Introduction Bio-products

28/01/2013 1

Dr. Mohammed Saleem

P. O. Box: 836 Amman 11821 Jordan

Tel.: 00962-6-5512561/2

Fax: 00962-6-5512563

contact@sipsmena.com

www.sipsmena.com

Science forum for Research Studies & Consultancies

(SIPS)

2.4 2.6 2.8 3.2 3.5 4.1

34

30

27

24

22

5.3

4.7 4.3

3.3 3.7 5.4

4.7 4.2

3.2 3.6

7.2

4.6 5.1 5.7 6.4 1.5 1.7 2.2 2.2 2.3

2.9 1.5 1.8

2.2 2.5 2.8

3.0

0%

5%

10%

15%

20%

25%

30%

-

5

10

15

20

25

30

35

2006 2007 2008 2009 2010 2011 2012F 2013F 2014F 2015F

Source: IMS Global Market Prognosis, Forecasted updated Mar.2012 *Marked countries pharma market size is estimated, based on trade statistics to represent 23% of MENA total by 2016

2016F

US

$ B

illi

on

s

Bahrain

* Qatar

* Oman

* Yemen

* Syria

* Iraq *

Jordan

Libya *

Lebanon

Tunisia

Morocco

Iran *

United Arab

Emirates Israel *

Egypt

Algeria

Saudi

Arabia

MENA

Growth

Global

Growth

20

18

16 14

12

10

MENA market size has grown by $10 bn in past 5 years, by doubling its size and forecasted to grow another $14bn until 2016…

Prospects for MENA

Saudi, Algeria, Egypt and UAE represent ca.60% of MENA market

MENA Pharma Market Size (US$Bn) & Growth%

Source: IMS MIDAS, 12/2011

(It includes old generation insulins which are unlikely to be targeted by biosimilar players)

0%

2%

4%

6%

8%

10%

12%

14%

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

Oncology

Enzyme

Epos

Vaccine

MS

Insulin

Other Biologics

RA/Crohn/Psoraisis

Source: Brogan Inc. Pay Direct Drug Plan Database

Biologics are major growth items within selected therapy areas

Share of Annual

Expenditure

0

Source: Brogan Inc. Pay Direct Drug Plan Database

1

2

3

4

5

6

0M

1M

2M

3M

4M

5M

6M

Patients are starting on biologics earlier in life & staying on them through old age

2001 2011 2021

$/ Claimant Population (M)

Age

Group

Chart notes Biologics include single identified components, whole cells, and include some forms of polymers. They can be purified from human, animal, plant or micro-organism sources. Biologics can be produced by recombinant DNA technology or chemically synthesized. Biosimilar products are biologic products approved in a country which has an abbreviated approval process for biologic products that reference an originator biologic in the regulatory submission.

• Biologics will account for an increased share of spending by 2016, as important clinical advances continue to emerge from research, and patients around the world are treated.

• Spending on biosimilars will increase from $693Mn in 2011 to $4-6Bn by 2016, which represents 2% of biologic spending.

• Adoption is expected to remain modest through 2016, largely because most biologic medicines will stay protected by patents or market exclusivity in many countries.

2016

2011

2006

Source: IMS Consulting Group, May 2012

Global Biologics Spending

Biologics usage will drive many therapeutic areas

$93Bn

$157Bn

$200-210Bn

99.98

%

Total Biologics Market

The Global Use of Medicines: Outlook Through 2016

Report by the IMS Institute for Healthcare Informatics 7

GLOBAL SPENDING ON MEDICINES

Treatment will be transformed by new and existing mechanisms

• Between 32-37 innovative products are expected to be launched per year over the next five years.

• These developments include new mechanisms of action in several disease states such as Alzheimer’s, autoimmune diseases and various types of cancer, which have the potential to transform disease treatment, though not every therapy will become available or achieve its ultimate clinical aims.

• There are also further developments in areas of research where some therapies have already launched including hepatitis C, multiple sclerosis and prostate cancer, which offer the potential to deliver better efficacy, safety or convenient administration.

