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Biopharmaceuticals
Prof. S. N. JOGDAND
M. Sc.• M. Phil.
Department of Microbiology,
Karrnaveer Bhaurao Patil College,
Vashi,
Nav; Mumbai.

( FIRST EDITION 2006 )

Hat
GJlimalaya GpublishingGJIouse
MUMBAI • DELHI • NAGPUR • BANGALORE • HYDERABAD
e AUTHOR
No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means,
electronic, mechanical, photocopying, recording and/or otherwise without the prior written permission of the author
and the publisher.

ISBN: 97 8-81-83186-20-9
FIRST EDITION : 2006

Published by Mrs. Meena Pandey

for HIMALAVA PUBLISHING HOUSE,
NRamdoot", Dr. Bhalerao Marg, Girgaon,
Mumbai - 400004.
Phones: 23860170 & 23863863,
Fax: 022-23877178
Email: [email protected]
Website: www.himpub.com
Branch Offices:
Delhi NPooja Apartments", 4-B, Murari Lal Street,
Ansari Road, Darya Ganj,
New Delhi -110 002.
Phone: 23270392, Fax: 011-23256286
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Ghat Road, Nagpur - 440018.
Phone: 2721216, Telefax: 0712-2721215
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Nextto Hotel Highlands, Madhava Nagar,
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Phones: 22281541 & 22385461, Fax: 080-22286611
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Phone: 55501745, Fax: 040-27560041
Printed by Geetanjali Press Pvt. Ltd.,
Kundanlal Chandak Industrial Estate,
Ghat Road, Nagpur - 440 018.
 CONTENTS

1. Introduction 1 - 23
2. Market for Biopharmaceuticals 24-34
3. Biopharmaceuticallndustry 35-41
4. Biopharmaceutical Sector in India 42-52
5. Drug Discovery and Drug Designing 53-66
6. Pharmacokinetics 67-79
7. Clinical Trials 80-89
8. Regulatory 90-101
9. Therapeutic Proteins 102-136
10. Blood Products 137-153
11. Monoclonal Antibodies 154-181
12. Hormone Therapy 182-195
13. Vaccine Production - New Developments 196-220
14. Transgenic Production of Biopharmaceuticals 221-243
15. Human Gene Therapy 244-265
16. Antisense Technology (DNA Medicines) 266-277
"This page is Intentionally Left Blank"
 INTRODUCTION

1.1 Introduction
1.2 Advantages of Recombinant Biopharmaceutica1 Products
1.3 Product Definition
1.4 Protein Production before the Biotechnology Revolution
1.5 Protein Glycosylation
1.6 Cost of Production, Pricing, Pharmacoeconomics
1.7 Biopharmaceutica1 Products
1.8 Classification of Biologics
1.9 Biotech Drugs in Development: Major Therapeutic Categories
1.10 Classification of Protein Drugs
1.11 Emerging Biopharmaceuticals
1.12 FDA-approved Biopharmaceuticals and Vaccines
1.13 Major Approved Protein Therapeutics
1.14 Products Facing Patent Expiration
1.1$ Companies Producing Biogenerics
1.16 Recent FDA Approvals
2 Biopharmaceuticals

1.1 INTRODUCTION
More than 20 per cent of new medicines launched in 2003 were produced using biotechnology,
and nearly 80 per cent of those under development either use biotechnology or are derived from it.
The discovery of recombinant DNA and monoclonal antibody technologies in the 1970s marked-
the birth of the biopharmaceutical industry. Biopharmaceuticals are complex macromolecules derived
from recombinant DNA technology, cell fusion, or processes involving genetic manipulation. They
include recombinant proteins, genetically engineered vaccines; therapeutic monoclonal antibodies; and
nucleic acid based therapeutics (i.e. DNA based drugs), including gene therapy vectors. While small
molecule drugs of traditional pharmaceuticals "re delivered orally, biopharmaceuticals are usually
administered by subcutaneous, intravenous, or intramuscular injection.
Proteins expressed by genes are the targets of most drugs on the market and in development.
Companies look for a compound that binds to a protein, either activating it or block it from working.
In some cases, a protein can also be used as a drug e.g. insulin or human growth hormone.
The first drug produced via genetic engineering was human insulin, which appeared on the
market in 1982. By mid-2000, 84 biopharmaceuticals had been approved for marketing with almost
half launched during the past three years. Worldwide sales have grown more than seven-fold over the
past decade to reach US $ 15 billion by 1998. The US represents 46% of the market; compared to
36% for conventional drugs, due to a combination of earlier regulatory approval, easier market
acceptance, and greater pricing flexibility than other countries. Although biopharmaceuticals comprise
only 5% of world prescription drug sales, they account for six of the top 50 selling drugs, 13% of
new medicines approved by the FDA in the 1990s and about 18% of all drugs in development. At
the end of 1999, there were 369 biotechnology drugs in US clinical development against 438 disease
indications with 25% in Phase III.
Despite decreases in regulatory approval times in the US and the EU, there has been a substantial
increase in the time biopharmaceuticals spend in clinical development, from 39.4 months for products
approved in the 1980's to 61.5 months in the late 1990's. A large number have also been discontinued
in Phase III. Many of the earlier products were recombinant versions of natural hormones with
relatively well-understood properties compared to newer biopharmaceuticals with more difficult
therapeutic targets and undefmed disease mechanisms. Also, the hurdle for discontinuing development
is higher for a biotechnology company because it only has a handful of products in clinical development
and its lead product largely determines its stock price.
The biopharmaceutical industry is a key part of the knowledge-based economy. Biopharmaceutical
development, however, is inherendy a high-risk activity.
BiopharmaceutiCalS are gaining significance worldwide as big pharma companies are realizing
the need to fill their depleted pipelines and due to unmet medical needs. About 300 bio-ventures are
in place in Korea, 200 in Australia, 170 in India, 140 in Japan, 100 each in China and Taiwan and
30 in Singapore alone. Asia is becoming a prominent region for the sector due to huge natural
resources for biotech development and a huge pool of bio-scientists, positive demographic trends,
market growth potential, favourable government policies and returning of scientists from the Western
countries. Asian countries lack first class scientists and scientific business managers, lack strong
research base, low R&D investments, shortage of private venture capital and financial skills to
efficiendy allocate early-stage capital. In the US, venture capital community has a critical role in bio-
entrepreneurship. This critical part of value chain needs to be addressed for the Asian biotech industry
to thrive. (Pharmabiz.com, 16th April 2005)
In the next 15 years more than 20 per cent of chemical drugs would be replaced by biologicals/
biotech drugs.
 Introduction 3

