Biopharmaceuticals, an industrial perspective (1999)

BIOPHARMACEUTICALS,
AN INDUSTRIAL PERSPECTIVE

Edited by

Gary Walsh
University of Limerick,
Limerick, Ireland
and

Brendan Murphy
Limerick Institute of Technology,
Limerick, Ireland

KLUWER ACADEMIC PUBLISHERS
DORDRECHT / BOSTON / LONDON


Library of Congress Cataloging-in-Publication Data

ISBN 0-7923-5746-9

Published by Kluwer Academic Publishers,
P.O. Box 17, 3300 AA Dordrecht, The Netherlands.
Sold and distributed in North, Central and South America
by Kluwer Academic Publishers,
101 Philip Drive, Norwell, MA 02061, U.S.A.
In all other countries, sold and distributed
by Kluwer Academic Publishers,
P.O. Box 322, 3300 AH Dordrecht, The Netherlands.

Printed on acid-free paper

All Rights Reserved
0 1999 Kluwer Academic Publishers
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
including photocopying, recording or by any information storage and
retrieval system, without written permission from the copyright owner.
Printed in the Netherlands


Contributors

Paschal Baker and Wael Allan, Raytheon Engineers & Constrqctors UK,
Validation and GMP Compliance Group;
Ronald E. Chance, N. Bradly Glazer and Kathleen L. Wishner, Eli Lilly
and Company, hdanapolis, USA
R. Stephen Crespi, European Patent Attorney, West Sussex, UK;
John Edwards, Neil Jh-by, Genetics Institute Inc., 87 Cambridge Park
Dnve, Cambridge, Mass.;
Maryann Foote and Thomas Boone, Amgen Inc., USA;
Maninder S. Hora and Bao-Lu Chen, Dept. of Formulation Development,
Chron Corporation, 4560 Horton Street, Emeryville, Ca 94608, USA;
R. Horowslu, J.-F. Kapp, M. Steinmayr, St. Stuerzebecher, Schering AG,
SBU Therapeutics, D- 13342, Berlin, Germany;
J.P Jenuth, D. Fieldhouse, J.C.-M. Yu, B a d , Bioinfomatics Inc.;
Robert E. Jordan, Marian T. Nakada, Harlan F. Weisman, Centocor Inc.,
Malvern, Pensylvania, USA;
Brendan Murphy, University of Limerick, Ireland;
Patricia ODonnell, University of Limerick, Ireland;

Henk J. Out, N.V. Organon, PO Box 20, 5340, BH OSS, The
Netherlands;
A. Rolland, S . Sullivan, K. Petrak, Gene Medicine Inc., 8301 New Trails
Drive, The Woodlands, Texas, USA
Stephen Slater, Raytherm Engneers and Constructors;
Scott Spinka, CareMerica Inc., 16508 Kingspointe, Lake Lane,
Chesterfield, Mo., USA;
Dr. John C. Stinson, Leo Laboratories Ltd., Crumlin, Dublin 12, Ireland;
V


vi

Biopharmaceuticals, an overview

Dr Wchael Waller and Dr Ulrich Kohnert, Boehringer Mannheim
Therapeutics, Mannheim and Penzberg, Germany;
K.F. Williams, Validation Technologies (Europe) Ltd., Sutton Place, 49
Stoney St., Nottingham, NG1 lLX, UK and C.J.A. Davis, Tanvec Ltd.,
Alexandra Court, Carrs Road, Cheadle, SK8 2JY, UK;
Dr. Gary Walsh, University of Limerick


Acknowledgements

The editors wish to thank the authors of individual chapters for
providing such excellent contributions, and for their cooperation during
the post writing phase of the publication process. A special word of
thanks to Sandy Lawson, for her professionalism and efficiency in
reformating the chapters to comply with publication requirements.

Finally, thank you to Janet Hoffman and her colleagues at Kluwer for all
their help.

vii


Preface

The bwnning of the modern biotech era can be traced to the mid-l970s,
with the development of recombinant DNA technology and hybridoma
technology. Thus far, the most prominent applied impact of these
technologes has been the successful development of biotech-derived
therapeutic agents - the biopharmaceuticals. T h ~ sclass of pharmaceutical
product has rapidly become established. The first such product, Humulin
(recombinant human insulin, Eli Lilly) was approved in the USA in 1982.
Today there are in excess of 50 biopharmaceutical products approved for
medical use, with almost another 400 undergoing clinical trials. While all the
biopharmaceutical products approved to date are protein-based, nucleic acidderived products are likely to gain regulatory approval w i t h the next decade.
Gven the undoubted scientific and commercial prominence of t h l s sector,
relatively few books detailing biopharmaceutical products or issues of
practical relevance to the biopharmaceutical industry have been published
thus far. l k s book aims to complement the previously published texts whch
focus upon t h s area. The initial chapters are largely concerned with specific
biopharmaceutical products, whch have, in the main, gained regulatory
approval in the relatively recent past. Subsequent chapters focus upon
various issues of practical relevance to the biopharmaceutical industry, such
as product stabilization, patenting and regulatory issues. The final two
chapters focus upon gene therapy, a therapeutic approach currently at the
cutting edge of pharmaceutical research and development.
The book, whose contributors are largely drawn from industry, is