Selected Product Launches 2012-2016 DISEASE AREA EXISTING MECHANISMS NEW MECHANISMS

Autoimmune • JAK inhibitor (tofacitinib )

Alzheimer’s disease • MAb (bapineuzumab, solanezumab)

The Global Use of Medicines: Outlook Through 2016

Report by the IMS Institute for Healthcare Informatics 9

Breast cancer • MAb (pertuzumab, trastuzumab emtansine)

Diabetes • GLP-1 (albiglutide, dulaglutide, lixisenatide)

• DPP IV (anagliptin, gemigliptin, teneligliptin, trelagliptin)

• SGLT inhibitor (canagliflozin, dapagliflozin, empagliflozin, ipragliflozin, tofogliflozin)

Hepatitis C • NS3/4A proteinase inhibitor (asunaprevir, BI 201335, simeprevir)

• NS5A inhibitor (daclatasvir)

HIV • Reverse transcriptase inhibitor (elvitegravir + emtricitabine + tenofovir disoproxil + cobicistat)

Lupus • Fusion protein (atacicept) • MAb (epratuzumab, tabalumab)

Melanoma • BRAF kinase inhibitor (dabrafenib) • Oncolytic HSV vector (talimogene laherparepvec)*

Multiple sclerosis • Immunomodulator (laquinimod, teriflunomide)

• MAb (daclizumab, ocrelizumab)

Ovarian cancer • Folate-targeted drug conjugate (vintafolide)

• Kinase inhibitor (nintedanib)

Obesity • Appetite suppressants (lorcaserin, phentermine/topiramate)

Thrombosis/ ACS

• Blood clotting factor Xa inhibitor (idraparinux sodium, otamixaban)

Prostate cancer • Antiandrogen (enzalutamide/MDV3100) • Kinase inhibitor (cabozantanib) • Radiotherapeutic (radium-223 chloride)

Chart notes Table includes selected not yet launched New Molecular Entities (NME) expected to be launched by 2016. An NME is the first commercial launch of a novel therapeutic entity. Abbreviations: JAK: janus-like kinase inhibitor; MAb: monocloncal antibody; GLP-1: glucagon-like peptide-1; DPPIV: dipeptidyl peptidase-IV inhibitor; SGLT: sodium-glucose cotransporter inhibitor; HSV: 5-HT2C: immunotherapeutic cancer vaccine, 5-hydroxytryptamine 2C; Xa: Xa coagulation factor inhibitor; NS5A: non-structural protein 5A; HSV: herpes simplex virus; ACS: acute coronary syndrome.

Source: IMS Institute for Healthcare Informatics, May 2012

TRANSFORMATIONS IN DISEASE TREATMENT

PROTEINS!

The Building Blocks of Proteins:

AMINO ACIDS!

R-groups affect Reactivity & Solubility

Amino Acids as Buffers:

How do Amino Acids Link Together to Form Proteins?

DIRECTIONALITY!

Protein Primary Structure

Hydrogen bonding!

Secondary Structure Secondary Structure !

Secondary Structure Tertiary Structure

Tertiary Structure Quaternary Structure (in some proteins)

Hemoglobin’s Chemical Formula: C3032H4816O872N780S8Fe4

Change in primary structure = Change in function!

Mutated Cells

Change in

structure =

Change in

function!

When Protein’s Go Awry = PRIONS!

Normal Misfolded (Prion)

• Scrapie

• Kuru

• Mad Cow

• Insulin (diabetes)

• Interferon b (relapsing MS)

• Interferon g (granulomatous)

• TPA (heart attack)

Protein Pharmaceuticals

• Actimmune (If g)

• Activase (TPA)

• BeneFix (F IX)

• Betaseron (If b)

• Humulin

• Novolin

• Pegademase (AD)

• Epogen

• Regranex (PDGF)

• Novoseven (F VIIa)

• Intron-A

• Neupogen

• Pulmozyme

• Infergen

Protein Pharmaceuticals

Protein Pharmaceuticals

• 77 FDA approved protein drugs

• 66/77 are recombinant proteins

• Protein pharmaceutical sales currently approach $25

billion/yr

• End of 2013 they are expected to exceed $60 billion/yr

Poorly characterized

• Traditional vaccines

• Whole blood

• Blood derivatives

• Blood components

• Allergenic extracts

• Stem cells

• Somatic cell and gene therapies

• Toxins

Well-characterized

• Natural proteins

• rDNA-derived proteins

• Monoclonal antibodies

• rDNA-derived vaccines

Types of Biological Drugs

Origins

Recombinant DNA

Recombinant proteins

recombinants Vaccines

Monoclonal Antibodies

Cytokines; Enzymes;

Hormones

Nucleic acids

& siRNA

DNA

Vaccines

Gene

therapies

Culture & purifications Techniques

Vaccines

Polyclonal Ab; Hormones;

Enzymes

Blood derivatives: Albumin, clotting

factors

Organs; Cells & Tissus

Biopharmaceutical products

Proteins have expected:

• Size, charge, hydrophobicity

• Correct folding (S-S bonds)

