The document discusses the complexities in developing biopharmaceuticals compared to traditional small molecule drugs. Biopharmaceuticals are much larger and more structurally complex, consisting of folded protein chains rather than single molecules. This increased complexity impacts many aspects of development, including determining the active structure, developing purification processes, assessing stability and immunogenicity, and performing comparability testing. While superficially similar regulatory filings are required, biopharmaceutical development faces unique challenges due to this complex three-dimensional structure of proteins.

1. both the product and the establishment that manufactured
the product were licensed; the latter licensed to produce the
specific product using a defined and unchangeable manufac-
turing process. This approval strategy was modified by the
FDA in 1996 and, as described in the 1996 PDA letter, John
Geigert stated “While the FDA is moving away from the prod-
uct being defined by the process for well-characterized biotech
products, it was clearly pointed out that a well-characterized
process is necessary for a well-characterized product.” In 2002,
FDA moved regulation of a number of biopharmaceuticals to
the Center for Drug Evaluation and Research (CDER) in rec-
ognition of the greater control manufacturers could exercise
over the manufacture and testing of biopharmaceuticals. Even
though industry now has greater control of manufacture of bio-
pharmaceuticals, it remains costly and difficult.
Determining Active Structures
Determination of the active structures of a biopharmaceu-
tical is not as simple as it is for a traditional small molecule
therapeutic. Biopharmaceuticals are, most often, comprised
of a linear sequence containing some or all of the 20 com-
mon amino acids (Figure 3) in a unique order for each pro-
tein. The sequence of these amino acids defines the primary
structure of the molecule but, unlike small molecules, the
primary structure of a biopharmaceutical does not by itself
define the active form. In a protein, the amino acid chain
folds into three-dimensional structures that can interact
with other structures within the protein that can be located
at a great distance in the primary structure but are in close
proximity after the protein folds into its lowest energy form.
The structures that interact may even be incorporated into
different protein chains (Figure 4). It is the complex, three
D
eveloping a biopharmaceutical is just like devel-
oping a traditional small molecule therapeutic…
except it is not. While the types of data needed
for regulatory filings is the same, the signifi-
cantly increased size and molecular complexity of biophar-
maceuticals compared to small molecule therapeutics has a
significant impact on the strategies and activities required
for development. Biopharmaceuticals can consist of proteins
or other types of products such as peptides, nucleic acids,
viral gene therapy vectors, carbohydrates, and lipids, alone
or in combination. The majority of biopharmaceuticals on
the market today are proteins, and therefore this article
focuses on those activities required primarily for develop-
ment of protein-based therapeutics and may not apply to
the other categories of biopharmaceuticals.
One might wonder why a pharmaceutical company that
has been successful in developing and marketing traditional
small molecule therapeutics would want to take on the chal-
lenge of developing a biopharmaceutical. Biopharmaceuticals
are a widely successful and profitable pharmaceutical class
of products, and one driver for companies to enter this area
is certainly the very attractive market opportunity. As shown
in Figure 1, even though small molecule therapeutics repre-
sent nearly 90% of the current total sales of pharmaceuticals
world-wide, biopharmaceutical sales are increasing at a rate
of 11% per annum while small molecule therapeutic product
sales are increasing at a significantly lower rate. The attrac-
tion of this strong market opportunity is further supported
by the reduced development risk and the increasing regula-
tory approval rate of biopharmaceuticals. In fact, the ap-
proval rate for the 50 largest pharmaceutical firms from the
mid-1990s through 2007 was 32% for biopharmaceuticals and
only 19% for small molecule drugs1
. According to a BIO study
published in 2011 and based on data from 2004-2010, the
probability of a biopharmaceutical in Phase 1 studies obtain-
ing regulatory approval is 15% compared to 7% for traditional
small molecule therapeutics.2
Financial Incentives Attract More Players
The clear financial incentive for biopharmaceutical devel-
opment, along with the increased opportunity in biosimilars
due to the large number of successful biopharmaceuticals
that are coming off patent in the near future, is attracting
more players to compete in this market segment; however
with this attractive opportunity comes the unique challenge
of developing these larger, complex products. Many pharma-
ceutical companies that only have small molecule experience
may not be familiar with the complexity of proteins and may
not have the equipment or in-house expertise to perform the
required activities. Superficially, the development pathway
for biopharmaceuticals and traditional small molecules may
appear to be quite similar, especially given the fact that the
same type of information describing the product’s physical
properties and biological activity is needed for regulatory fil-
ings; however there are a multitude of differences as to how
this key information is obtained for each product type.