primarily aimed at an industrial audience. However, it should also prove a
useful reference source to research and educational personnel with a direct
interest in t h t s field.
1x


x

Biopharmace uticals, an ove wiew

In conclusion, the editors wish to thank all those who have contributed to
the successful completion of t h s book. Chief amongst these are the various
chapter authors (and their employers), as well as Kluwer Academic
Publishers, whose professionalism was much in evidence at all stages of the
publication process. A special word of thanks is reserved for Sandy Lawson,
whose patience and word processing slulls yet again proved to be second to
none.
Gary Walsh
Brendan Murphy
Limerick
September 1998


Contents

Contributors

V

Acknowledgements

vii

Preface

ix
1

Biopharmaceuticals, an overview
GARY WALSH

Abciximab: The First Platelet Glycoprotein IIb/IIIa Receptor Antagonist 35
ROBERTE. JORDAN, MARIAN T. NAKADA,
HARLAN F. WEISMAN
Recombinant Coagulation Factor IX (BeneFixO)
JOHN EDWARDS,
NEILm y

73

Biopharmaceutical Drug Development: A Case History
MARYANN
FOOTE,AND THOMASBOONE

109

Follitropin beta (Puregon)
HENK J. OUT

125

Insulin Lispro (Hmalog)
RONALD E. CHANCE, N. BRADLY GLAZER AND
KATHLEENL. WISHNER

149

i


11

Contents

Interferon beta-lb - the first long-term effective treatment of relapsing173
remittug and secondary progressive multiple sclerosis (MS)
R. HOROWSKI, J.-F. KAPP, M. S"MAYR, ST.STUERZEBECHER
Reteplase, a recombinant plasminogen activator
MICHAEL WALLER AND ULRICH KOHNERT
Stabilisation of biopharmaceutical products and finished product
formulations
M A " D E R S. HORA AND BAO-LUCHEN

185

217

Patent Law for Biopharmaceuticals
R.STEPHEN
CRESPI

249

The development of new medicines: an overview
JOHNC. STINSON

269

The EMEA and regulatory control of (bio)pharmaceuticals within the
European Union
GARY WALSH

289

Biopharmaceutical Validation: an overview
STEPHENSLATER

311

Validation of Biopharmaceutical Chromatography Systems
K.F. WILLIAMS (*) AND C.J.A. DAVIS(**)

337

Validation of Water for Injections (WFI) for Biopharmaceutical
Manufacture
PASCHAL
BAKERAND WAELALLAN

363

Information retrieval and the biopharmaceutical industry: an
introductory overview
PATRICIA O'DONNELL

3 89

Information technology and the internet as a resource of
biopharmaceutical information
J.P. JDWTH, D. FIELDHOUSE,J.C.-M.YU

405

Marketing Issues for the (Bi0)pharmaceutical sector
SCOTT SPINKA

42 1


Contents

Viral mediated gene therapy

iii

443

BRENDANMURPHY

Pharmaceutical gene medicines for non-viral gene therapy

A. ROLLAND,
S. SULLIVAN,K. PEnwC

47 1

Index

505


Chapter 1

Biopharmaceuticals, an overview

Dr. Gary Walsh
Lecturer, Industrial Biochemistry Programme, University of Limerick, Ireland

Key words:

Bipharmaceuticals, drug, therapeutic agents, blood products, cytokines, gene
therapy

Abstract:

The modem pharmaceutical industry is barely 100 years old. Amongst the
most recent product types developed are the biopharmaceuticals; therapeutic
substances produced by modem biotechnological techniques. Thus far, in
excess of 50 such substances have gained regulatory approval for medical
use. All are proteins produced by recombinant DNA technology or (in the
case of monoclonal antibodies) by hybridoma technology.

Biopharmaceuticals approved to date include blood factors, anticoagulants
and thrombolytic agents, therapeutic enzymes, hormones and haemopoietic
growth factors. Also approved are a number of interferons and an
interleukin. Recombinant vaccines and several monoclonal antibody based
products are also now on the market.
In addition to these, in excess of 350 potential biopharmaceutical products
are currently under evaluation in clinical trials. Prominent amongst these is
a new sub-class of biopharmaceutical - nucleic acid. Nucleic acid based
products find application in the emerging therapeutic techniques of gene
therapy and anti-sense technology. These techniques will likely provide
medical practitioners with an additional powerful tool with which to treat
conditions such as genetic diseases, cancer and infectious diseases.
The biopharmaceutical sector will continue to grow strongly for the
foreseeable fbture. Its current global market value of $7-$8 billion is likely
to triple within the next 5-6 years. This sector, born less than 20 years ago,
is quickly reaching maturity.

