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, March 2007

Definitions
Low-molecular-weight drug

Classical medicinal pharmaceutical

product

Generic drug

Chemical and therapeutic equivalent of

low-molecular-weight drug with
expired patent

Biopharmaceutical

A medicinal product developed by

means of one or more of the following
biotechnology practices: rDNA,
controlled gene expression, antibody
methods1

Biosimilar, or similar
biological medicinal
product

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 expired1

EMEA = European Medicines Agency.

1. EMEA definition.

Biopharmaceuticals
Introduced in the beginning of the 1980s
More than 150 products marketed around
the world
Over 370 products in development for a
wide range of serious conditions

Biopharmaceuticals represent a fast-growing and important

group of drugs mainly for the treatment of severe diseases

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

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

Most biopharmaceutical
proteins have small
markets, but high value
< 10 kg/yr, > US$10,000/g

Biopharmaceuticals will outperform the total pharmaceutical market

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

Knaeplein, 2007

Market & Technology

Maturity

Technology Evolution in
Pharma Industry

Chemistrybased
compounds

Extraction-based
biological compounds

Therapeutic
recombinant
proteins
Vaccinal
recombinant
proteins

Gene
Therapy
Immature

Vaccine
cell
Cell
therapy therapy

Emerging

Growing

Mature

Decreasing

Common
Fully
Mastering
Non validated Validated
technologies technologies technologies mastered and technologies
opmized
Ongoing
First products More
technologies Manufacturing
alternatives required
optimization
entering
products in
Many
development development
Decreasing number
Many
products in
phases
Few products products in
development of products in
development and,
No products
in the market development and in the
eventually, in the
and in the
in market
market
market
market
Source: Paulo Barbanti

Why biopharmaceuticals are different

High molecular weight

Complex three-dimensional structure
Complex 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 integrity
Inherent risk of immunogenicity

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

Five expression technologies for protein production

Bacteria

Yeast

Transgenic Animals

Sheep, goat, cow

Mammalian Cells

Saccharomyces

CHO

Transgenic Plants

Tobacco, moss

Escherichia coli

knaeplein

Complex production and downstream purification process

Bottom line: complete characterization: mission impossible

International Journal
of Pharmaceutics

Th
e
qu
in
the ali
t
p r y is
oc
es
s

2003, November,
266, 3-16

Two main IFN alpha-2 preparations

Generic
name

Commercial Aa position Natural

name
23
alelle

Hu IFN
alpha-2a

Roferon

Lys

No

Hu IFN
alpha-2b

Intron

Arg

Yes

PEGylated proteins
PEG-Interferon alfa
PEG-G-CSF
.

Strategies for improved protein delivery

PEGylation: coupling of PEG molecules to protein

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

Different epo products on the market

outside EU/US (epoetin alfa)
2 studies: qualitative and quantitative analyses on
11/12 non-FDA/EMEA approved epoetin alfa
samples
Conclusions
Significant deviations from standard specifications
In vivo bioactivity 71226% of the EPREX/ERYPO
standard
Isoform patterns variable, even between samples
from the same manufacturer
Level of bacterial endotoxins was unacceptable
in 3 samples

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

Epo: isoform distribution (IEF) of epo products

Isoform distribution
B

Cathode

IA

IB IIA

IIB

IIIA IIIB

IV

VII

VIII

VI

Anode

Sample

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)

Schellekens

Brief history of the protein immunogenicity problem

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

recombinant human interferon beta

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

Immunogenic biopharmaceuticals and clinical

consequences
Loss of efficacy
Insulin
Streptokinase
Staphylokinase
ADA
Calcitonin
Factor VIII
Interferon alpha 2
Interferon beta
Interleukin-2
GnRH
TNFR55/IgG1
Denileukin diftitox
HCG
GM-CSF/IL3

Enhancement of efficacy
Growth hormone

Neutralization of
endogenous protein
Megakaryocyte-derived growth
factor (MDGF)
Epoetin

General immune effects

Allergy
Anaphylaxis
Serum sickness, etc

Sequence variation
A
L
C
N
A
T

human
F K

K
T
K

A
L
S
N
A
I

Glycosylation

non-human
F K

K
F
K

Immunogenicity
Contaminants &
Formulation
impurities

Length of
treatment

Assay technology

Application
route

Patient features

Dose

Unknown
factors

February

Schellekens, Nature Reviews Drug Discovery, 2002

Neutralizing antibodies standard serum

in different laboratories
Neutralizing Activity

12
10
8

Assay validation!

6
4
2
0
400

200

100

50

25

12,5

Serum Dilution
Hanover
Uppsala

Rijswijk
Rome

Basel
Copenhagen

6,25

3,1

Interferon alfa
1996

Therapeutic effect versus antibody level in

interferon- treated patients
16

Response

14
12
10
8
6
4
2
0
Negative

< 2000

> 2000

Two main IFN alpha-2 preparations

Generic
name

Commercial Aa position Natural

name
23
alelle

Hu IFN
alpha-2a

Roferon

Lys

No

Hu IFN
alpha-2b

Intron

Arg

Yes

Antibodies and type of interferon

Type of
interferon

% Antibodies

r-IFN alpha 2a

20

r- IFN alpha 2b

Immunogenicity differences between IFN-

formulations
2000

IFN neutralizing units

1800

A (n = 190)

1600
1400
1200
1000
800

B (n = 86)

600
400

C (n = 110)

200

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

0
0

Duration of treatment (months)

Ryff, J Interferon Cytokine Res, 1997

RP-HPLC profile of a highly immunogenic batch

of interferon (IFN)-alfa2A

Mo is an oxidized form, which enhances immunogenicity and contributes

contributes to aggregate formation,
From Schellekens, Nature Reviews, 1, 2002, 457