The size of the molecule is, perhaps, the most obvious dif-
ference between traditional small molecules and biopharma-
ceuticals, but this obvious difference masks a greater disparity
–three dimensional complexity. Figure 2 shows an illustration
of aspirin (A), a traditional small molecule drug, and two bio-
pharmaceuticals, human Growth Hormone (B) and a mono-
clonal antibody (IgG molecule) (C). The aspirin molecule com-
prises 20 atoms and can readily be illustrated. In contrast, the
hGH and IgG molecules are more complex, comprising 3,000
and 20,000 atoms, respectively. While small molecules exist in
single molecular compositions, even pure preparations of bio-
pharmaceuticals contain multiple forms with different forms of
the protein such as slightly different folding and different and
multiple post-translational modifications.
The complexity of biopharmaceuticals and the lack of suffi-
cient analytical tools to characterize their structure in enough
detail to meet regulatory requirements led to the early regula-
tory approach of “the process is the product.” Prior to 1996
Development of a Biopharm aceutical is … Complex
n By Sheila G. Magil, Julia Adam, Dawn Ecker, and Susan Dana Jones, BioProcess Technology Consultants, Inc.
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dimensional structures that, primarily, determines the sta-
bility and biological activity of the molecule. The interac-
tion of structures that are distant in the primary structure
but close in the three dimensional structure means that a
change in an area quite distant from the active center may
still have a significant impact on the biological activity of
the molecule.
Because the folded structure of a protein is critical to de-
termining the biological, and therefore therapeutic, activity
of the molecule, a suite of analytical methods are required
in order to test a biopharmaceutical product that has been
manufactured for human clinical use and to release it if it
meets the previously agreed-upon specifications. For example,
the definition of purity for a biopharmaceutical is a function of
the assay used to measure it, which is in sharp contrast to the
release of traditional small molecule therapeutics. The entire
structure of a small molecule can be determined using one or
more analytical methods that measure first principles, such
as Infrared Spectroscopy, whereas the complexity of most
biopharmaceuticals requires measurement of its many varying
forms through the use of several chromatographic and other
methods and comparison to the behavior of a reference prepa-
ration in the same system. No single method will be able to
separate and quantify the multiple variant forms present.
The importance of the three dimensional structure in the
activity of a biopharmaceutical increases the need for a potency
assay that has been shown to be related to the biological activ-
ity of the molecule. Since the ‘one-dimensional’ structure is not
directly indicative of potency, it is important to demonstrate
activity of a biopharmaceutical in a system that closely models
the biological activity. Assume, for instance, a monoclonal an-
tibody product’s biological activity is to prevent the growth of
Pharmaceutical Processing | october 2012 33 n
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If it's so complex - why is everyone jumping in?
Figure 1. Current Sales for Small Molecule Therapeutics and Biopharmaceuticals
Figure 2. Illustration of Traditional and Biopharmaceutical Molecules. Left to right: A) Aspirin, 21 Atoms, MW 180 Da. B) Human Growth Hormone, ~3000
Atoms, MW ~22 kDa . C) IgG Molecule, ~20000 Atoms, MW ~150 kDa
A CB

2. Figure 3. Twenty Common Amino Acids
blood vessels that supply oxygen and nutrients
to tumors in a cancer patient. It is insufficient
to simply demonstrate that the antibody binds
to its target, such as human vascular endothe-
lial growth factor (VEGF), with an ELISA assay.
It is also necessary to demonstrate that bind-
ing of the monoclonal antibody to this target
also reduces VEGF binding to and activation
of the VEGF receptor molecule, and therefore
reduces blood vessel formation and growth, in
a dose-dependent manner.
Complexity Issues
The structural complexity of a biophar-
maceutical also impacts the development of
the purification process that will be used to
manufacture the product for human clinical
use. Impurities in a purified preparation of
a protein can be related to the production
process (process related impurities) or to
the molecule (product related impurities).
In general the process related impurities are
quite different from the product and are more
easily removed by chromatographic methods.
Product related impurities are closely related
to the product and therefore can be much
more difficult to remove during purification.
Product-related impurities can consist of, in
part, misfolded forms, aggregates, or trun-
cated versions that were not completely syn-
thesized or that were cleaved by a protease
during production. An additional constraint
on the purification of biopharmaceuticals is
the need to maintain the structure, which
often requires working in an aqueous environ-
ment under relatively mild conditions.