1


2

1.

Dr. Gary Walsh

DEVELOPMENT OF THE PHARMACEUTICAL
INDUSTRY

The modern pharmaceutical industry is a premier global industry, both

commercially and technologtcally. It employs several hundreds of thousands
of people, and its annual global sales value exceeds $200 billion. Currently,
there are over 10,000 pharmaceutical companies in existance, manufacturing
over 5,000 different medicinal products. The majority of the 100 or so large
multinational companies in t h l s sector origtnated from Europe and the USA,
with many of the remainder having been founded in Japan.
At the turn of the century, there were only 4 drugs available whch had
been scientifically proven to be effective in treating their target indications:
Digitalis, which consisted of extracts of foxglove, was shown to stimulate heart
muscle and, hence, proved effective in treating various heart conditions. The
active ingredients were subsequently shown to be two cardiac glycosides:
digoxin and digitoxin.
Quinine, an alkaloid obtained from the bark and roots of the fever tree
(Cinchona species), was found to be effective in treating Malaria.
Pecacuanha, obtained from the bark and roots of the plant species, Cephaelis,
was effective in treating dystentry. (The active ingredients of this preparation
turned out to be a mixture of alkaloids).
Mercury, which was used to treat syphilis.
From such modest begtnnings, the pharmaceutical industry has grown
rapidly.
Most medicines now available can be categorized into one of four groups,
depending upon their method of manufacture. The majority of medicinal
substances are relatively low molecular weight organic compounds
manufactured by direct chemical synthesis. Others (e.g. taxol and semisynthetic antibiotics) are obtained by semi-synthesis, whle a smaller, but
important, group of drugs are obtained by direct extraction from their native
biologtcal source (Table 1). The fourth group are ‘products of biotechnology’
or ‘biopharmaceuticals’. By and large, these are protein-based therapeutic
agents (Table 2). However, several nucleic acid-based biopharmaceuticals
are likely to gain regulatory approval wittun the next few years.

Biopharmaceuticals, an overview

3

Table 1. Some pharmaceuticals which may be obtained by direct extraction from biological
source material. Note that, in some cases, recombinant versions of the same product are

Substance
Blood products (e.g. clotting factors)
Vaccines
Antibodies
Insulin
Enzymes

Antibiotics
Plant extractives (e.g alkaloids)

Medical application
Treatment of blood disorders such as
haemophilia A or B
Vaccination against various diseases
Passive immunization against various
diseases
Treatment of diabetes mellitus
Used as thrombolytic agents, digestive aids,
debriding agents (i.e. cleansing of wounds),
etc.
Treatment of various infectious conditions
Various, including pain relief

Table 2. Most biopharmaceuticals approved or in clinical trials are proteins. Functionally,
they may be classified as belonging to one or other of the families of proteins listed below
Blood clotting factors
Monoclonal antibodies
Colony stimulating factors
Neurotrophic factors
Enzymes
Polypeptide anticoagulants
Growth factors
Polypeptide hormones
Interferons
Thrombolytic agents
Interleukins
Vaccines

1.1

The birth of the biopharmaceutical industry

Over the years, advances in biomedical research has identified various
biomolecules synthesized naturally by the body whose therapeutic potential
was obvious. Early examples include insulin and various blood clotting
factors, More recently discovered examples include interferons, interleuluns
and other cytokines whch regulate aspects of immunity, inflammation and
other processes of central importance to maintaining a healthy state.
As the majority of these substances were complex macromolecules
(predominantly proteins), their direct chemical synthesis proved to be
techcally challengnghmpossible and economically unattractive. Some (e.g.
blood products and various hormones) are produced naturally in quantities

sufficient to facilitate their direct extraction from biologcal source material in
medically useful quantities. In many cases, however, (e.g. most cytolunes),
these biomolecules are produced in exceedingly low concentrations in the
body. T h ~ smade their isolation difficult and routine large scale production
impossible.
In addition to such problems of source availability, extraction from natural
sources carried with it the possibility of accidential transmission of disease.


4

Dr. Galy Walsh

Well publicized examples include the accidental transmission of HIV and
other blood borne viruses via infected blood products and the transmission of
Creutzfeldt-Jacob disease via human growth hormone extracted from the
pituitaries of deceased human donors.
The development in the 1970s of the twin technologes of genetic
engneering and hybridoma technology largely overcame these problems of
source availability and accidental transmission of disease.
Genetic
engneering essentially facilitates the production of limitless quantities of any
protein of interest, whtle hybridoma technology allows production of limitless
quantities of a chosen monoclonal antibody.
These biotechnologcal innovations, along with an increasing
understanding of the molecular mechanisms underlining both health and
disease, rendered possible the development of a new generation of biotech.derived drugs - the biopharmaceuticals. By the late 1970s, hundreds of startup biotechnologcal companies had been formed to develop such products.
Most such ventures were founded in the USA, mainly by academics and
techrucal experts in the biotech. arena. These companies were largely
financed by speculative monies. Whde they boasted sigmficant techmcal

