Performance of biotech products (biopharmaceuticals):

Other sources of variability?

Daan J.A. Crommelin

EUFEPS meeting, Verona

– Dept Pharmaceutics, Utrecht University, NL– Scientific Director Dutch Top Institute Pharma,

Leiden, NL

Based on presentation at the EAHP Annual Meeting Bordeaux, MarchBased on presentation at the EAHP Annual Meeting Bordeaux, March 20072007

DefinitionsDefinitions

“A biological medicinal product referring to an existing one and submitted to regulatory authorities for marketing authorization by an independent applicant after the time the original product expired”1

Biosimilar, or similar biological medicinal product

“A medicinal product developed by means of one or more of the following biotechnology practices: rDNA, controlled gene expression, antibody methods”1

Biopharmaceutical

Chemical and therapeutic equivalent of low-molecular-weight drug with expired patent

Generic drug

Classical medicinal pharmaceutical product

Low-molecular-weight drug

1. EMEA definition.EMEA = European Medicines Agency.

BiopharmaceuticalsBiopharmaceuticals

Introduced in the beginning of the 1980sMore than 150 products marketed around the worldOver 370 products in development for a wide range of serious conditions

Biotechnology Industry Organization. 2004. www.bio.org.

Biopharmaceuticals represent a fast-growing and important group of drugs mainly for the treatment of severe diseases

With the highest growth rates within the entire With the highest growth rates within the entire pharmapharma market, market, biopharmaceuticals will reach > US$ 92 billion revenues in 2011biopharmaceuticals will reach > US$ 92 billion revenues in 2011

Biopharmaceuticals will outperform the total pharmaceutical market

– With over 1/3 of ALL pipeline products the market forecast is US$ 92 billion in 2011

Most biopharmaceutical proteins have small

markets, but high value < 10 kg/yr, > US$10,000/g

Knaeplein, 2007Knaeplein, 2007

Emerging

Mar

ket &

Tec

hnol

ogy

Mat

urity

Gene Therapy

Vaccinal Vaccinal recombinant recombinant

proteinsproteins

Extraction-based biological compounds

Immature

• Non validated technologies

• First products entering development phases

• No products in market

Growing Mature Decreasing

• Validated technologies

• More products in development

• Few products in the market

• Mastering technologies

• Ongoing optimization

• Many products in development and in the market

• Fully mastered and opmized technologies

• Many products in development and in the market

• Common technologies

• Manufacturing alternatives required

• Decreasing number of products in development and, eventually, in the market

Cell therapy

Vaccine cell

therapy

Therapeutic Therapeutic recombinant recombinant

proteinsproteins

Chemistry-based

compounds

Technology Evolution in Technology Evolution in Pharma IndustryPharma Industry

Source: Paulo Barbanti

Why biopharmaceuticals are differentWhy biopharmaceuticals are different

High molecular weightComplex three-dimensional structureComplex manufacturing process *Produced by living organisms; therefore often heterogeneous *Difficult to characterize completely by physico-chemical analytical methods or bioassays *Dependence of biological activity on reproducibility of the production process, in-house standards, and maintenance of cold-chain integrityInherent risk of immunogenicity

Crommelin DJA, et al. Int J Pharm. 2003;266:3-16.

Five expression technologies for protein productionFive expression technologies for protein production

Sheep, goat, cow

Transgenic Animals

Yeast

Saccharomyces

Transgenic Plants

Tobacco, moss

Bacteria

Escherichia coli

Mammalian Cells

CHOCHO

knaeplein

Complex production and downstream purification processComplex production and downstream purification process

International Journal International Journal of Pharmaceuticsof Pharmaceutics

2003, November, 2003, November, 266, 3266, 3--1616

Bottom line: complete characterization: mission impossibleBottom line: complete characterization: mission impossible

The quality is

The quality is

in the process

in the process

Two main IFN alpha-2 preparations

Generic Commercial Aa position Naturalname name 23 alelle

Hu IFN Roferon Lys Noalpha-2a

Hu IFN Intron Arg Yesalpha-2b

PEGylatedPEGylated proteinsproteins

PEG-Interferon alfaPEG-G-CSF ……………………….