• Subunits

• Glycosylation

• Bioactivity

& Unexpected:

• Aggregation (side effects)

• Incorrect folding

• Amino acid modifications

– ox, deam, cys • Truncation, proteolysis

Statin

Therapeutic protein ~5,000 - 300,000 Da

~400 Da *

x

Chemical Vs Biological Drugs

Biological

drugs

Size

Structure

Stability

Modification

Manufacturing

Characterization

Large

Complex

Unstable

Many

options

Unique line of living cells

Impossible to make identical copy

Impossible to characterize fully

due to a mixture of related

molecules

Qu

alit

y A

ttri

bu

tes

Chemical drugs

Small

Simple

Stable

Well-defined

Predictable chemical process

Identical copy can be made

Easy to fully

characterize

Chemical Vs Biological Drugs

mAbs Glyco-Protein Polypeptide Chemical

InfliximAb Epoetin Insulin Simvastatin

Immunologicals

Virus Like

~144,190 Da ~34,000 Da ~5,808 Da ~419 Da ~22,000,000 Da

Complexity of Structures

Chemical Vs Biological Drugs

O

H

O

O

HO O

O

H

O

H O

HO O

Simvastatin

Simvastatin Acid

Structural Changes mAbs

Chemical Vs Biological Drugs

O

O

O

HO O

H O

O

O

H

H

O

O

H

Simvastatin-

Closed Lactone Ring

Simvastatin Acid-

Opened Lactone Ring

Identification of Structural Changes

Chemical Vs Biological Drugs

O 2. 1.25

O O

H

2.0 HO O

H

5.97

H

5.50

H

4.11 1.53

1.18.52

9 2.33

2.13

1.53

1.16

1.16

4.25 1.64

1.29

2.01

0.96

3.63

2.5

5

53

5.47

0 1 2 3 4 5 6 PPM

O

O

O

HO O

H

O

O

OH

HO O

H

O 2.53

O

OH

11.0

HO

3.54

O

H

H

5.97

H

5.50

H

4.11 1.53

1.851.29

1.25

2.33

2.13

1.29

1.16

1.16

1.29

1.64

1.29

1.44

0.96

2.38 2.0

5.47 0 2 4 6

PPM 8 10 12

1H-NMR

1 H-NMR

Analytical Identification of Structural Changes

Chemical Vs Biological Drugs

O

O O

HO O

172.

0

H

40.9

145.

3

73.

7

35.

9 22.0

412.29.

0

128.

0

124.

7

24.

5

31.

5

174.0

43.3

25.

1 133.

6

33.

2

18.

0

20.

2

71.

6 31.

0

44.

1

8.4

66.

7

43.

4

0 20 40 60 80 100 PPM

12

0

14

0

16

0

18

0

174.

0

O

O

OH 24.1

HO

66.

7

O

H

40.9

73.7

25.1

145.3

35.9

41.922.

0

128.

0

124.

7

30.5

31.5

133.

6

29.2

18.0

43.3

20.2

31.0

22.0

37.3

8.4

177.

0

43.4

0 20 40 60 80 100 PPM

120 140 160 180

O

O

O

HO O

H

O

O

OH

HO O

H

13C-NMR

13C-NMR

Analytical Identification of Structural Changes

Chemical Vs Biological Drugs

•Monoclonal antibodies are complex molecules

– High level of microheterogeneity, there will always be

differences

– The mode of action is complex and may involve contributions

from multiple mechanisms

– Even small differences may have significant effects.

–Need to combine physicochemical results with functional

assays (e.g. antigen-antibody binding assays and cell-based

assays) and the qualification in preclinical and clinical studies

Increasing complexity of Bioproducts

Theoretical Molecular Mass ~150,000 Da

–>200 AA residues light chain

–>450 AA residues heavy chain

–More than 1 predominant mass

Glycosylated

Complex Structure Biantennary

+/- Core Fucose

Sialylation Disulfide Bonds: – Contain Inter and intra-chain bonds

– C-terminal Lysine Heterogeneity

– Additional Post-translations

Modification

– (deamidation, methionine oxidation,

etc.)