Anti-epoetin antibody-related pure red cell

aplasia cases

Number of Epoetin Alfa

PRCA Cases

Removal of human serum albumin stabilizer

from epoetin alfa (outside USA)
80
70
60
50
40
30
20
10
0
<1997 1998

1999 2000

2001

2002 2003

Year

EPO alfa (Epogen /Procrit ) in USA

EPO alfa (Eprex ) outside USA

Main Stabilizers Used in Epoetin

Formulations
Epogen/Procrit Eprex
(US)
(pre 1998)

Eprex
(post 1998)

NeoRecormon
(1990 launch)

HSA

Polysorbate 80

Polysorbate 20

Glycine

Glycine

HSA

Complex of
5 other
amino acids
Calcium chloride
Urea
26

Factors Potentially Contributing to

the Immunogenicity of Eprex
Formation of micelles associated with

Epoetin (Hermeling et al, 2003)

Silicon droplets in the pre-filled syringes
Leachates from rubber stoppers
Mis-handling

27

Neutralizing antibodies in patients receiving

interferon
(same production process, different site)

30

Avonex

NAb (% of patients)

25

BG9015
20
15
10
5
0
6

12

18

Treatment (months)

24

Antigenicity of identical Hu IFN beta produced

at different sites
%
patients

months

Most therapeutic proteins induce antibodies

Two mechanisms:
Reaction to neo-antigens
(classical immune response)

Breakdown of immune tolerance

Types of immune reaction against therapeutic proteins

Classical immune response

Breaking of self-tolerance

Type of product

Foreign proteins (microbial or

animal origin)

Human homologues
(recombinant human proteins)

Immune
response
(antibody
production)

Fast, often after a single

injection

Slow, after long treatment

Cause

The presence of foreign

antigens

Disappear after treatment

Long duration

Impurities, aggregates, ...???

Dogma: protein aggregates are immunogenic

Monomeric proteins
non-immunogenic

Aggregated proteins
immunogenic

Mechanisms for breaking B cell tolerance

Repetitive epitopes (T cell independent)
Optimum spacing ~ 5-10 nm
Minimum valency ~ 10 epitopes
Shown for small haptens (Dintzis et al, 1976)

5-10 nm

Not
immunogenic

Immunogenic

~60 nm

Not
immunogenic

Sequence variation
A
L
C
N
A
T

human
F K

K
T
K

A
L
S
N
A
I

Glycosylation

non-human
F K

K
F
K

Immunogenicity
Contaminants &
Formulation
impurities

Length of
treatment

Assay technology

Application
route

Patient features

Dose

Unknown
factors

February

Schellekens, Nature Reviews Drug Discovery, 2002

Patent expiration of Biopharmaceuticals

Pioneer
company

Product

Indication(s)

Genentech

Nutropin (somatropin)

Abbott

EU patent/market
exclusivity expires

US patent/market
exclusivity expires

Growth disorders

Expired

Expired

Abbokinase (eudurase
urokinase)
Humulin (recombinant insulin)

Ischaemic events

Expired

Expired

Diabetes

Expired

Expired

Ceredase (alglucerase);
Cerezyme (imiglucerase)

Gaucher disease

Expired

Expired

AstraZeneca Streptase (streptokinase)

Ischaemic events

Expired

Expired

Expired (France)
2007 (Italy)

Expired

Eli Lilly
Genzyme

Biogen/
Roche

Intron A (interferon-alpha-2b)

Hepatitis B and C

Serono

Serostim (somatotropin)

AIDS wasting

NA

Expired

Eli Lilly

Humatrope (somatropin)

Growth disorders

NA

Expired

Amgen

Epogen, Procrit, EPREX

(epoetin)

Anaemia

Expired

2013

Roche

NeoRecormon (epoetin)

Anaemia

Expired

NA

Genentech

TNKase (tenecteplase TNK-tPA)

Acute myocardial infarction

Expired

Expired

InterMune

Actimmune
(interferon-gamma-Ib)

Chronic granulomatous disease,

malignant osteopetrosis

Expired

2006/2012

Genentech

Activase, Alteplase (tPA)

Acute myocardial infarction

Expired

Expired/2010

Chiron

Proleukin (interleukin-2)

HIV

Expired

2006/2012

Amgen

Neupogen (filgrastim G-CSF)

Anaemia, leukaemia, neutropenia

2006

2015

G-CSF = granulocyte colony-stimulating factor;

tPA = tissue plasminogen activator.

Adapted from Schellekens H. Trends Biotechnol. 2004;22:406-10.

Biosimilars available on the market

outside the USA and the EU

Limited amount of pre-clinical data published

In general, clinical studies involve small patient
populations1

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

Regulatory 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 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

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

Biosimilars guidelines
(EMEA concept papers)

Biosimilars

Product-class-specific annexes
(concept papers)

Somatotropins

Epoetins

Insulins

G-CSF

Others?

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

Biosimilars with marketing approval in EU:

hGH
Omnitrope, Sandoz (ref. Genotropin, Pfizer)
Valtropin, Biopartners (ref. Humatropin, Lilly)
Epo
Binocrit, Sandoz
Epoietin alfa Hexal, Hexal Biotech Forschung
Abseamed, Medice Arzneimittel Putter
Not approved
Interpheron alpha 2a
Alpheon, Biopartner (ref. Roferon A Roche)

Innovators..
Eprex, Procrit, Janssen-Cilag
Epogen, Amgen
NeoRecormon, Roche

Interchangeability..
United States (U.S. FDA Considerations on
Possible INN Policies for Biosimilars)
U.S. FDA believes that the only way to
establish pharmacologic interchangeability
is 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

Wim Jiskoot

Identification of relevant differences between products