Strategies for improved protein deliveryStrategies for improved protein delivery

PEGylation: coupling of PEG molecules to protein

http://www.roche.com/pages/facets/10/pegasyse.htm

Different Different epoepo products on the market products on the market outside EU/USoutside EU/US ((epoetin alfa)epoetin alfa)

2 studies: qualitative and quantitative analyses on 11/12 non-FDA/EMEA approved epoetin alfa samples

ConclusionsSignificant deviations from standard specificationsIn vivo bioactivity 71–226% of the EPREX®/ERYPO®

standardIsoform patterns variable, even between samples from the same manufacturerLevel of bacterial endotoxins was unacceptablein 3 samples

Schmidt CA, et al. Arq Bras Endocrinol Metab. 2003;47:183-9; Schellekens H. Eur J Hosp Pharm. 2004;3:43-7.

Isoform distribution

Isoform patterns: deviations displayed by 9 of the 11 samples (including additional basic and acidic isoforms, and increased

bar intensity) compared with the EPREX® standard (E)

E IA IB IIA IIB IIIA IIIB IV V VII VIII E

BA

Cathode

AnodeE VI

Sample

EpoEpo: : isoformisoform distribution (IEF) of distribution (IEF) of epoepo productsproducts

SchellekensSchellekens

Brief Brief historyhistory of the of the proteinprotein immunogenicity immunogenicity problemproblem

Proteins of animal origin (> 1920s)(e.g. equine antisera, porcine/bovine insulin)

Human derived proteins (> 1950s)(e.g. growth hormone, factor VIII)

Recombinant human proteins (> 1980s)(e.g. insulin, interferons, GM-CSF, epo)

Selected papers (2004) on the immunogenicity of Selected papers (2004) on the immunogenicity of recombinant human interferon betarecombinant human interferon beta

In the EU ca. 100 million euro/year is spent on useless IFNIn the EU ca. 100 million euro/year is spent on useless IFN--ββ therapytherapy

Immunogenic biopharmaceuticals and clinical Immunogenic biopharmaceuticals and clinical consequencesconsequences

Loss of efficacyLoss of efficacyInsulinInsulinStreptokinaseStreptokinaseStaphylokinaseStaphylokinaseADAADACalcitoninCalcitoninFactor VIIIFactor VIIIInterferon alpha 2Interferon alpha 2Interferon betaInterferon betaInterleukinInterleukin--22GnRHGnRHTNFR55/IgG1TNFR55/IgG1DenileukinDenileukin diftitoxdiftitoxHCGHCGGMGM--CSF/IL3CSF/IL3

Enhancement of efficacyEnhancement of efficacyGrowth hormoneGrowth hormone

Neutralization of Neutralization of endogenous proteinendogenous proteinMegakaryocyteMegakaryocyte--derived growth derived growth factor (MDGF)factor (MDGF)EpoetinEpoetin

GeneralGeneral immune immune effectseffectsAllergyAllergyAnaphylaxisAnaphylaxisSerum Serum sicknesssickness, etc, etc

Immuno-genicity

AL

CNA

TF K

KTK

AL

SNA

IF K

KFK

Sequence variation Glycosylation

human non-human

Contaminants & impurities Formulation

Application route Dose

Length of treatment Assay technology Patient features Unknown

factorsFebruary

Schellekens, Nature Reviews Drug Discovery, 2002

0

2

4

6

8

10

12

400 200 100 50 25 12,5 6,25 3,1

Neutralizing antibodies standard serum in different laboratories

Neu

tral

izin

gA

ctiv

ity

RomeHanoverUppsala

BaselRijswijkCopenhagen

Serum DilutionInterferon alfa

1996

Assay validation!Assay validation!

TherapeuticTherapeutic effect versus antibody effect versus antibody levellevel in in interferoninterferon--αα treatedtreated patientspatients

Resp

on

seR

esp

on

se

0

2

4

6

8

10

12

14

16

Negative < 2000 > 2000

Two main IFN alpha-2 preparations

Generic Commercial Aa position Naturalname name 23 alelle

Hu IFN Roferon Lys Noalpha-2a

Hu IFN Intron Arg Yesalpha-2b

Antibodies and type of interferon

Type of interferon

% Antibodies

r-IFN alpha 2a r- IFN alpha 2b

20 7

Immunogenicity Immunogenicity differencesdifferences betweenbetween IFNIFN--ααformulationsformulations

DurationDuration of of treatmenttreatment ((monthsmonths))

0200400600800

100012001400160018002000

0 1 2 3 4 5 6 7 8

IFN

neu

tral

izin

gun

its

A (n = 190)A (n = 190)

B (n = 86)B (n = 86)

C (n = 110)C (n = 110)

D (n = 81)D (n = 81)E (n = 74)E (n = 74)