Heterogeneous in both size and

charge

Increasing complexity of Bioproducts

1. Binding to antigen : Inhibition of binding of ligand to receptor

2. Binding to cellular receptors

Fc-gamma-RI Fc-gamma-RII Fc-gamma-RIII FcRn

3. Complement binding

4. Relative contribution of effector functions mostly unknown

Multi-functionality of Monoclonal Antibodies (mAbs)

Subtle changes to the structure can result in profound effects on the activity

The structure of specified molecule is related to the function (biological activity) and susceptible to:

- Genetic Instability of Cell Banks

- Issues within Fermentation and Purification

Post Translational Modifications

Product Degradation or Modification

Purity

Biological activities

Immunogenicity

Multi-functionality of Monoclonal Antibodies (mAbs)

Focus on immunogenicity

Small Chemical Molecules Biopharmaceuticals

• Variable structure

– Living-organisms process

– Process-dependent variability

• Immunogenicity

– Leads to cellular- and

humoral immune response

that can be neutralizing

– Immune response may

hamper endogenous

molecules functionalities

• Single structure

– Chemical synthesis process

– Reproducible under controlled

environment

• Non- or poor immunogen

– Commonly non

immunogenic molecules: hapten properties

Consequences of immunogenicity

- Clinical consequences include loss or reduction of efficacy, local reactions, and systemic reactions - Response can be regained by introduction of an alternative biological

agent of the same or different class (Yanai & Hanauer Am J Gastroenterol. 2011; 106:685-698)

- The risk of immunogenicity needs to be considered individually for each

indication/patient population - Serum antibodies can be used as markers to monitor the safety of

therapeutic biopharmaceuticals

- Acute and delayed consequences - Pathogenic mechanisms of Mabs-related adverse reactions include

hypersensitivity, IgE- and non-IgE-mediated events and cytokine release syndrome (Maggi et al. Expert Rev Clin Immunol. 2011; 7:55-63)

- Autoimmunity (EPO > Pure² Red-Cell Aplasia)…

Immunogenicity assessment

- Important parameters:

- Aggregation

- Protein structure (secondary modifications)

- Excipients and impurities

- Prediction:

- Unwanted immune responses are dependent on the molecule,

manufacturing process, treatment duration and individual patient’s

susceptibility

- In-silico modeling may help to identify T-cell epitopes but does not predict

immunogenicity

- The incidence of immune response is too low to be fully identified during clinical

studies; therefore…

- Systematic post-marketing monitoring may be necessary to capture clinical signs

that could be related to immunogenicity

• Continue benefit risk assessment

• Risk assessment, pharmacovigilance, and post-marketing studies are essential

Immunogenicity

• Immunogenicity can not be fully determine due to limitations of clinical trials

• May not be detected during preapproval

clinical testing – Evaluated through postmarketing surveillance or

studies

Immunogenicity

Immunogenicity

• Immunogenicity is influenced by many factors such as the nature of the active substance and product- and process related impurities

•An immune response to the product may have a

significant impact on its clinical safety and efficacy – Irrelevant for therapy, to serious and life-threatening

What are Biopharmaceuticals

• Biopharmaceuticals are defined as pharmaceuticals manufactured by biotechnology methods, with the products having biological sources, usually involving live organisms or their active components

• Biopharmaceuticals are protein or nucleic acid based pharmaceuticals (substance used for therapeutic or in vivo diagnostic purpose), which are produced by mean other than direct extraction from a native biological source.

43

• The methods and techniques that involve the use of living organisms (such as cells, bacteria, yeast and others) are tools to perform specific industrial or manufacturing process are called biotechnology

• Pharmaceutical Biotechnology will continue to provide new breakthroughs in medical research in the years to come, leading to treatment in field which have previously eluded us (including AIDS, cancer asthma, Parkinson’s disease, Alzheimer disease)

44

Pharmaceutical Biotechnology

Pharmaceutical Biotechnology

• Biotechnology offers better product-targeting for specific diseases and patient groups, through the use of innovative technologies, in particular, genetics. Examples include, amongst others, treatment for rare diseases and cancers.

• Some products are not naturally created in sufficient quantities for therapeutics purpose.

• Biotechnology makes large-scale production of existing substances possible, for example, insulin in the field of diabetes treatment

45

Majority of Biotech Products Use Living Cells to Produce a Protein Product

• Insert gene encoding the protein of interest

• Cells require proper conditions for optimal growth (temp, pH, oxygen, feeds, etc.)