expertise, most of these companies lacked practical experience in the drug
development process. In the earlier years, most of the established large
pharmaceutical companies failed to appreciate the potential of biotechnology
as a means to produce drugs and, consequently, were slow to invest in t h ~ s
technology. As its medical potential became apparent, many of these
companies did diversify into t l x s area. W l e some initiated biotech. efforts in
house, most either acquired small established biopharmaceutical firms, or
entered stratwc alliances with them. An example of the latter was the
alliance formed between Genentech and Eli Lilly with regard to the
development and marketing of recombinant human insulin.
Many of the original biopharmaceutical companies set up in the late 1970s
and 1980s no longer exist. In addition to mergers, acquisitions and alliances,
many were forced out of business due to lack of capital, or disappointing
clinical trial results. However, a number of the early start-up companies have
successfully developed products and are now well established withn the
biopharmaceutical sector. Major examples include Genentech and Amgen. A
list of pharmaceutical companies who now manufacture and/or market
biophannaceutical products is provided in Table 3 ,


Biopharmaceuticals, an overview

5

Table 3. (Bio)pharmaceutical companies which manufacture andor market
biopharmaceutical products which have gained regulatory approval in the USA andor the
EU. (Note: several of these companies have a presence in both regions)
Company
Company
Company

A m g e n (CA, USA)
Cytogen (NJ, USA)
Novo-Nordisk (NJ, USA)
N.V. Organon (The
Eli Lilly (IN, USA)
Bay& Corp. (CT, USA)
Netherlands)
Baxter Healthcare
Galenus Mannheim
Ortho-biotech (NJ, USA)
(Germany)
(MA, USA)
Behringwerke A.G.
Genentech (CA, USA)
Ortho McNeil
Pharmaceuticals (NJ, USA)
(Germany)
Genetics Institute
Pharmacia & Upjohn (ML,
Berlex Labs (NJ, USA)
(MA, USA)
USA)
Schering Plough (NJ, USA)
Biogen (MA, USA)
Genzyme (MA, USA)
Bio-Technology General
Hoechst AG (Germany)
Serono Labs (MA, USA)
(NJ, USA)
Boehringer-Mannheim

Hoechst Marion Roussel
SmithKline Beecham (PA,
(MO, USA)
USA)
(Germany)
Boehringer-Ingelheim
HoEinan La Roche
Sorin biomedica diagnostica
(Germany)
(NJ, USA)
(Italy)
Centocor (PA, USA)
Immunex (WA, USA)
Chiron (CA, USA)
Immunomedics (NJ, USA)
Interferon Sciences
Ciba Europharm (UK)
(NJ, USA)
CISbio (France)
Merck (NJ, USA)

1.2

Biopharmaceuticals; market value

From a zero starting point in the edarly 1980s, the world-wide sales value
of biopharmaceuticals reached US$5 billion by 1993. (The first
biopharmaceutical to gain marketing authorization was recombinant human
Insulin in 1982). By 1997, the global market value had surpassed the $7
billion mark. By 2003, t h ~ sfigure is projected to be in the regon of $35

billion, whch will represent some 15% of the total global pharmaceutical
market (1-3). Biopharmaceuticals are amongst the most expensive of
therapeutic agents. The annual cost of erythropoietin, for example, per
patient per year is in the regon of $4000-$6000, whle that of human growth
hormone can be $12,000-$18,000. In monetary terms, erythropoietin is the
single largest selling biopharmaceutical product, and was the first such
product to surpass an annual sales value of $1 billion. The estimated sales
value of some notable biopharmaceutical products is presented in Table 4.


6

Dr. Gary Walsh

Table 4. Some major biopharmaceuticals currently on the market. The value of each
product quoted represents its estimated annual global sales value
Biopharmaceutical
Indication
Year first approved
Value ($ million)
1986
1,000
a-Interferon
Cancer,
Viral infection
Multiple sclerosis,
1993
35
p-Interferon
Viral infection

1990
45
y-Interferon
Chronic
granulomatous
disease
1,800
Erythropoietin
Anaemia
1989
Factor WI
Haemophilia
1993
445
Granulocyte-colony
Neutropenia
1991
870
stimulating factor
1985
660
Human growth
Growth deficiency
hormone
Insulin
Diabetes mellitus
1982
1,000
Interleukin 2
Cancer

1992
50
OKT 3 Monoclonal
Kidney transplant
1986
160
antibody
rejection
Tissue plasminogen
Cardiovascular
1987
120
activator
disease

2.