1.2 ADVANTAGES OF RECOMBINANT BIO-PHARMACEUTICAL

PRODUCTS
Conventionally proteins and other biological products, processed from human or animal serum
or tissues, often are of low purity. On the other hand, rDNA promises around 99 per cent purity,
higher efficacy (therefore lower dosage requirement) and lower side effects. The other advantages with
recombinant technology are that the final protein can be changed by a slight modification of the amino
acid sequence. In other words, gene expression can be modified easily with faster product innovations.
Recombinant drugs can be manufactured without encountering shortfall of raw materials. Besides;
companies do not have to depend on factors like shortage of donors or animals. Many products like
plasma derived hepatitis vaccines, human growth hormone, erythroprotein are dependent on donors
who have to pass through stringent tests to ensure good quality proteins. Recombinant technology is
free from such issues. The unlimited supply of recombinant drugs means constant and steady flow of
products, hence is likely to be a driving factor in the coming years.
Since recombinant drugs can be produced en masse, they achieve economies of scale much faster
than chemically developed/animal derived drugs. This in turn results in lesser costs of the final product.
The lower price, combined with the benefits offered by them make them value for money products.
For instance, recombinant erythropoietin is about 30 per cent less expensive than the currently
available imported products in India. Similarly, many Asian companies (predominantly Indian &
Chinese) manage to sell Hepatitis B vaccines at low prices. This has encouraged local governments
to include Hepatitis B vaccme in its list for compulsory vaccines.
Biotech attempts at understanding individual response to drugs. Traditional methods focused on
having the same drug for the entire population. This meant savings for the manufacturer. Genetic
testing could let pharmaceutical companies segment people into four groups, according to how they
react to a given drug: high responders (those who respond especially well), low responders, non
responders, and adverse reactants.
Warner-Lambert's revenues from Rezulin would have grown much more quickly had it been
possible to identify the patients susceptible to the liver problem, who could then have taken the drug
provided their Jiver enzyme levels were monitored. Instead, the company lost about $200 million in
potential sales while it investigated the problem. (Naik Nitin, 'Asian Biotech Industry' A checklist on
challenges ahead, Pharmabiz.com, 18th April 2002).
With approximately 350 products in clinical testing and more than 1,000 others in early development
phases, biopharmaceuticals are growing at almost twice the rate of total pharmaceuticals.