The complexity of a biopharmaceutical also
impacts determination of stability, making this
evaluation more difficult than for traditional
small molecule therapeutics. The composi-
tion of a traditional small molecule is a single
molecule with a single structure; and as stated
previously, a pure biopharmaceutical can con-
tain multiple forms. Small molecule degrada-
tion, involving one kind of molecule proceeds
according to first order kinetics and the deg-
radation rate at standard storage conditions
can be estimated with good accuracy by the
Arrhenius equation. Unfortunately, degrada-
tion pathways and degradation rates are not
as simple for proteins. A pure biopharmaceuti-
cal may contain a large number of molecular
types and therefore it degrades according to
second or higher order kinetics. Each of these
forms may degrade according to slightly differ-
ent kinetics and pathways, and the interaction
of the multiple forms may influence the degra-
dation of each. The stability of a biopharma-
ceutical can only be determined, according to
current regulatory guidelines, using real time
studies conducted under real, intended stor-
age conditions. In addition, since there may be
some slight differences in impurities present
at full commercial production scale compared
to those present at lab or pilot scale, stability
information from batches produced at smaller
scales are considered to be supporting infor-
mation and not sufficient for expiry dating of
product produced at full commercial scale.
More Challenges
Other challenges in developing a biophar-
maceutical include immunogenicity and com-
parability testing. In contrast to biopharma-
ceuticals, traditional small molecules are not
considered to be immunogenic due to their
small size, and therefore immunogenicity test-
ing is not part of the development pathway
for a traditional small molecule therapeutic.
Biopharmaceuticals are largely considered to
be immunogenic, even if completely equiva-
lent in sequence to a native human protein,
because the potential for differences in struc-
ture and mode of administration may give
rise to an immune reaction. Consequently,
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evaluation of immunogenicity during clinical
trials is a requirement for biopharmaceutical
development. Determination of comparabil-
ity for biopharmaceuticals is of significant
concern during development. During scale up
of the manufacturing process it is necessary
to demonstrate that production changes do
not result in critical changes in the molecu-
lar structure that could impact potency or
immunogenicity. With the recent legislation
(Biologics Price Competition and Innovation
Act) allowing for the development of biosimi-
lars, demonstration of structural comparabil-
ity between the innovator biopharmaceutical
and the biosimilar will be crucial.
Differences in the development of biophar-
maceuticals and traditional small molecule
therapeutics can be traced to the disparate
complexity of these two molecule classes.
Demonstration of small molecule structure
can be done using analytical methods whose
first principles are well understood and
the testing results are readily interpreted.
Structural analysis of biopharmaceuticals is
not simple and the testing results are more
difficult to interpret. The majority of the
challenges in the development of a biophar-
maceutical result from its complexity. This
same complexity will also make development
of biosimilars much more challenging than
the development of generic versions of suc-
cessful small molecule therapeutics.
References
1 DiMasi JA, et al. Trends in risks associ-
ated with new drug development: success
rates for investigational drugs. Nature.
2010 Mar;87(3):272-7.
2 Hay M, et al. BIO / BioMedTracker: Clinical
trial success rates study. Presented at the
Biotechnology Industry’s 14th Annual CEO
and Investor’s Conference; 2011 Feb 14-15;
New York, New York.
3 Figure adapted from: Bioprocess
Technology Consultants [Internet]. Woburn
(MA): Bioprocess Technology Consultants,
Inc. Biomanufacturing supply pipeline
product databases; 2011 [cited 2012 Aug
31]; [about 2 screens]. Available from:
http://www.bptc.com/pipeline-databases.
php and IMS Health Inc. Total unaudited
and audited global pharmaceutical market,
2003 – 2011 [Internet]. Parsippany (NJ):
IMS Health, Inc.; 2012 May [cited 2021
Aug 31]. 1p. Available from: http://www.
imshealth.com/deployedfiles/ims/Global/
Content/Corporate/Press%20Room/Top-
Line%20Market%20Data%20%20Trends/
2011%20Top-line%20Market%20Data/Global_
Pharma_Market_by_Spending_2003-2011.pdf.
4 Aspirin [Internet]. San Francisco (CA):
Wikipedia, The Free Encyclopedia; 2001
Nov 6 [updated 2012 Aug 28]. File:Aspirin-
B-3D-balls.png; 2008 May 15 [cited 2012
Aug 29]; [about 4 screens]. Available from:
http://en.wikipedia.org/wiki/File:Aspirin-B-
3D-balls.png.
5 Growth hormone [Internet]. San Francisco
(CA): Wikipedia, The Free Encyclopedia;
2001 Nov 6 [updated 2012 Aug 28]. File:
Somatotropine.GIF; 2005 Aug 25 [cited
2012 Aug 29]; [about 4 screens]. Available
from: http://en.wikipedia.org/wiki/File:
Somatotropine.GIF#file
6 Protein structure [Internet]. San Francisco
(CA): Wikipedia, The Free Encyclopedia;
2001 Nov 6 [updated 2012 Aug 28]. File:
Main_protein_structure_levels_en.svg; 2008
Oct 7 [cited 2012 Aug 29]; [about 4 screens].
Available from: http://en.wikipedia.org/wiki/
File:Main_protein_structure_levels_en.svg n
Figure 4. Structural Motifs of Biopharmaceuticals
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