SOURCES AND MANUFACTURE OF
BIOPHARMACEUTICAL PRODUCTS

The vast majority of biopharmaceutical products currently on the market
are produced by recombinant DNA technology in either E. coli or Chinese
Hamster ovary (CHO) cell lines (4). Most monoclonal antibody based
products are predictably still produced by hybridoma technology, although the
t e c h c a l methodology now exists to facilitate production of antigen-binding
antibody fragments by recombinant means ( 5 , 6).
E. coli represents a popular recombinant expression system for a number
of reasons (7, 8). In addition to its ease of culture and rapid growth rates, E.
coli has long served as the model system of the prokaryotic geneticist. Its
genetic characteristics are thus exceedingly well-characterized and reliable

standard protocols for its genetic manipulation have been developed.
Appropriate fermentation technology is well established, and hgh expression
levels of recombinant proteins are generally attained. E. coli, however, does
display some disadvantages as a recombinant production system.
Recombinant proteins generally accumulate intracellularly, complicating
downstream processing and (often more critically) E. coli lacks the ability to
glycosylate proteins (or carry out any other post-translational modifications).


Biopharmaceuticals, an overview

7

Many proteins of therapeutic interest are naturally glycosylated and lack of
the carbohydrate component can, potentially, adversely affect its biologcal
activity, solubility, or in vivo half-life.
Recombinant proteins may be expressed in a number of other microbial
systems which do contain the enzymatic activities to facilitate posttranslational processing. Various proteins have been expressed, both in yeast
(particularly Saccharomyces cerevisiae) and fungi (especially various
Aspergilli) (9-1 1). W l e such microorganisms are capable of glycosylating
recombinant therapeutic proteins, the pattern of glycosylation usually differs
to that associated with such proteins when expressed naturally in the human
body. Such microbial expression systems exhlbit a number of characteristic
advantages and dsadvantages in terms of recombinant protein production.
Thus far, however, few recombinant biopharmaceuticals developed are
produced in either yeast or fungal systems.
Two approved
biopharmaceuticals are produced in Saccharomyces cerevisiae: Refludan
(recombinant hrudin, an anticoagulant marketed by Behringwerke AG) and
recombinant hepatitis B surface antigen, incorporated into various

combination vaccines by SmithKline Beecham.
More recently, a number of recombinant therapeutic proteins produced in
various animal cell lines have gained marketing approval. Chmese hamster
ovary (CHO) cells have become popular recombinant production systems, as
have baby hamster ludney (BHK) cell lines (12). Patterns of glycosylation
associated with recombinant glycoprotein biopharmaceuticals produced in
such systems resemble most closely the native glycosylation pattern when the
protein is produced naturally in the body.
The production of recombinant therapeutic proteins in the milk of
transgenic animals has also gained much publicity over the last few years (13,
14). A variety of therapeutically sipficant proteins, including tissue
plasminogen activator, al-antitrypsin, interleulun 2 and factor IX have been
produced in t h s matter (15, 16). It is likely that therapeutic proteins
produced in such systems will gain regulatory approval w i t h the next few
years.

2.1

Upstream processing

After its initial construction, the recombinant producer cell line is
thoroughly characterized and its genetic stability verified. The cell line is then
used to construct a ‘master’ and ‘worlung’ cell bank system (4). Irutial stages
of upstream processing invariably involves lab-scale culture of the contents of
a single vial from the worlung cell bank. l h s , in turn, is used to innoculate a
larger volume of media which (after cell growth) is, in turn, used to innoculate
the production scale bioreactor. The scale of fermentation depends upon the


Dr. Gary Walsh

8

level of production required, but generally production scale bioreactors would
vary in capacity from one thousand litres to several tens of thousands of litres,

2.2

Downstream processing

All biopharmaceutical products must be exhaustively purified in order to
remove virtually all contaminants from the product stream.
Such
contaminants include proteins (related or unrelated to the protein product),
DNA, pyrogens, viral particles and microorganisms.
Downstream processing is initiated by recovery of the crude protein
product from the fermentation media (if produced extracellularly) or cell paste
(if produced intracellularly). The crude product is then usually concentrated
(often by ultrafiltration, but ammonium sulphate precipitation or ion exchange
chromatography may also be used). It is next subjected to hgh resolution
chromatographc purification (17, 18). Generally, at least three different
chromatographc steps (e.g. ion-exchange, gel filtration, hydrophobic
interaction chromatography or affinity chromatography) are employed,
yelding a product whch is 98-99% pure.
Whlle chromatographc fractionation is designed to remove contaminant
proteins from the protein of interest, several chromatographc steps are also
quite effective in removing additional potential contaminants from the product
stream, Gel filtration chromatography, for example, is usually quite effective
in removing any contaminant viruses.
After chromatography, excipients are added (19) and the product potency

is adjusted by dilutiodconcentration as necessary. As therapeutic proteins are
heat labile, product sterilization is by filtration and t l u s is followed by aseptic
filling into final product containers. Although some products may be
marketed in liquid format, most are freeze dried (20, 21). Freeze dried
products generally are more stable, exhibiting a longer shelf life than
An example of a generalized
analogous liquid formulations.
biopharmaceutical production procedure is provided in Figure 1.