1.3 PRODUCT DEFINITION

Biopharmaceutica1 Products: Although small molecule drugs remain the standard trea.tment for
disease, new strategies based on the engineering of proteins, genes and cells as therapeutic agents will
revolutionize medicine in the coming decades. Areas may include gene therapeutics, tissue engineering,
stem cell biology, cell-based therapeutics and gene correction technologies.
There is a growing conflict between the development of small-molecule and large-molecule
therapies-between the pharmaceutical industry's traditional synthetic organic chemicals and the complex
proteins and antibodies that are emerging from biotechnology. Many new drug targets key on protein-
protein interactions, and traditional small molecule approach is getting abandoned. Industry sources
estimate that about 30% of the drugs in the development pipeline are biopharmaceuticals-the majority
at pre-clinical stages. Industry expects that the 40% failure rate for small-molecule drug candidates in
late-stage clinical trials will probably translate into a bleak prospect for success in biotech drugs.
4 Biopharmaceuticals
30 biotech drugs missed critical milestones in phase IT or phase ill trials. Still, 20 biopharmaceuticals
received final approval last year, and 15 received approvals for new indications. From their small
base~urrently 8% of the $ 390 billion worldwide drug market-biopharmaceuticals are expected to
reach 15% of a $ 550 billion market by 2006.
Barriers to developing large-molecule therapies are: (i) The mainstream pharmaceutical industry
is still geared to the production of small-molecule drugs and lacks the infrastructure to produce major
biopharmaceutical products, (ii) Small-molecule drugs can be administered in oral dosage form,
whereas large-molecule drugs are administered via injection-a less popular option with patients and
(iii) The cost of producing biotech drugs. is much higher than that of manufacturing small-molecule
drugs.
Biologics: Biologics, include products, which may be regarded as conventional biologics and
recombinant biologics. Conventional biologics consist of blood derived polyclonal antibodies and
clotting factors, antibiotics, and classical vaccines based on live or killed viruses, and are frequently
classified as biopharmaceuticals, but these products long predate the emergence of recombinant DNA
and monoclonal antibodies.
As per an estimate, there are over 1100 biologicals in pre-clinical stage, over 220 biologicals in
phase I stage, over 340 in phase II stage, about 80 in phase III and about 380 are approved or waiting
for approvals.
Biopharmaceuticals: Chemically synthesized small molecule drugs have been the mainstream of
the traditional pharmaceutical industry. Biophannaceutica1s are complex macromolecules created through
the genetic manipulation of living organisms using gene cloning, recombinant DNA (gene splicing),
or cell fusion technologies. In terms of product type, these include: .
• Recombinant proteins;
• Recombinant antigen vaccines and vaccines crafted from genetic material such as DNA;
• Therapeutic monoclonal antibodies; and
• Oligonucleotides (short sequences of DNA or RNA) such as antisense molecules which
interrupt the production of disease causing proteins by inhibiting gene function and gene
therapy which can enhance the production of a missing protein through the addition of a
synthetic gene.
Conventional drugs have molecular weights of the order of several hundred daltons but
biopharmaceuticals are 100 times or more larger e.g. interferons (20,000), interleukins (15,000), tPA
(70,000), and Factor VIII and several monoclonal antibodies (close to a million).
The first recombinant protein (human insulin) was launched in 1982, the first recombinant
vaccine (against hepatitis B) in 1986, the first therapeutic monoclonal antibody (against kidney
transplant rejection) also in 1986, and the first and only oligonucleotide in 1998 (against Cytomegalovirus
retinitis in AIDS patients). No gene therapy product has yet been approved. Recombinant proteins
dominate the biopharmaceutical market accounting for the bulk of sales to date.
Generics (or, more appropriately, multisource pharmaceuticals) are typically defined as
pharmaceutical preparations that:
• are essentially similar to an original product;
• involve an active substance with expired patent protection;
• are approved through a simplified registration process; and
• sell under a common name typically with very little (if any) promotional activities.
 Introduction 5
Essentially, generic pharmaceutical companies offer consumers a cheaper version of a product
for which patent protection has expired. Within this framework, a critical success factor is represented
by the ability to develop the generic copy with relatively modest investments, in order to make the
economics work.
Generics are prescription medicines that have lost patent protection and are being marketed by
one or more companies that did not originally develop the drug. They are essentially similar to the
original product, are approved through a simplified regulatory process and are sold under a common
name with very little promotion.
By extension, the term 'BiogenericsJ is used for pharmaceutical preparations (involving a biologically
active substance) stemming from modern biotech tools. As with pharmaceutical generics, these
biogenerics are essentially similar to an original biopharmaceutical whose substance patent has expired,
are approved through a simplified abbreviated registration process, and are sold under the generic
substance name as opposed to a brand name (e.g., EPO as opposed to Epogen).
Biogenerics refers to therapeutic products based on genetically engineered or recombinant
technologies.
Biogenerics are generic versions of pharmaceutical preparations involving a biologically active
substance that has generally been created using modern biotech tools. These products are still in their
infancy due to the relatively recent patent expirations of the first branded set of biotech products.
Biotech-engineered drugs are products based on large molecule proteins. On the other hand,
generics generally refer to non-biologi<;al products, also called small molecule drugs.
Approximately 1,049 million grams of bulk drug proteins were produced worldwide, generating
sales of nearly $ 30 billion in 1999. Production is expected to rise to 1,150 million grams by 2004
with sales of over $ 43 billion. In dollar terms, the market is expected to grow at an average annual
growth rate (AAGR) of 7.6% during the 5-year forecast period, whereas in grams produced, it will
grow at an AAGR of 1.9% during the same period. Human proteins make up the largest share of
the current _and forecast production, while animal, plant and microbial proteins make up the rest.
Production for non-therapeutic applications (laboratory research, in vitro diagnostics, in vivo diagnostics
and veterinary) is excluded from these estimates.
Regular biological methods overwhelmingly predominate in the manufacture of drug proteins,
including natural source extraction from human, animal and plant materials, regular (non-recombinant)
microbial fermentation and regular (non-recombinant) cell culture. Production of proteins for human
therapeutic use by human-source extraction and by natural animal source extraction, is declining for
reasons of viral contamination risk, the vagaries of source material supply and, most significantly, for