Ryff, J Interferon Cytokine Res, 1997

RPRP--HPLC profile of a highly immunogenic batchHPLC profile of a highly immunogenic batchof interferon (IFN)of interferon (IFN)--alfa2Aalfa2AMo is an oxidized form, which enhances immunogenicity and contriMo is an oxidized form, which enhances immunogenicity and contributes to aggregate formation, butes to aggregate formation, From Schellekens, Nature Reviews, 1, 2002, 457From Schellekens, Nature Reviews, 1, 2002, 457

AntiAnti--epoetin antibodyepoetin antibody--related pure red cell related pure red cell aplasiaaplasia casescases

0

10

20

30

40

50

60

70

80

Num

ber o

f Epo

etin

Alfa

PR

CA

Cas

es

<1997 1998 1999 2000 2001 2002 2003Year

EPO alfa (Epogen /Procrit ) in USAEPO alfa (Eprex ) outside USA

Removal of human serum albumin stabilizer from epoetin alfa (outside USA)

2626

Main Stabilizers Used in Epoetin Main Stabilizers Used in Epoetin FormulationsFormulations

Eprex®

(post 1998)

Polysorbate 80

Glycine

NeoRecormon®

(1990 launch)

Polysorbate 20

Glycine

Complex of5 other amino acids

Calcium chloride

Urea

Epogen®/Procrit®

(US)

HSA

Eprex®

(pre 1998)

HSA

2727

Factors Potentially Contributing to Factors Potentially Contributing to the Immunogenicity of the Immunogenicity of EprexEprex®®

•• Formation of micelles associated with Formation of micelles associated with Epoetin (Hermeling et al, 2003)Epoetin (Hermeling et al, 2003)

•• Silicon droplets in the preSilicon droplets in the pre--filled syringesfilled syringes•• Leachates from rubber stoppersLeachates from rubber stoppers•• MisMis--handlinghandling

0

5

10

15

20

25

30

6 12 18 24

Treatment (months)

NA

b (%

of p

atie

nts)

Avonex

BG9015

Neutralizing antibodies in patients receiving Neutralizing antibodies in patients receiving interferoninterferon––ββ

(same production process, different site)(same production process, different site)

Antigenicity of identical Hu IFN beta producedat different sites

monthsmonths

%%patientspatients

Most Most therapeutictherapeutic proteinsproteins induceinduce antibodiesantibodies

Two mechanisms:

Reaction to neo-antigens(classical immune response)

Breakdown of immune tolerance

Breaking of self-toleranceClassical immune response

The presence of foreignantigens

Fast, often after a single injection

Long duration

Foreign proteins (microbial oranimal origin)

Impurities, aggregates, ...???Cause

Slow, after long treatment

Disappear after treatment

Immune response (antibody production)

Human homologues(recombinant human proteins)

Type of product

Types of immune reaction against therapeutic proteinsTypes of immune reaction against therapeutic proteins

Dogma: protein aggregates are immunogenic

Monomeric proteinsMonomeric proteinsnon-immunogenic

Aggregated proteinsAggregated proteinsimmunogenic

Repetitive epitopes (T cell independent)– Optimum spacing ~ 5-10 nm– Minimum valency ~ 10 epitopes

– Shown for small haptens (Dintzis et al, 1976)

Not Not immunogenicimmunogenic

Not Not immunogenicimmunogenic ImmunogenicImmunogenic

55--10 nm10 nm ~60 nm~60 nm

MechanismsMechanisms forfor breakingbreaking B B cellcell tolerancetolerance

Immuno-genicity

AL

CNA

TF K

KTK

AL

SNA

IF K

KFK

Sequence variation Glycosylation

human non-human

Contaminants & impurities Formulation

Application route Dose

Length of treatment Assay technology Patient features Unknown

factorsFebruary

Schellekens, Nature Reviews Drug Discovery, 2002

Patent expiration of BiopharmaceuticalsPatent expiration of Biopharmaceuticals

AstraZeneca

Genzyme

Roche

Eli Lilly

Serono

Genentech

AmgenChironGenentech

InterMuneGenentech

AmgenEli Lilly

Biogen/ Roche

Abbott

Pioneer company

Expired

Expired

NA

Expired

Expired

Expired

20152006/2012

Expired/2010

2006/2012Expired

2013Expired

Expired

Expired

US patent/market exclusivity expires

Ischaemic events

Gaucher disease

Anaemia

Diabetes

AIDS wasting

Growth disorders

Anaemia, leukaemia, neutropeniaHIVAcute myocardial infarction

Chronic granulomatous disease, malignant osteopetrosis

Acute myocardial infarction

AnaemiaGrowth disorders

Hepatitis B and C

Ischaemic events

Indication(s)