• Culture and fermentation can take weeks

• Complex Purification Steps

• Safe product with desired potency

Bar Charts, Inc. 2003

Is our protein of interest glycosylated? needs post-translational modifications

HOST

CELLS

Procaryotic cell

(e.g E. coli) Eucaryotic cell

Yeast cell Mammalian cell

Chinese Hamster Ovary (CHO) Cells)

Better chance to reach the reference API quality by using the same host cells

Technical Development of Biotechnological Drugs

Environmental

Monitoring

Excipients

Environmental

Moniotoring

Media

Components

Environmental

Moniotoring

Media

Components

Cell

Bank

Inprocess

Tests

Production

Cells

InProcess

Tests

Process

Materials

Active

Substance

Container

Closures

InProcess

Tests

Drug Product

Basic Steps of Biological Drugs Manufacturing

Envirnmental Raw Purification InProcess Resins Process

Monitoring Materials Intermediate Tests Materials

Envirnmental Raw Media Fermentation InProcess Process

Monitoring Materials Components Intermediate Tests Materials

Media Prep

Working Cell

Bank

Sub-

Culture

Inoculum

Sub-

Culture

Sub-

Culture

Sub-

Culture

Sub-

Culture

Large Scale Bioreactor

Wave

Bag

Seed Bioreactors

Fermentation

150L

Bioreactor

750L

Bioreactor

5,000L

Bioreactor

26,000L

Bioreactor

Depth

Filtration

Collection

Centrifuge

Harvest/Recovery

Harvest

Collection

Tank

1,500L

Filter

Chromatography

Anion Exchange Chromatography

(QXL)

Column Eluate

Hold

Tank

8,000L

Eluate

Hold

Tank

6,000L

Filter

Chromatography

Protein A

Chromatography

Column

Chromatography

Column

Eluate

Hold

Tank

20,000L

Hydrophobic Interaction Chromatography

(HIC)

Viral

Inactivation

Eluate

Hold

Tank

5,000L

Filter

Chromatography

Anion Exchange Chromatography (QFF - Fast

Flow)

Column

Post-viral

Hold

Vessel

3,000L

Viral Filtering Ultra Filtration

Diafiltration

Bulk

Fill

Purification

24 days 31 days

8 days

1 day

Technical Development of Biotechnological Drugs

Cell Bank System

50

Cell Bank System

51

Starting material

Parental cell line

Master cell bank (MCB)

Manufacturer working cell bank (MWCB)

Production substrate

Late expanded cell bank (LECB)

Genetic transformation

expand

expand

expand

A Cell Bank

• A collection of ampoules of uniform composition

stored under defined conditions, each containing

an aliquot of a single pool of cells

52

The Master Cell Bank (MCB)

• Generally derived from the selected cell clone

containing the expression construct.

• The MCB is used to derive all working cell banks.

53

The Manufacturer Working Cell Bank (MWCB)

• Derived by expansion of one or more ampoules of

the MCB under defined culture conditions

• The working cell bank is used for the production

of the batches.

54

The Late Expanded Cell Bank (LECB)

• Obtained in multiplying the cells used for the

production of the recombinant protein, several

passage after the passage of production.

• It is used to reveal a potential low viral

contamination and to study genetic stability of

the transgene.

55

Intracellular

(microbial fermentation)

Bulk Formulation

Purification Purification

Isolation/Recover

y Isolation/Recovery

Cell Disruption/Refold

Cell Harvesting Cell Removal

Bioreactor Conversion

Bulk Formulation

Working Cell Bank Extracellular (microbial fermentation &

mammalian cell culture)

“Downstream” Process

“Upstream” Process

General Scheme for Biotechnological Drugs Production

Ammonium sulfate fractionation

Homogenization Organelle

Size Charge Polarity Affinity

Gel filtration,

SDS-PAGE,

Ultrafiltration

Ion exchange,

Chromatofocusig,

Disc-PAGE,

Isoelectric

focusing

Reverse phase chromatograpy,

HIC,

Salting-out

Affinity

chromatography,

Hydroxyapatite

Small molecule Macromolecule Cell

Debris Amino acid, Sugar,

Nucleotides, etc

Nucleic

acid

Protein

Carbohydrate

(Lipid)

Protein Purification

Peptide

variants

Desired

Product

Post-translational

variants

IMPURITY PROFILE

??? PROFILE

Degradation

Process & Product-related impurities

Challenges with Proteins

• Very large and unstable molecules

• Structure is held together by weak non-covalent forces

• Easily destroyed by relatively mild storage conditions

• Easily destroyed/eliminated by the body

• Hard to obtain in large quantities

Manufacturing processes involved in the production of Biotechnology

Large Molecules compared with Chemical Small Molecules

Technical Development of Biotechnological Drugs

Noncovalent Covalent

- Denaturation - Deamidation

- Aggregation - Oxidation

- Precipitation - Disulfide exchange

- Adsorption - Proteolysis

Problem with Proteins (in vivo – in the body)

Problem with Proteins (in vivo – in the body)

• Elimination by B and T cells

• Proteolysis by endo/exo peptidases

• Small proteins (< 30 kD) filtered out by the kidneys very quickly

• Unwanted allergic reactions may develop (even toxicity)

• Loss due to insolubility/adsorption

General requirements

1. A highly similar protein molecule to reference product

2. Same mechanism of action(s)

3. Same strength, dosage form and route of administration

Biosimilar biological products

• No clinically meaningful differences

• To be considered as a new active ingredients

Not interchangeable

Interchangeable biological products

• Risk of safety or diminishing efficacy of alternation and switching is not greater than the risk of continued use of reference product.