rapid progress in recombinant technology. During the 5-year forecast period, this market is expected
to grow at an AAGR of 1.8%.
Recombinant biological methods provided only 0.1 % of the worldwide total bulk drug protein
production in 1999, but are projected to provide 0.6% of the total production in 2004 as this market
grows at an AAGR of 41% over the next five years. Recombinant DNA technology focuses mainly
on proteins of human origin (as well as mouse-derived monoclonal antibodies). Current recombinant
biological methods include recombinant microbial fermentation and recombinant mammalian cell
culture. In the future, we anticipate transgenic animals and plants will contribute as manufacturing
methods, once proteins now being developed by transgenics attain regulatory approval. Transgenically
sourced proteins will still call for the application of extraction processes.
Chemical peptide synthesis is a relatively minor method of manufacture, in terms of the bulk
quantities produced. However, this technology could get a boost, perhaps, from research efforts
6 Biopharmaceuticals
exploring shortened versions of large protein molecules that still retain biological activity. Also possibly
giving impetus to the role of chemical synthesis in the future are current research efforts to deliver
peptide drugs orally, through a "stomach overdosing" approach.
The complexities of biogenerics will change with the formulations strategies. Formulations
strategies feature low dose, reduced dose, increased stability (increase half life), safety to humans
(immunogenecity), and protected IPR for a longer period. Technologies that include pegylated,
hydrogel, microsphere, nanotechnology and complexing with lipid are examples of new formulation
strategies. One way for the US market to beat the biogenerics market is to hold the patents rights
by changing the formulations strategy.
Biogenerics not only will provide patients the same health benefits as their brand counterparts,
but also yield tremendous cost savings for consumers and healthcare providers by creating healthy
competition. Competition also will foster innovation in the biopharmaceutical industry. And while the
US is debating the manufacture of generic biopharmaceuticals, citizens of other nations are already
enjoying access to more affordable generic biopharmaceutical products.
As patent protection expires for the first wave of biopharmaceutical products, the potential
marketplace for generic substitutes looms large. Biogenerics. offer a multi-billion dollar marketplace,
one, which has yet to be exploited.
The USFDA is actively seeking comment in order to draft"a policy for "biogenerics", "follow
on biologicals", "biosimilar" products. The European Medicines Evaluation Agency (EMEA) is also
actively working towards such guidelines.
Potential global bio-generics market is estimated at US dollar two billion. With revenue from
protein based therapeutics expected to grow at 15 per cent, twice that of the pharma industry, a global
capacity crunch is forecast. India with strengths in process engineering, trained manpower in plant
management is perhaps best positioned to leverage on the bio-generics opportunity and can be
expected to repeat its success in pharma-generics"
Regulatory hurdles facing biogenerics are expected to be resolved and the first of such products
is likely to be introduced in United States and Europe in the 2006-2008 timeframe. Total worldwide
generic and biogeneric prescription volume is expected to rise from $ l.80 billion in 2003 to $ 3.27
billion in 2010 (rise mainly due to ageing population in U.S., Europe and Japan), reflecting CAGR
of 8.9%.
Issues Related to Biogenerics
Hatch-Waxman rule cannot be applied to biogerics. The rule allows generic manufacturers to
submit Abbreviated New Drug Application, ANDA in US for approval to market generic product.
The process does not require firms to replicate extesnsive clinical trials with generics, but they must
prove the essential similarity and conduct tests to prove its bioequivaence to the brand name drug.
In case of biogenerics, such comparisons are difficult to make. Most biogenerics being proteins
essential similarity need not only be in composition but also has to be in conformation.
Usually the biopharma product is process specific. The expression/vector system, production and
purification process, facility, equipment, analytical techniques etc. used by the copying firm are usually
different from those of the brand manufacturer. Such changes may affect amino acid sequence,
physico-chemical and biological properties of the end product. Even minor modifcations in a bioprocess
such as aeration, agitation, reactor size, operating conditions or culture media, changes in cell or
microbial system, purification, post translational modifications like glycosylation, folding, impurity
proftles may change the functioning of molecule and could result in altered. safety and efficacy. Hence
 Introduction 7
for proper evaluation extensive and expensive clinical trials become necessary for biogenerics. This
defeats the purpose of making biogeneric. An abbreviated process for generic biopharmaceuticals thus
does not exist in US and Europe.
However, the dogma that the "process makes the biotech product" has been challenged and the
concept of "product comparability" is gradually more accepted than "essential similarity" for biogeneric
products.
Today, it is possible to demonstrate through pharmacokinetic, pharmacodynamic and clinical
studies that the biopharmaceutical versions are comparable and bioequivalent to original product. This
is best examplified by HGH products of different companies like Pharmacia, Ferring, Lilly, Nova and
Serono based on distinct processes and involving different expression systems showing same proftles
in terms of amino acid sequence, potency, safety and efficacy.
Biosimilars
Recently, the European Parliament approved new pharmaceutical legislation that, among other
things, set out a legal framework for the registration of so-called 'biosimilars', biological therapies that
are therapeutically the same as existing, approved biological products.
Swiss biopharmaceuticals company BioPartners has ftled its first application in Europe for a
generic version of interferon alpha, a biological drug. If approved, the 'brosimilar' could be on the
market as early as next year. This development comes shortly after the US government turned down
an application to market Omnitrop/Omnitrope (somatropin), a generic version of recombinant human
growth hormone developed by Sandoz, citing problems in establishing an appropriate regulatory route
for 'biogeneric' products. Sandoz had also ftled for approval with the European Medicines Evaluation
Agency for Omnitrop, but was unable to progress the application. BioPartner's filing represents the
first occasion that the EMEA has accepted an application for a biosimilar drug for review. To date,
the development of the market for biosimilar drugs outside regions with less stringent intellectual
property protection such as Asia has been little.
1.4 PROTEIN PRODUCTION BEFORE THE BIOTECHNOLOGY
REVOLUTION
Major route of obtaining proteins of therapeutic value, before the advent of biotechnology was
extraction from various sources.
Extract from Tissues: Insulin from Guineapig pancreas, Growth hormone from cadaver pituitary
glands
Extra~ from Blood: Factor VIII for haemophilia, Albumin for IV solutions and media

