonproprietary cell

line available from commercial sources. The chapter will

contain the animal test and the cell assay.

Future:

– <123> Glucagon Bioidentity Tests – USP is working with a

sponsor to add a cell-based assay to this chapter and

distribute the necessary cell line as a Reference Standard

– <121> Insulin Assays – USP has reached out to sponsors for

suitable cell-based assay for insulins and hopes to revise the

chapter with one or more in the future

Case Study: Somatropin Bioidentity Test

Official in the Somatropin monographs since 2005 for

bioidentity (to assure potency of the material now assigned by

an HPLC Assay method; previously the rat method was used

to assign potency)

Currently official method uses hypophysectomized rats

injected with somatropin for 10 days. Body weight change is

measured and potency calculated relative to the USP

Somatropin RS

Two sponsors came forward with new cell-based methods.

– Both validated and accepted by FDA for use with US-approved products

– Bridging to the animal methods was submitted by the sponsors

– Verification of method performance and suitability performed in USP laboratory

– Most robust method was chosen for development of a new chapter <126>Somatropin Bioidentity Tests, using commercially available, nonproprietary cell line

Cell-based Bioidentity Test Format

Uses the cell line Nb2-11 derived from a rat

lymphoma

Proliferation of the cells is dependent on factors

such as prolactin, IL-2, or hGH

The method is based on measurement of ATP

production in response to Somatropin (rhGH)

treatment relative to the USP Somatropin RS. The

amount of intracellular ATP is directly proportional

to the proliferation of the cells due to the hGH

treatment.

Cell-based Bioidentity Test Format

On the day of the assay, cells are harvested, washed, and

seeded in 96-well assay plates in a minimal medium

containing 1% horse serum for one hour.

A dilution series of either Somatropin test sample or USP

Somatropin RS (or positive and negative controls) is

delivered and the cells are incubated for ~30 hours

A luminescent cell substrate solution is added for ~30 min

then the luminescence in the wells is measured

Relative potency is calculated using parallel line analysis

relative to the USP Somatropin RS

To pass bioidentity, a somatropin sample must possess not

less than 2 USP Somatropin Units/mg

Representative Data from Cell-based Method*

USP Somatropin Dose Response Triplicate Plates Passage #10

Concentration

(ng/mL) Col 2 Col 3 Col 4 Col 5 Col 6 Col 7 Col 8 Col 9 Col 10 MEAN STD RSD

10 4185 4513 4577 4731 4564 4834 4667 4619 4586.042 191.4806 4.18%

1 4074 4127 4064 4279 4061 4132 4266 4180 4176 4151.037 81.8658 1.97%

0.5 4506 4494 4566 4536 4434 4492 4450 4541 4551 4507.593 45.3449 1.01%

0.25 3934 3994 4299 4180 4126 4113 4109 4143 3979 4097.593 113.0542 2.76%

0.125 3238 3284 3398 3258 3301 3343 3346 3441 3558 3352.111 101.0542 3.01%

0.0625 2892 2625 2709 2622 2671 2710 2666 2585 2711 2688 88.4498 3.29%

0.03125 2088 2135 2169 2145 2057 2151 2119 2243 2277 2153.815 69.5885 3.23%

0.015625 1540 1586 1611 1595 1505 1586 1523 1555 1627 1569.852 41.1275 2.62%

0.0078125 1162 1230 1283 1222 1210 1284 1249 1296 1257 1243.741 42.5375 3.42%

0 804 861 884 886 918 968 906 897 890.375 47.0213 5.28%

S-N

Ratio 5.15 3.08%

y = 826.73ln(x) + 5094 R² = 0.9919

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0.001 0.01 0.1 1

Lu

min

escen

ce V

alu

es

Somatropin Concentration in ng/mL

Somatropin Dose-Response Curve

*Data courtesy of Michele Virjee, USP Laboratories.

Data Summary and Next Steps

The cell-based method has good signal-to-noise ratios,

averaging between 5 and 6

Percent RSD’s were generally below 5%.

Little plate-to-plate variability

Plate uniformity analyses performed in the USP laboratory

suggested a slight edge effect in the left-most column and

upper-most rows of the microtiter plates, but based on the

analyses there was no significant impact on the results. The

sponsor did not see any uniformity issues.

The sponsor has successfully used the Nb2-11 cells for the

procedure through 50 passages. The USP confirmed the

method consistency through passage 16 (which was 11

passages post-thaw).

Data Summary and Next Steps, cont’d.