• Substitution with the innovator's products permitted

Innovator Bio-products VS Bio-similar

Upstream

- Cell line development

- Fermentation

Downstream

– Purification

– Fill and Finish

Comprehensive analytical characterization

and comparability study

Clinical study

–Preclinical

–Clinical

Bio-products Development

In-p

rocess c

on

trol finis

hed

-pro

duct

co

ntr

ols

Release

Tests

Extended

Characterization

Manufacturing

Process

Quality data combined with

preclinical and clinical

experience provide the full

picture

Clinical

Assessment

Bioproducts Dvelopment

Physicochemical characterisation

- Mass spectrometry techniques (e.g., MALDI-TOF)

- Nuclear magnetic resonance

- Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF)

Primary structure

- peptide map

Secondary structure detection

- Circular dichroism in near- and far-UV spectra

Comprehensive set of orthogonal analytical methods

“Increasing sensitivity”

Product related variants and impurities

- SEC, CE-SDS, CZE, CEX, etc.

Antigen-antibody interaction

- Surface plasmon resonance

- FACS

Combination of methodologies

- liquid chromatography combined with mass

spectrometry

Comprehensive set of orthogonal analytical methods

“Increasing sensitivity”

Multiple bioassays elucidate structure

function relationships

Analytical Advances have demystified the Complexities

Analytical methods are sensitive to differentiate between:

• Batch to batch variation

• Batches before and after a change of the manufacturing process

• Batches from different sites

Analytical methods can determine whether batches sourced in different

countries are identical or not microheterogeneity of protein structure

• Purity profiles

• Glycan distribution

Analytical method are sensitive enough to even differentiate between individual batches

Complete quality range

for justification of limits

and claiming

biosimilarity

ADCC

basic variants

Variability is significant in Reference Products

Monitoring batches of an

approved mAb revealed a shift in

quality

Shift in glycosylation (structure)

pattern results in different

potency in cell-based assays

(function)

Indication of a change in the

manufacturing process

Such shifts observed in several

original products

A biosimilar mAb can sometimes be more similar to its reference

product than a post-change version to a pre-change version of a

single product

Variability is significant in Reference Products

Expected Heterogeneity- experience with monoclonal antibodies

C terminal lysine variability occurs in most monoclonal antibody products

Manufacturers set acceptable ranges for each species

Can be measured by various techniques, wCEX-HPLC, IEF, others

Doesn’t seem to impact potency or safety profile

Monoclonal antibodies Unacceptable, stress induced heterogeneity

Detectable by various methods

Manufacturers set stability specifications

Can compromise potency if OOS

Heterogeneity- experience with other recombinant products

• Case 1: protein terminus heterogeneity

– Traced to metaloprotease

– Minimal impact on potency

• Case 2: product clipping

– Minimal impact on potency

• Case 3: N-terminal glutamine cyclization

– Cyclized form had increased activity

Carbohydrate Profiles & Comparability

Nature Biotechnology 26, 592 (2008)

FDA balks at Myozyme scale-up by George Mack

“Genzyme ran into a snag in April when the US Food and Drug Administration (FDA) rejected its application to produce Myozyme (alglucosidase alfa, rhGAA) in its 2,000–liter-scale facility under the same approval authorization given for its 160-liter-scale plant. The FDA says the carbohydrate structure of the products manufactured at each scale differs and thus the 2,000-liter product requires a new biologic license application”

* Genzyme has been acquired by Sanofi-Aventis.

Epoeitin Alfa

Erstellt von Jamiri

30.4 Kda

2 disulfide linkages

165 amino acids

4 glycosylation sites

Monoclonal Antibodies

K

pyro-E O

D

G

G

D

O D

O

O pyro-E

D

D

D

G

G

150 kda

Pyro-Glu

Methionine oxidation

deamidation

High mannose,

G0, G1, G1, G2

Sialylation S. Kozlowski, OBP, CDER

Erstellt von Jamiri

Glycoform Profiles and Comparability Testing

(Dr. Stephan Fischer, Roche ) Schellekens H., Nephrol Dial Transplant 2005; 20 [Suppl 4]:iv31–iv36 (H. S. has contributed to meetings and publications sponsored by Amgen, Roche, Johnson & Johnson and Shire.)