9

Biopharmaceuticals, an overview

Ifprotein is expressed
intracellularly
Removal of cells from media
(centrifugation or filtration)

Removal of
cellular debris
(centrifugation or
filtration)

ultrafiltrationiion exchange or precipitation)

I

Concentration of product-containing
extracellular media (ultrafiltration or

precipitation)

Chromatographic
purification; usually 2-4
chromatographic steps

1
Sterile filtration and
aseptic filling

Freeze drying

Sealkg offmal product container.
labelling and packing

Figure 1. Overview of a generalized downstream processing procedure employed to produce

a finished-product (protein) biopharmaceutical. Quality control also plays a prominent role
in downstream processing. QC personnel collect product samples duringafter each stage of
processing. These samples are analysed to ensure that various in-process specifications are
met. In this way, the production process is tightly controlled at each stage. (Reproduced
&om ‘Biopharmaceuticals: Biochemistry and Biotechnology’, J. Wiley & Sons, 1998, with
kind permission of the publisher)


Dr. Gary Walsh

10

3.

SPECIFIC BIOPHARMACEUTICAL PRODUCTS

Thus far, in excess of 50 biopharmaceutical products have gained
regulatory approval in the USA and/or the EU. All are proteins, although
several nucleic acid-based therapeutic agents are currently in clinical trials.
Most of the products approved may be categorized into specific families,
depending upon their biologcal activity, or mode of action. These approved
products are briefly reviewed below.

3.1

Blood products

Blood and blood products constitute a major group of traditional biologcs
(22). The major blood products which find therapeutic application include
red blood cell and platelet concentrates, plasma and plasma protein fraction,
albumin and blood clotting factors. W l e all these products are still sourced
from healthy blood donations, associated with tlzls practice is the potential for
inadvertent transmission of blood-borne pathogens. Infectious agents whch
can be accidently transmitted via infected blood/blood products include: HTV,
hepatitis B & C viruses, cytomegalovirus, human T cell lymphocytotrophic
viruses (possible causative agents of lymphoma), as well as Treponema
pallidum (causes syplulis), Plmmodium protozoa (causes malaria) and
Tvpanosoma cruzi (causes Chagas’ disease).
A number of protein-based products, particularly blood clotting factors
are now also produced by recombinant DNA technology. Thls essentially
eliminates the risk of disease transmission and ensures a regular supply of
product.
3.1.1

Blood clotting factors

The human body naturally produces 12 blood clotting factors, generally
designated by Roman numerals (factors I-XIIt; there is no factor VI). All but
one are proteins and most are proteolytic precursors whch become
sequentially activated during the blood coagulation cascade. Any defect
whch impedes the biological activity of any blood factor can result in a
severely retarded clotting ability. Genetic defects in all factors (except factor
IV,i.e. calcium) have been characterized. However, up to 90% of such
defects relate to factor VIII, whle most of the remainder relate to factor D(.
Poorly functional/dysfmctional factors VIII and I
X result in haemophlia A
and B, respectively, conditions treatable only by perio&c adrmnistration of the
appropriate clotting factor. Some recombinant blood clotting factors whch
have been approved for general medical use, or whch are in clinical trials, are
listed in Table 5 .


Biopharmuceuticals, an overview

11

Table 5. Recombinant blood factors which have gained marketing approval (in the US
and/or the EU), as well as such products which are currently being assessed in clinical
trials. Data sourced fiom PhRMA (na,org) and the EMEA
(ra. org/emea.html).
Product name
Company
Indication

Status
Benefix
Genetics Institute,
Haemophilia B
Approved 1997 (US)
(r factor
(MA, USA)
Genetics Institute,
Haemophilia B
Approved 1997 (EU)
(Europe, France)
Haemophilia A
Approved 1993
KoGENate
Bayer Corp
(CT, USA)
(USA)
(r factor WI)
Recombinate
Baxter Healthcare
Haemophilia A
Approved 1992
(r factor WI)
(CA, USA) and
(USA)
Genetics Institute
(MA, USA)
NovoSeven
Novo-Nordisk,
Prevents bleeding in Approved 1995 (EU)

(r m a )
Denmark
patients with
inhibitors to
coagulation factor
VIII or factor IX
Novo-Nordisk,
Haemophilia A & B
Phase III trials
(NJ, USA)
(USA)

3.1.2

Anticoagulants

The inappropriate formation of a blood clot (thrombus) w i t h a diseased
blood vessel can have serious, if not fatal, medical consequences such as heart
attacks and strokes. Anticoagulants are substances which can prevent blood
clot formation and, hence, are applied therapeutically in cases where hgh risk
of inappropriate blood clot formation is diagnosed (23). Traditional
anticoagulants include heparin, dicoumarol and warfarin.
Heparin is a proteoglycan (highly glycosylated polypeptide) whch is
sourced commercially from beef lung or pig gastric mucosa. It functions by
binding - and thus activating - a plasma protein: antithrombin III. The
heparin-antithrombin IJI complex then binds a number of activated clotting
factors. Thls binding inactivates the clotting factors, thus preventing clot
formation. Although heparin is an effective and inexpensive anticoagulant, it
can display a poorly predictable dose response and it can display a narrow
benefit : risk ratio.