Extract from Urine: Erythropoietin (made in kidneys)

Today cost of the development is $ 700 to $ 800 million per drug. Average time of development
is U.S years. 2/3 of the drugs that make it to IND never make it to market. 2/3 of the drugs that
make it to market do not pay for their R&D costs. Decision for expression system has to be made
in first three years of R&D. 61 products were approved between 1998 and 2003.
1.5 PROTEIN GLYCOSYLATION
Interestingly many of the functional proteins like therapeutic proteins, regulatory proteins, hormones
and antibodies exist as glycoproteins in our body system.
• Many proteins of therapeutic and diagnostic interest have chains of sugars attached.
• Sugar chains are attached via specific asparagine, serine, or threonine residues.
8 Biopharmaceuticals
• Unlike the primary amino acid sequence, only the potential for protein glycosylation is
genetically determined.
• The location and sequence of sugar addition depends on the type of cell and culture conditions.
Bacteria do not glycosylate proteins. Yeast and insect cells glycosylate differently than animal
cells. Animal cells must be used for proteins that require human-like glycoslyation. Modulation of
glycosylation in yeast and insect cells (via incorporation and deletion of key enzymes) is an active
research area.

1.6 COST OF PRODUCTION, PRICING AND PHARMACOECONOMICS

The average cost to bring a new drug to market currently is about $ 802 million. Manufacturing
costs are crucial to overall profit margins. Production costs appear large in management planning.
Intense product competition, patent expirations, the introduction of more second-generation therapeutics,
and pricing constraints are forcing close scrutiny of the economics of bringing drugs to market.
The sales price (S/g) of the different drug products generally decreases with increasing production
volume. Biogen's Interferon, a small-volume drug, sells at $ 2,940/g. Large-volume drugs such as
intravenous immune globulin (IVIG) typically sells at S 40 to S 60/g. Also, the split between the cost
of upstream and downstream unit operations reportedly depends on the source organism. The upstream
processing costs are lower (14 to 19% of total) for a recombinant E. coli or Streptomyces derived
product and significantly higher (40% of total) for a mammalian cell culture product.
Process design can be modified to increase productivity. For example, use big bead resins for
faster chromatography and appropriately sized equipment and utilities. Also, a model could be used
to evaluate the cost difference between a single large bioreactor and multiple small bioreactors.
• Using ion-exchange (lEX) chromatography as an alternative to Protein A chromatography.
lEX is cheaper (media cost/g processed is $ 0.30 for High S Macroprep vs. S 16.25 for
rProteinA Sepharose) and is also free of contaminating immunoglobulins. However, additional
steps may be required if lEX is used and may require process development time.
• Using membrane chromatography (MC) as an alternative to conventional resin column
chromatography. Me requires higher medium and equipment costs, but lower buffer, labour,
and validation costs might make it a more economical option.
• Using fully disposable, presterilized, and prevalidated components in the bioprocessing plant
instead of conventional stainless steel equipment. A comparison of two cases showed that the
disposable option substantially reduced capital investment (60% of the conventional plant).
The running costs for the disposable option were 70% higher than for the conventional
plant; however, there was a nine-month reduction in time-to-market with the disposable
option.
Optimize the facility to minimize downtime and improve efficiency of changeover and maintenance
procedures. Some facility changes meeting these objectives could be: addition or upgrade of manufacturing
area or equipment; upgrade of utility systems; modernization of control systems; and coupling or
decoupling of several process steps to improve operational flexibility and maximize equipment utilization.
Focus process development efforts on increasing the titer of fermentation and cell cultures.
A fivefold improvement in titer combined with a 40% improvement in downstream recovery resulted
in a sevenfold reduction in processing volume plus a fourfold reduction in COGS and associated capital
requirements.
 Introduction 9
Manufacturing Costs of Biologics
(US $/gram of protein) (a) Scale (kg)
0-10 10-100 100-1,000 > 1,000
Bacterial fermentation (b) $ 22,000 $ 40-65 $ 32-55
Mammalian cell culmre 10,500 $ 300-3,000 1,000-5,000
Transgenic animal (milk) 300-500 100-300 30-100 < 40
(a) These costs are for bulk active purified proteins but do not include the fill and finishing costs associated
with the production of biopharmaceuticals.
(b) Many complex proteins cannot be expressed in active form by bacterial fermentation.
Source: Nexia Biotechnologies Prospectus 2000.

Economics of Protein Production

CHO Cells (lg/litre) Transgenic (Sg/litre)

Capital Investment ($ MM)

100kg $ 20 $5
500kg $ 75 $ 10
COGS ($/g)
100kg $ 500 $ 100
500kg $ 200 $ 40
Source: SG Cowen, Genzyme Transgenics.