A Case of MAb Biosimilar or Not?

• Reditux by Dr.Reddy’s approved in India in 2007

• Analysis by Genentech:

Identical amino acid sequence and molecular weight

Glycoforms not comparable

Charge distribution not comparable

Aggregate content not comparable

Effector function not comparable

Higher host cell protein content

Clinical data with Reditux in NHL comprised 17 patients only

Reed Harris, Genentech, Presentations at FABIAN

2008”Biopharma, Biosimilar, Biogenerics? Bioanalysis”,

Groningen, the Netherland, 2008 and “Biogenerics 2008”.

Insulin Case: Marvel Rapid failure

From EMEA Withdrawal Assessment Report EMEA/CHMP/317778/2007

redrawn by Heinemann & Hompesch

J Diabetes Sci Technol 2011;5(3):741-75

Combined Clamp (PD) and PK in 24 healthy subjects

•Early insulin AUCs bioequivalent (95% CI at 1 hour: 103 %-124%), •NOT Early Glucose Infusion Rate AUCs (95% CI at 1 hour: 100%-145%);

- 58 -

Marvel (insulins) 16h January 2008

• Did mot meet PK/PD equivalence margin

• Clinical equivalence margin too broad

• Many CMC deficiencies

Biograstim, Ratiograstim,Tevagrastim: 21st

February

2008

• Many more in the pipeline!

Monoclonal Antibodies

• Next wave of biosimilars

• Data pending

Interpretation of findings Experience in the EU

Alpha interferon case: Alpheon failure

From EMEA Withdrawal Assessment Report Procedure No. EMEA/H/C/000585

”Clinically and statistically significant difference in virological

relapse rates found between the end of therapy and the end of the

observation period.”

- 56 -

Omnitrope (somatropin): 26th January 2006

• Literature reliance not allowed

Valtropin (somatropin): 23rd February 2006

• Different Host Cell Allowed

• Data had to be re-analysed excluding US sourced reference product

Alpheon (alpha interferon) 28th June 2006

• More patients had return of disease after treatment when Alpheon

stopped than for reference medicine and more side effects with

Alpheon

Binocrit, Abseamed, Epo- α Hexal (epoetin α): 28th Aug07

• Slight differences in glycosylation acceptable

• Single comparative study acceptable (guidelines require 2)

• Lack of immunogenicity data in renal patients not acceptable = iv only

Interpretation of findings Experience in the EU

CASE: Silapro EPO Biosimilar Bioassay reflects difference in bioactivity

Found to mirror difference in protein concentration

•Erypo (Eprex) batches were found on average to have 8% higher bioactivity than SB309 (Silapo) batches, although all batches remained within the Ph. Eur. limits, namely 80%-125% (with error limits of 64 to 156%).

•There was a correspondingly higher protein content in the Erypo batches which contained on average 9% over the labelled amount of protein, compared with on average 1% over the labelled amount with SB309.

•The average specific activities for both products were remarkably similar (130.80 for test vs. 130.75 units/μg for reference).

CASE: Silapro EPO Biosimilar

The dosage of the test drug within the last four weeks was approximately 10%

higher than the dosage of the reference product.

Clinical trial does not detect difference during correction

phase - does during maintenance phase

(Silapo EPAR)

Goals of Quality, Non-clinical, and Clinical Studies • Quality

To demonstrate of comparability of the product to a reference product- the most critical step.

• Pre-clinical toxicology

To confirm therapeutic index and safety profile.

To qualify impurities by short-term animal studies .

Full animal toxicity studies are not necessary.

• Non-clinical PK/PD studies

To confirm dosing regimen by PK profiles.

To confirm the mechanism of actions by biomarkers (PD).

• Clinical safety

To compare immunogenicity and/or hypersensitivity with the reference products.

• Efficacy

To conduct confirmatory trials or other clinical trials for interchangeability.

Use of complementary biomarkers,or surrogate endpoints in some cases.

Comparability

Quality

Studies Physicochemical tests

Bioactivity/Potency assays

Stability

Non-clinical

Studies PK/PD studies

Toxicology

Clinical

Studies Efficacy

Immunogenicity

Quality attributes are highly similar( not necessary identical).