Expired

Expired

Expired

Expired

NA

Expired

2006Expired

ExpiredNA

Expired

Expired (France)2007 (Italy)

Expired

Expired

Expired

EU patent/market exclusivity expires

Streptase® (streptokinase)

Ceredase® (alglucerase); Cerezyme® (imiglucerase)

NeoRecormon® (epoetin)

Humulin® (recombinant insulin)

Serostim® (somatotropin)

Nutropin® (somatropin)

Neupogen® (filgrastim G-CSF)Proleukin® (interleukin-2)

Epogen®, Procrit®, EPREX®

(epoetin)

Humatrope® (somatropin)

TNKase® (tenecteplase TNK-tPA)

Intron A® (interferon-alpha-2b)

Activase®, Alteplase® (tPA)

Actimmune®

(interferon-gamma-Ib)

Abbokinase® (euduraseurokinase)

Product

Adapted from Schellekens H. Trends Biotechnol. 2004;22:406-10. G-CSF = granulocyte colony-stimulating factor; tPA = tissue plasminogen activator.

Biosimilars available on the marketBiosimilars available on the marketoutsideoutside the USA and the EUthe USA and the EU

Limited amount of pre-clinical data publishedIn general, clinical studies involve small patient populations1

1. Combe C, et al. Pharmacotherapy. 2005;25:954-62.

Regulatory framework developmentRegulatory framework development

Regulatory authorities in the EU (EMEA) and the USA (FDA), and the scientific community have recognized that biosimilars differ from generic low-molecular-weight drugs in several ways Not possible at present for 2 different manufacturers to produce 2 identical biopharmaceuticals (similar at best)

The legal and regulatory principles of “essential similarity” (EU) or “bioequivalence” (USA) cannot be applied to biosimilars

FDA = Food and Drug Administration.

Regulatory status of biosimilars in the EURegulatory status of biosimilars in the EU

Since 2001 review of pharmaceutical legislation in EU

Part of the new medicines legislation – came into effect in EU member states in

October 2005

There is a clearly defined legal/regulatory framework in the EU

European Commission: DG Enterprise and Industry. http://pharmacos.eudra.org/F2/review/index.htm.

Key aspects of the new EU legislation Key aspects of the new EU legislation on biosimilars on biosimilars

The centralized procedure is obligatory for biopharmaceuticals and biosimilarsHigh-standard data package on quality, safety, and efficacy to assess application of biosimilarsReference product must be authorized in the EU Strong emphasis on safety Framework for general and product-specific, case-by-case approach

Biosimilars guidelinesBiosimilars guidelines(EMEA concept papers)(EMEA concept papers)

Biosimilars

Product-class-specific annexes (concept papers)

Somato-tropins Epoetins Insulins G-CSF Others?

EMEA guidelines. www.emea.eu.int/pdfs/human/biosimilar/4283205en.pdf.

BiosimilarsBiosimilars with marketing approval in EU:with marketing approval in EU:hGHhGH

OmnitropeOmnitrope®®, , SandozSandoz (ref. (ref. GenotropinGenotropin, Pfizer), Pfizer)ValtropinValtropin®®, , BiopartnersBiopartners (ref. (ref. HumatropinHumatropin, Lilly), Lilly)

EpoEpoBinocritBinocrit®®, , SandozSandozEpoietinEpoietin alfaalfa HexalHexal®®, , HexalHexal Biotech Biotech ForschungForschungAbseamedAbseamed®®, , MediceMedice ArzneimittelArzneimittel PutterPutter

Not approved Not approved InterpheronInterpheron alpha 2aalpha 2a

AlpheonAlpheon®®, , BiopartnerBiopartner (ref. (ref. RoferonRoferon A Roche)A Roche)

InnovatorsInnovators……....

EprexEprex®®, , ProcritProcrit®®, Janssen, Janssen--CilagCilagEpogenEpogen®®, Amgen, AmgenNeoRecormonNeoRecormon®®, Roche, Roche

InterchangeabilityInterchangeability……....

United States (U.S. FDA Considerations on Possible INN Policies for Biosimilars)“U.S. FDA believes that the only way to establish pharmacologic interchangeabilityis through scientific data”

Europe (Thomas Lonngren media interview)“It is not possible we would guarantee a biosimilar is interchangeable (with its originator) … the decision is based on clinical experience that you could switch …”

Identification of Identification of relevantrelevant differences between productsdifferences between products

Wim JiskootWim Jiskoot