There is also the risk factor to consider. The drug discovery value chain can be broken down
into five stages: from target discovery (5000-10,000) to lead discovery (500-1000) to clinical trials,
one has about five from the original 10,000, making it to phase III and, possibly, one goes to the
manufacturing stage, to the market.
Biopharmaceuticals with exception of insulin are much more expensive than small molecule
drugs. Monoclonal antibodies, for example, are in the range of US $ 15,000 to $ 25,000 per year,
estimates for ex vivo gene therapy are over US $ 100,000, while certain orphan drugs e.g. genetically
engineered blood clotting factor for heamophiliacs can cost as much as US $ 150,000 per year. The
new generation of vaccines will not be affordable outside industrialized countries.
Governments and third party payers are increasingly demanding evidence that pharmaceuticals
generate benefits commensurate with their high costs .(e.g. reduced public health care expenditures,
decreased mortality, functional improvement, increased quality of life, or shorter length of therapy).
Ontario, BC, a number of countries (Australia, New Zealand, Finland, UK, France, Italy, Norway
and the Netherlands), and several US managed care groups (e.g. Blue Cross, Blue Shield) have
instituted mandatory cost-effectiveness submission guidelines. However, comparisons are difficult
because of the lack of standard study methodologies, the use of placebos in clinical trials, problems
in measuring quality of life benefits and improved workplace productivity, and in accounting for
differences in the cost of health care inputs from country to country. Clinical trials have generally not
been aimed at demonstrating long-term health outcomes and cost savings so the need to capture this
additional information will increase trial costs and extend development times.
10 Biopharmaceuticals

1.7 BIOPHARMACEUTICAL PRODUCTS

NfJ. Examples

1. Antianaemic Agents Erythropoietin (EPO)

2. Antihaemophilic Agents Factor VII, Factor VIII, Factor IX, Tissue Factor, von
Willebrand Factor

3. Anti-inflarrunatory Agents Angiogenic Factors, Inhibitors of Proteolysis, Colony

Stimulating Factors, Transforming Growth Factors, Heparin-
Binding Growth Factors, Interferon-Gamma, Interleukins,
Lipocortins, Lysozyme, Superoxide Dismutase, Tumor
Necrosis Factor

4. Antineoplastic Agents Angiogenic Factors, Aproliferin, Inhibitors of Proteolysis,

Colony Stimulating Factors, Interleukin-3, Granulocyte/
Macrophage-Colony Stimulating Factor, Granulocyte-
Colony Stimulating Factor, Macrophage-Colony Stimulating
Factor, Endothelial Cell Growth Factors, Epidermal Growth
Factor, Erythroid Differentiation Factor, Interleukins,
Interferons, Transforming Growth Factors, Tumour Necrosis
Factors, Laminin, Monoclonal Antibodies

5. Antithrombotic and Fibrinolytic Agents Hirudin, Protein C, Protein S, Thrombomodulin,

Pro-Urokinase and Urokinase, Streptokinase, Tissue
Plasminogen Activator, Antithrombin III

6. Antiviral Agents Interferons, Tumour Necrosis Factors, Antisense Nucleic

Acids, Recombinant Vaccines

7. Cardiovas~lar System-Related Agents Alpha-I-antitrypsin, Angiogenic Factors, Apolipoproteins,

Atrial Natriuretic Factor, Calcitonin Gene-Related Peptide,
Lipid Exchange Proteins, Superoxide Dismutase

8. Growth Factors Nerve Growth Factor, Osteogenic Factors

9. Hormone Replacement Therapies Insulin, Proinsulin, Growth Hormone, Calcitonin

10. Immunomodulators Colony Stimulating Factors, Interferons, Interleukins, Tissue

Necrosis Factors, Monoclonal Antibodies, Catalytic
Antibodies

11. Wound Healing Accelerators Epidermal and Fibroblast Growth Factors (EGF and FGF),
Platelet-Derived Growth Factor, Transforming Growth
Factors, Somatomedinsflnsulin-Like Growth Factors

12. Other Biopharmaceuticals Analgesics, Gastrointestinal Agents, Anti-obesity Agents,

Respiratory Agents, Elastase Inhibitors, Superoxide
Dismutase, Human Pulmonary Surfactant Protein, Atrial
Natriuretic Peptide, Sex Hormones and Related Products,
Gonadotrophins and their Releasing Factors, Inhibins,
Vasoactive Intestinal Polypeptide
 Introduction 11

1.8 CLASSIFICATION OF BIOLOGICS

No Class Category

(I) Blood and Blood Derivatives

• Cellular Components.
• Stem Cells.
• Platelets.
• Packed Red Blood Cells.
• Plasma Fractions - Human Serum Albumins.
• Plasma Fractions - Immune Gloulins.
• Plasma Fractions - Other Plasma Products.
• Whole Blood.
• Blood Substitutes.

1. Conventional Biologics (II) Conventional Protein Drugs.

• Anticoagulants.
•Hormones.
•Antineoplastic Agents.
•Enzymes.
• Other Conventional Proteins.

(III) Conventional Vaccines

•Pneumococcal Vaccine.
•DtaP Vaccine.
•MMR Vaccine.
•Influenza Vaccine.
• Hepatitis A Vaccine.
• Vercella (Chickenpox) Vaccine.
•Cancer Vaccine.
•AIDS Vaccine.
•Other Conventional Vaccines.
•Veterinary Vaccines.

2. Recombinant Proteins (IV) Diagnostic Reagents

• Enzymes.
•
Hormones.
•
Antigens and Antibodies.
•
Hematology Reagents.

(I) Blood Related Agents.