Dicoumarol and warfarin are low molecular weight, coumaran-based
anticoagulants whch can prevent the post-translational modification of
various clotting factors, thus also rendering them inactive.


Dr. Gary Walsh

12

More recently, a protein-based anticoagulant has been developed.
Refludan (lepirudin) is a hlrudin-based anticoagulant whch gained a
marketing licence in the EU in 1997, and in the USA in 1998. It is approved
for the treatment of adult patients with heparin-associated thrombocytopenia
type II,and thromboembolic disease.
firudin was first noted in the 1880s as a major anticoagulant present in
the saliva of leeches (24). It was purified in the late 1950s and found to be a
65 amino acid polypeptide containing a tyrosine residue at position 63 whch
is normally sulphated. Its anticoagulant activity is due to its ability to bind
(and induce inactivation of) thrombin (factor IIa). The hirudin gene was
cloned in the 1980s and expressed in various microbial systems. Refludan is
produced commercially in yeast cells (Saccharomyces cerevisiae) transfected
with an expression vector containing the hrudin gene. It is presented as a
freeze dried powder whch also contains the excipient mannitol as a bullung
and tonicity agent. Unlike the native molecule, the recombinant form does not
exhibit a sulphate group on tyrosine 63, but this has no major impact upon its
anticoagulant activity.
3.1.3

Thrombolytic agents

In cases of inappropriate clot formation in a blood vessel, the level of
tissue damage induced often depends upon how long the clot deprives the
effected area of oxygen. Rapid clot removal can limit t h ~ sdamage, and a
number of thrombolytic (clot degrading) agents are used medically for t h s
purpose (25, 26). (In the USA alone, an estimated 1.5 million people suffer
acute myocardial infarction each year, whle an additional 0.5 million suffer
strokes). Traditional thrombolytic agents include streptokinase (a protein
produced by several strains of Streptococcus haemolyticus Group C ) , and
urokinase (a serine protease produced in the ludney and whlch can be purified
from urine).
The thrombolytic process, as it occurs naturally, is triggered by a 527
amino acid proteolytic enzyme, tissue plasminogen activator (tPA). tPA
proteolytically converts the inactive protease plasminogen into active plasmin.
Plasmin then proteolytically degrades fibrin, the major structural protein
found in clots. The medical potential of tPA was obvious for many years, but
its low levels of synthesis in the body precluded its medical use. The tPA
gene was cloned in 1983, facilitating large-scale production of the protein.
The gene has been expressed in both procaryotic systems (e.g. E. coli) and in
an animal (CHO) cell line. Several recombinant tPA products have now
gained marketing approval (Table 6) (27).


Biopharmaceuticals, an overview

13

Table 6. Recombinant tissue plasminogen activator-based products which have gained
marketing approval, or are in clinical trials. Data sourced fiom PhRMA
() and the EMEA (ra,org/emea.html).
Product name

Company
Indication
Status
Activase
Genentech (CA,
Acute myocardial
Approved 1987
USA)
infarction
(USA)
Acute massive
Approved 1990
pulmonary
(USA)
embolism
Acute myocardial
Approved 1995
infarction
(USA)
(accelerated
infusion)
Ischemic stroke
Approved 1996
(USA)
Retevase
Boehringer
Acute myocardial
Approved 1996
Manheim (MD,
infarction

(USA)
USA) and
Centocor (PA, USA)
Ecokinase
Galenus Mannheim
Acute myocardial
Approved 1996 (EU)
(Germany)
infarction
Rapilysin
Boerhinger
Acute myocardial
Approved, 1996
Mannheim
infarction
(EU)
(Germany)
Lanoteplase
Bristol-Myers
IIclinical
Acute myocardial
Phase I
infarction
trials
Squibb (NJ, USA)
TNK
Genentech (CA,
Acute myocardial
Phase I
IIclinical

USA)
infarction
trials

3.2

Therapeutic enzymes

A variety of enzymes are used for therapeutic purposes (28, 29). Some
(e.g. tPA and urolunase) have already been discussed. Traditional (nonrecombinant) enzymes used for medical purposes includes asparagmase (used
to treat some forms of leukaemia) as well as lactase, pepsin, papain and
pancrelipase used as digestive aids. Proteolytic enzymes, such as trypsin,
collagenase and pepsin, have also gained limited use as debriding and antiinflammatory agents.
In the last few years, a number of recombinant enzymes have also gained
marketing applications. These include DNase (Pulmozyme; dornase a ) and
glucocerebrosidase (cerezyme).
Pulmozyme, produced by Genentech, was first approved for treatment of
Cystic Fibrosis in 1993. The most notable clinical symptom of Cystic
Fibrosis (CF) is the production of an extremely viscous mucus in the lungs,
whch compromises respiratory function. The physiologtcal changes induced