Reference Products Step-wise testing based

on:

Biosimilar Products Comprehensive studies

due to:

• Molecular complexity

• Manufacturing process

• Degree of

characterization

• Clinical indications

• Production history

• Availability of

safety and clinical

data

• Complex structure

• Different cell line

and production

process

• Different characterization

and release tests

• Extensive safety and

clinical data not available

Comparability Exercises

• Molecular complexity

• Manufacturing process

• Degree of characterization

• Clinical indications

• Production history

• Availability of safety and clinical data

Quality

Studies Physicochemical tests

Bioactivity/Potency assays

Stability

Non-clinical

Studies PK/PD studies

Toxicology

Clinical

Studies Efficacy

Immunogenicity

Quality attributes are highly similar( not necessary identical).

Reference Products Step-wise testing based

on:

Biosimilar Products Comprehensive studies

due to:

• Different cell line

and production

process

• Different characterization

and release tests

• Short production history

• Extensive safety and

clinical data not available

Comparability Exercises

LMH

W Heparin-induced

thrombocytopenia type II, anaphylactoid and anaphylactic reactions

Safety data should be collected from a cohort of patients representing all approved therapeutic indications

Post-marketing studies

Specific requirement for specific product

G-C

SF

Antibodies to the currently marketed rG-CSF occur infrequently

Attention should be paid to immunogenicity and potential rare serious adverse events, especially in patients undergoing chronic administration

Lack of efficacy should also be monitored, especially in individuals undergoing hematopoietic progenitor cell mobilization

Specific requirement for specific product

Specific requirement for specific product

Eryt

hro

po

ieti

n Rare serious adverse events such

as immune mediated Pure red cell aplasia “PRCA” and immune- related adverse events

Hypertension/aggravation of hypertension and thromboembolic events

Safety data should be collected from a cohort of patients representing all approved therapeutic indications

Regulatory trends

• Equivalence trials

– EU to consider non-inferiority for phase III

• Reference Product

– EU to change requirements on EU sourcing

• Risk-based approach

– FDA Totality-of-the-Evidence

• Step-wise approach from CMC via N-C to Clinical parts

EU Specific requirements – Clinical

(*) = Low end total subjects in studies where known from EPARs

Product PK PD Efficacy Safety

Somatropin HV (25*) HV (PK) Juvenile def. (90) 12 m Efficacy pop (90)

Insulin T1D Clamp No need 6 m appr., 12 m follow-up

G-CSF HV (2X28) NC (4X36) Chemo prot (140)

Not spec (160; 6 cycles)

IFNα 2a/2b HV (2x25) Markers (2x25)

Viral elim w12 (200/arm)

48 wk Efficacy pop (400)

LMw heparin By PD HV marker Thrombosis prev

“Sufficient number”

EPO HV (24) HV (PK) 1 route (200/arm)

(bridge w PK/PD)

12 m Efficacy pop (600)

mAb HV/pat ? HV/pat (PK) ? Risk based Risk-based – SciAdv!

Scientific advice

EU product specific Clinical Requirements

Directive 2001/83/EC

General guideline CHMP/437/04

Quality Issues CHMP/BWP/49348/200

5

Non-Clinical & Clinical Issues CHMP/BMWP/42835/2005

Revision concepts 2011

Revision draft2012

Changes planned? Legal level

Overarching

guidance

Level(s)

Detailed

guidance

level

Som

Ins

G-CSF

LMW Hepar

EPO IFN α 2a/b

mAb

IFN β FSH

Safety data

Risk Management Program

– Within the authorisation procedure the applicant should present an updated of the risk management plan in accordance with current EU legislation and pharmacovigilance guidelines. This should take into account risks identified during product development and potential risks.

Safety data

Immunogenicity

– If high levels of neutralising antibodies associated with comparator, then similar immunogenicity easier to investigate

– If serious immunogenic events rare than may need very large trial e.g. 30 000 patients to detect PRCA with Eprex

– Impact of neutralising antibodies – critical for epoetin, acceptable for interferon.

– Impact of non-neutralising antibodies - complex, may even enhance efficacy

– Need testing plan

– Need validated assay

Safety data

Extent of Data Base

• Not the detection of adverse events per se, but the evaluation of differences in occurrence.

• Study duration and sample size calculation should consider both frequency and magnitude of reasonably expectable adverse events

• Look for possible differences in clinical presentation (duration, magnitude, reversibility, response to treatment etc.).

• Acute/chronic use

• See also ICH guideline ICH E1

– 300 – 600 patients for 6 months

– 1500 patients

Do we need all the pieces to fully understand the picture?

Do we need all the pieces to fully understand the picture? to know it is a women – NO to know if she is smiling? – we need more information and a smile can make the difference!