• Eryhropoietins.
•Colony Stimulating Factors.
•Antihemophilic Factor.
• Thrombolytic Agents.

3 Recombinant Vaccines (II) Recombinant Hormones.

• Human Insulin.
•Huffian Growth Hormone.
•Fertilty Hormones.
•All Other Hormones Interferons.
12 Biopharmaceuticals

No Class
(III) Other Recombinant Proteins.
• Enzymes.

Recombinant Hepatitis B Vaccine

4 Monoclonal Antibodies Other Recombinant Vaccines.
• In Vitro Diagnostic Monoclonal Antibodies.
• In Vivo Diagnostic Monoclonal Antibodies.
• Therapeutic Monoclonal Antibodies.
• Anti-cancer Monoclonal Antibodies.
• Anti-inflammatory monoclonal Antibodies.
• Other Therapeutic Monoclonal Antibodies.
- Anti-transplant Rejection Monoclonals
Thrombolytic Agents - Monoclonal
Antibodies.

1.9 BIOTECH DRUGS IN DEVELOPMENT: MAJOR THERAPEUTIC

CATEGORIES
Therapeutic Area

Oncology 175
Infectious Diseases 45
Autoimmune Diseases 25
AIDSjHIV 20
eNS Disorders 20
Respiratory Disorders 18
Hear Disease 15
Skin Disorders 15

1.10 CLASSIFICATION OF PROTEIN DRUGS

No Category
1 Antibiotics
2 Antibodies
3 Blood Proteins
4 Enzyme inhibitors
5 Enzymes
6 Hormones
7 Lymphokines
8 Other Protein drugs
9 Vaccines
 Introduction 13
Proteins in living organisms are classified according to their biological roles. These include:
• Enzymatic - proteins that trigger all the chemical reactions that occur in the cells of living
organisms.
• Transport - proteins that carry other substances throughout the body or molecules aC{"Qss
cell membranes. For instance, the protein haemoglobin carries oxygen from the lungs to
other parts of the body.
• Structural- proteins that help in supporting functions in the body. For instance, Keratin
is the protein that is important for supporting hair and other skin parts.
• Storage - proteins that store amino acids. For instance, casein is the protein in milk that
provides a source of amino acids for baby mammals.
• Hormonal - proteins that coordinate bodily activities. For instance, insulin is the protein
hormone secreted by the pancreas that regulates the level of sugar in the blood.
• Receptor - proteins that are built into the membrane of a cell and detect chemical signals
released by other cells. They contribute to the cell's response to the chemical stimuli.
• Contractile - proteins that help in movement. For example, actin and myosin are responsible
for the movement of muscles.
• Defensive - protect the body against diseases. For example, antibodies are proteins that
protect the body against viruses and harmful bacteria.
• Immunoregulators - Proteins that act as molecular messengers in cell-cell interactions for
immune response. For example interleukins.
Protein-based drugs are:
Cytokines, Hormones, Clotting Factors, Vaccines, Monoclonal Antibodies.

1.11 EMERGING BIOPHARMACEUTICALS

Mimetics and peptidomimetics (small molecule compounds that mimic proteins and peptides)
have advantages over proteins in terms of ease of delivery and cost, but there are considerable technical
challenges in mimicking the large and intricate protein-protein interacting surfaces and overcoming
the weak binding forces of small molecules, a major cause of side effects. Examples include mimetics
of insulin and granulocyte colony stimulating factor (in early stage development), peptide based
mimetics of erythropoietin (late stage), and Montreal based Neurochem's mimetics of glycosaminoglycans
(GAGs - complex carbohydrates that promote amyloid fibril formation characteristic of Alzheimer's)
which compete with naturally occurring GAGs to inhibit deposition of amyloids (early stage).
Glycotherapeutics (complex sugars/carbohydrates) have significant potential both as drugs and
drug targets because of their diverse biological roles (cell surface carbohydrates act as binding sites
for other molecules, playing structural roles in cancer transformation, immune system regulation, tissue
repair, and anti-infection responses, as well as genetic disorders such as Gaucher's and Fabry'S
diseases) and their enormous structural diversity (more than 10 million polysaccharides can be formed
from nine common monosaccharides compared to 16,000 peptides from 20 amino acids). However,
their application as biopharmaceuticals has lagged far behind protein drugs because they are difficult
molecules to analyse and synthesize.
Fully Human Monoclonal Antibodies First generation mouse-based antibodies provoked a
strong immune reaction in humans limiting their clinical utility. Strategies to generate more human
compatible antibodies include mouse-human chimeras (60%-70% human protein) first introduced in
1995, followed by humanized antibodies (90%-95% human protein) in 1998 in which certain mouse
antibody amino acid fragments are grafted onto a human antibody. The next generation now in clinical
14 Biophannaceuticals
development are fully human antibodies produced via methods such as transgenic mice containing
human antibody genes, phage display, or collection of human B cells from infected individuals which
are then fused with human tumor cells to create antibody producing hybridomas. Because of their high
costs and large dose requirements, advances in manufacturing capability are needed to meet market
demand.
Vaccines Conventional vaccines based on inactivated (killed) viruses; live, attenuated (weakened)
viruses; microbial toxoids; or antigenic fragments (subunit vaccines), have a number of disadvantages.
11 New vaccine' strategies include:
• recombinant antigen vaccines where the antigen is produced in bacteria or yeast rather than
extracting it from chronic human or animal carriers (e.g. hepatitis B);
• recombinant vector vaccines using weakened viruses such as poliovirus, vaCCInIa virus or
salmonella bacterium as carriers of the disease causing organism to overcome the inability
of inactivated whole antigens or recombinant antigen vaccines.to enter cells and express the
desired gene product (hepatitis B, HIV, herpes simplex, in development);
• DNA vaccines, produced from a core gene of a virus, are effective against those pathogens
that change their exterior protein coat by attacking the interior proteins of a virus (HIV,
malaria, and a number of anticancer applications are the closest to commercialization); and
• RNA vaccines (particularly applicable to yellow fever and polio caused by RNA viruses).
Genomics has the potential to improve the effectiveness of existing vaccines and ot:sign vaccines
for diseases for which no protection currently exists: by sequencing invading bacterial and viral
organisms, those critical genes that encode the proteins that cause disease can be identified, leading
to the development of novel and more effective antigens. Complete DNA sequences of most human
pathogenic viruses and more than 20 bacterial pathogens have already been determined. The first
genomics derived vaccine (Group B meningitis) developed by Chiron is expected to enter clinical trials
in 2001. Vaccines are also being developed as anti-cancer agents, involving attempts to activate
immune responses against antigens in the tumour to which the immune system has already been
exposed. Research is also being undertaken on edible vaccines e.g. transgenic bananas in order to offer
an improved mode of delivery.
Gene therapy Whereas recombinant proteins and antibodies are produced outside the body and
administered to the patient,gene therapy has the potential for the body to produce the desired protein
on its own if a synthetic gene with the correct DNA sequence for that protein can be inserted into
targeted defective cells. A number of major challenges have to be solved: design of a safe and effective
gene delivery system, the short duration of gene expression, control over where the transferred gene
is expressed, and possible immune reaction caused by the vectors, genes or new proteins. Many efforts
now concentrate on conditions that require only transient gene ~xpression, e.g. cancer and coronary
artery disease, because sustained gene expression is not necessary once the targeted tumor cells die
or new blood vessels grow to bypass blocked arteries.
 Introduction 15