Dr. Gary Walsh

14

in the lung of CF patients makes it susceptible to frequent, recurrent microbial
infection. This, in turn, attracts phagocytes and other immune elements. The
resultant destruction of the microbial (and some immune) cells results in a
build-up of large quantities of free DNA which is extremely viscous. Until

recently, the only way to successfully dislodge the mucus was by percussion
therapy (physical pounding of the patient’s chest to dislodge the mucus,
allowing the patient to expel it). Delivery into the lung of recombinant DNase
by aerosol technology promotes degradation of the free DNA, reducing its
viscosity sigruficantly. T h ~ sallows the patient to expel it with greater ease
(30, 3 1). The annual cost of treatment varies but often falls in the $10,000 $15,000 range.
Gaucher’s disease is a relatively rare genetic condition in whch sufferers
lack the enzyme, glucocerebrosidase. T h ~ scompromises their ability to
degrade glucocerebiosides (a specific class of lipid). Clinical consequences
include enlargement and reduced function of the spleen and liver, bone
damage and, on occasion, mental retardation.
The effects of this disease can be minimized by enzyme replacement
therapy. Ceredase is a commercial glucocerebrosidase preparation extracted
duectly from plancentae obtained from maternity hospitals. Its low
expression level in the placenta renders t h ~ sproduct very expensive to
produce. In 1994, a recombinant version (Cerezyme, produced by Genzyme)
gained marketing approval. The current annual global market for th~sproduct
is estimated at $200 million.

3.3

Recombinant therapeutic hormones

A number of recombinant therapeutic hormones have now gained
marketing approval (Table 7). In fact, the first ever product of genetic
engmeering to gain regulatory approval as a medicine was Humulin
(recombinant human insulin). Marketed by Eli Lilly, it was first granted
regulatory approval in the USA in October 1982.
Insulin was first used medically in 1921 and, for the following 50 years or
more, it was sourced from either porcine or bovine pancreatic tissue. In the

1970s, a method was developed whtch allowed the enzymatic conversion of
porcine insulin into human insulin (insulin from these two species differ in
sequence only by a single amino acid). Irutially, recombinant insulin was
produced by separate expression of insulin A and B chains in 2 different E.
coli cells (both K12 strains) (32). After purification of the two chains, they
were co-incubated under oxidizing conditions. ThIs promotes interchain
disulphtde bond formation yelding mature insulin. Subsequently, an
alternative method was developed whch entails the expression in E. coli of a
nucleotide sequence coding for human proinsulin. Purification of proinsulin is


Biopharmaceuticals, an overview

15

followed by in vitro proteolytic excision of the connecting (C) peptide,
yelding mature insulin.
A more recently approved insulin product is Humalog (Eli Lilly).
Humalog consists of insulin lispro, a human insulin analogue produced by
recombinant DNA technology in E. coli. The amino acid sequence of insulin
lispro is identical to that of human insulin except for an inversion of the
natural proline-lysine sequence of the insulin B chain at positions 28 and 29.
l h s modification produces an insulin product of quicker and shorter duration
of therapeutic action. It is thus a short-acting insulin whch can be
administered to diabetics immediately before meals.
An additional recombinant hormone preparation which gained regulatory
approval in the 1980s was Protropin (recombinant human growth hormone,
hGH). It was approved by the FDA in 1985 for the treatment of growth
deficiency in chldren. Since then, various additional recombinant hGH
preparations have gained approval for tlus and additional supplementary

indications (Table 7) (33, 34).
The approval of a recombinant form of hGH in 1985 coincided with the
banning of the use of hGH preparations extracted directly from the pituitaries
of deceased human donors. In that year, it was discovered that a young man
who had d e d from Creutzfeldt-Jacob disease contracted th~sfatal condition
from an infected batch of pituitary-derived hGH. (Unlike insulin, for
example, growth hormone is relatively species specific, so animal-derived
preparations e A b i t little or no biologcal activity when administered to
humans).
Recombinant follicle stimulating hormone (FSH) preparations have now
also gained marketing approval (Table 7). FSH is a prominent member of the
gonadotrophns, a family of hormones for whch the gonads represent the
primary target. The major activity of gonadotrophins is to regulate
reproductive function and additional members of t l u s family include
luteinizing hormone (LH), (human) chorionic gonadotrophn (hCG), pregnant
mare serum gonadotrophin (PMSG; horses only), irhbin and activin (35).
Table 7. Recombinant therapeutic hormones which have gained marketing approval or are in
clinical trials. Data sourced from PhRMA () and the EMEA
( h t t p : l l w . eudra. orglemea.htm1)
Product name
Company
Indication
Status
Insulins
Humulin
Eli Lilly (IN, USA)
Diabetes
Approved 1982
(USA)
Novolin

Novo-Nordisk (NJ,
Diabetes
Approved 1991
(various
USA)
(USA)
presentations)