Overall, the cell-based method provides a more precise,

consistent, and reliable method when compared to the in

vivo method

The new chapter <126>Somatropin Bioidentity Tests will be

proposed in the PF39(5) issue, appearing September 2013.

Public comments are open through November 30, 2013

Collaborative laboratory testing of the USP Somatropin RS

for this new purpose will occur in parallel and be complete

prior to the chapter moving to official

Epoetin – The Commutability Challenge

For epoetin special challenges exist in the transition

from in vivo to in vitro assays

Different types of in vitro assays have varying

degrees of sensitivity to and correlation with

glycosylation (esp. sialylation) differences

In vivo to in vitro transition, equivalence

determination and unit transition has to occur on an

assay-by-assay basis

<124> Epoetin Bioassays – What are the Options?

Option 1: include both in vivo and in vitro assays

– In vivo assay: include only normocythemic assay

– Potential misuse of in vitro assay to inappropriately assign units of

activity to EPO preparations.

– Which in vitro assay to include? The proposed cell assay is not the

actual assay used for product release

Option 2: include only the in vivo assay with guidance on

– How the standard can be used in an in vitro assay (acceptance

criteria for in vitro to in vivo activity ratios)

– How to assign an in vitro unitage to the USP standard, so that the in

vitro assay can be used to assign units to EPO preparations

Both options assume that the monograph refers to chapter

<124> for assay requirements

Path Forward and Future Opportunities

Recommendation for a Path Forward

– Move forward with option 2, and publish <124> in PF, with in

the in vivo assay only. This will provide strict guidance on the

use of both in vivo and in vitro bioassays to measure the

biological activity of erythropoietin (EPO) and will preserve the

traceability to the WHO International Standard for EPO

– In a second phase, develop a cell-based assay to be included in

the chapter. Other cell assays can be used provided that these

assays are validated against compendial assays

– Physico-chemical assays that can predict outcome of bioassays

can also be added in the future.

Expert Panel Discussions – Chapter <124>

USP EPO for bioassay RS calibrated against 3rd WHO

International Standard for EPO by in vivo mouse bioassay

– assigned unitage in International Units (IU) of EPO

Calibration strategy means that USP EPO for bioassay RS

can be used directly in in vivo mouse bioassay for

calibration of any process-specific EPO preparation.

An in vitro bioassay cannot be used to transfer the unitage

from the USP EPO for bioassay RS to a process-specific

EPO preparation

– Unless you demonstrate that the USP EPO for bioassay RS

and the process-specific EPO preparation exhibit an

equivalent ratio of in vitro to in vivo potency.

The ratio of in vitro to in vivo activity is generally

inversely correlated with the degree of terminal

sialylation, thus highly sialyated products have a

higher level of in vivo activity and as such, have a

relatively lower ratio of in vitro to in vivo activity.

Different manufacturing processes mean that the

degree of terminal sialylation can be highly variable

from one EPO preparation to another.

– To use an in vitro assay to measure the biological activity of

a preparation of EPO, you must have a full understanding of

the relationship between its in vivo and in vitro activity.

• Include with the USP EPO for bioassay RS, in both the in vivo

assay and in a specific in vitro assay.

Expert Panel Recommendations – Chapter <124>

The USP EPO for bioassay RS is assigned a unitage

which represents its activity in both in vivo assays and in

vitro assays.

– If the ratios of in vitro to in vivo potency for the material being tested and

the USP EPO for bioassay RS are equivalent, then the USP EPO for

bioassay RS can be used directly in the in vitro assay to calibrate the

material being tested.

– However, if these ratios are not equivalent, then the standard cannot be

used with its assigned in vitro potency in the in vitro assay.

– Instead, the ratio determined for the material being tested should then be

used to assign a process-specific, in vitro assay unitage to the USP EPO

for bioassay RS. The USP EPO for bioassay RS, with its adjusted in vitro

assay unitage, can then be used in the in vitro assay to transfer the

unitage from the USP EPO for bioassay RS to the material being tested.

Expert Panel Recommendations – Chapter <124>

Modernizing Compendial Bioassay/Bioidentity Tests: Challenges

For small proteins or peptides: when can a bioassay be

replaced by a physicochemical method? And, can the

method stand alone or is bioidentity assurance needed?

Assays for biological products with more than one activity

Commutability of assays and reference materials

Availability of public methods and data:

– Intellectual property barriers

Equivalence determination between assays:

– Challenges in establishing equivalent results between

different biological systems: animal vs. cell-based assays vs.

physico-chemical, etc.

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