1.12 FDA-APPROVED BIOPHARMACEUTICALS AND VACCINES

Product NR.me Generic NR.me Company DR.te ofApprovR.l Applimtion

Actimmune gamma Genentech, Inc. Dec. 1990 Management of chronic

interferon Nov. 1987 granulomatous disease
Activase Genentech, Inc. Treatment of myocardial
recombinant March,1990 infarction
alteplase Treatment of infants and
Adagen Enzon, Inc. children with severe
adenosine immunodeficiency
deaminase OctOber, 1989. Use in genital warts
Management of relapsing,
AlferonN Interferon Sciences remitting multiple sclerosis
recombinant Inc. Treating Type 1 Gauchefs
interferon beta 1-B Berb: August, 1993. disease
Betaseron Treating Type 1 Gaucher's
Alglucerase Laboratories/ April, 1991. disease
Ceredase hepatitis B vaccine Chiron Corp. Treating anemia associated
Genzyme, Corp. June, 1994. with chronic renal failure and
Engerix B epoetin alfa Cerezyme anemia in Retrovir-treated,
Epogen imiglucerase Sept., 1989. HlV-infected patients
Somatropin Genzyme, Corp. Treatment of diabetes
Humatrope SmithKline June, 1989. Treatment of hairy tell
alpha interferon Beecham leukemia, genital wa~ts,
Intron A October, 1982 AIDS-related Kaposi's
anti-haemophiliac Amgen, Ltd. sarcoma, non-A, non-B
KoGENate factor June, 1986 hepatitis, and hepatitis :6
Eli Lilly & Co. June, 1988 Use in the treatment lof
Leukine yeast derived GM- November, 1988 hemophilia A
CSF Schering-Plough Februaty, 1991 Use in autologous botne
Neupogen Corp. July, 1992 marrow transplantation '
Oncaspar pegaspargase Use in treating
Miles, Inc. Februaty, 1993 chemotherapy-inducFd
Orthocione OKT-3 Immunex, Corp. neutropenia and bone
March,1991 marrow transplant-
Procrit epoetin alfa Amgen,Ltd. associated neutropenia
Februaty,1991 Use in treating acute
Proleukin interleukin (IL-2) Enzone/Rhone- June, 1994 lymphoblastic leukemia
Poulenc Rorer Use in reversal of acut\e
Protropin somatotrem Februaty,1994 kidney transplant rejection
Ortho Biotech Approved for use in the
Pulmozyme Dnase June, 1986 treatment of anemia
December, 1990 associated with chronic renal
Recombinate rAHF recombinant Ortho Biotech April, 1993 failure and anemia in
haemophiliac factor Retrovir-treated, HIV-
Recombivax HB recombinant alpha Chiron Corp. May, 1992 infected patients and
interferon Genentech, Inc. May, 1985 chemotherapy-associated
RoferonA Approved for December, 1993 anemia
Treatment of kidney (renal)
Genentech, Inc. December, 1992 carcinoma
July, 1986 Treating human growth
Baxter Healthcare June, 1986 hormone deficiency in
November, 1988 children
Merck & Co. Use. in the ,?anagement of
cystiC fibrOSIS
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