This document discusses the differences between excipients used in small molecule and biotherapeutic drug formulations. For biotherapeutics, excipients help stabilize proteins and peptides, which are sensitive molecules. Common excipients include amino acids, sugars, polymers and detergents. Historically, human serum albumin and gelatin were used as excipients but there are now concerns over using animal or human derived materials due to risk of disease transmission. Recombinant versions of gelatin and human serum albumin produced in yeast are presented as safer alternatives that avoid these risks.

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FORMULATION REQUIREMENTS – SMALL MOLECULES
VERSUS BIOTHERAPEUTICS
The term excipient is defined as a raw material that is purposely
added to a pharmaceutical; it is an inactive material that can
perform a number of functions, but the ultimate aim is for them
to aid in the preparation of a stable drug formulation that has the
desired shelf life and bioavailability. There are significant
differences between the functions of excipients in small
molecule pharma and biotherapeutic formulations. In the
former, excipients can assist tablet formation by, for example,
affecting compressibility or acting as a lubricant, disintegrant,
filler or glidant. These functions differ from those required
for biotherapeutics (proteins, peptides and vaccines). For
biotherapeutics, the endpoint of a stable, safe formulation with
the desired bioavailability is still a necessity, but the challenges
offered by the formulation of proteins are different.
Proteins are often sensitive to heat, denaturation from liquid
shear, or denaturation at air-liquid interfaces; additionally,
solution pH and buffer components can inactivate these
Formulation of Biotherapeutics
Avoiding Human and Animal
Derived Excipients
400
300
200
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By Dr Steve Berezenko, Research Director at Delta Biotechnology Ltd
Dr Stephen Berezenko has worked in the biotechnology field for 20 years
and is currently Research Director at Delta Biotechnology, a subsidiary
of Sanofi Aventis. Previous positions at Delta included Head of Process
Development and Technology Transfer Manager. Dr Berezenko gained his PhD
at Heriot-Watt University in Edinburgh, researching in the field of affinity
chromatography matrix design and the application of affinity chromatography
to the purification of serum and recombinant proteins.
molecules. Biotherapeutics also have other mechanisms of
decomposition in addition to the usual drug degradation
pathways such as oxidation, racemisation and hydrolysis: they
can undergo disulphide exchange, beta elimination, aggregation
and deamidation. Whilst, as with small molecules, there is no
typical formulation for biotherapeutics, some generalities can
be considered.
BIOTHERAPEUTICS FORMULATION ISSUES
At a basic level, the formulation of a biotherapeutic will contain
salts and have an optimal solution pH. The pH of the
formulation has two significant effects; the obvious one being
that the pH has to be within a range in which the protein is stable
and active. The second is that deviation from physiological pH
will result in the patient suffering injection site pain during
administration of the drug. The salts present are often targeted
to a physiological level or isotonicity. Thereafter, if the protein
is not stable under these conditions, it is necessary to find
further excipients to create stability. Some commonly used
excipients are listed in Table 1. These include amino acids,
sugars, polyols and polymers.
These excipients can aid lyophilisation and
reconstitution of a protein as well as
stabilising the product in solution. One
specific problem associated
with proteins in liquid
formulations is denaturation
at the air-liquid interface;
to reduce this problem,

2. detergents, generally of a non-ionic nature, are
often used. A typical non-ionic detergent used in
many protein formulations is polysorbate. This
family of detergents is based on a polyoxyethylene
backbone with a side chain containing sorbitan and
a fatty acid, polysorbate 20, 40 and 80, having
laurate, palmitate or oleate respectively as the
side chain.
The mechanism of action is considered to be
that the amphipathic detergent molecules gather
at the air-liquid interface, with the hydrophobic
moiety in the air and the hydrophilic tail in
the aqueous environment, thus preventing
denaturation at this interface.
For many proteins, the combinations of pH,
detergent and low molecular weight excipients
may still not produce an optimal formulation.
One aspect of this is that biotherapeutics are most
often required in very small therapeutic doses and
can be denatured by surface adsorption to glass
containers or container closures such as butyl
septa. This has resulted in many formulations
requiring a bulking agent; two proteins in
particular have been used extensively – notably gelatin and
human serum albumin (HSA). These proteins can be added in
large excess over the active protein and thus reduce the risk of
protein denaturation by surface adsorption and also contribute
to the reconstitution of lyophilised products. In comparison
with expensive biotherapeutics (which have been prepared via
cell culture, cell separation and downstream processing) these
two proteins are relatively cheap and commercially available
in large quantities.
HSA AND GELATIN: HISTORY AND USE AS EXCIPIENTS
HSA is the most abundant protein in blood, being present at
a concentration of approximately 42g/l (1). Since 1940,
albumin has been fractionated from blood plasma (2) for use
as a blood volume replacement and to treat burns victims.
Today, HSA can be regarded as a low cost side-fraction
generated when purifying expensive blood components such
as IgG and factor VIII.
Gelatin is chemically extracted from animal hides and bones on
a massive scale; approximately 50,000 metric tonnes of gelatin
is produced annually for medical use, most of which is used in
oral drug delivery, such as capsule formation (3). Due to
differences in extraction procedures from company to company
and the relatively crude nature of the extraction procedure, there
is significant source and batch to batch variability that can
affect the physiochemical properties of the gelatin. This can in
turn affect the product in which the gelatin is used.
HSA has been widely used as an excipient to stabilise a number
of therapeutic proteins (see Table 2). Additionally, as it is a
human derived protein and there are instances of allergic
response to animal derived gelatins, it can be regarded as the
stabiliser of choice, immunologically speaking (4,5). However,
this simple approach of adding animal and human derived
proteins to formulations is now being challenged.
CURRENT CONCERNS REGARDING ANIMAL
AND BLOOD DERIVED PROTEINS
The drive to remove all animal and human derived products
from biopharmaceuticals was prompted by the advent of ‘mad
cow’ disease (Bovine Spongiform Encephalopathy) and the
prion disease in humans, variant Creutzfeldt-Jakob Disease
(vCJD), the latter of which has been linked to eating BSE-
infected products. This has raised the question as to whether
prions or other viral diseases could be transmitted via
gelatin (6) or blood (7) and the HSA derived from it. Although
there is no evidence that this can happen, it has forced
formulation scientists to think twice about using these proteins
as excipients.
Recently, a number of public statements, and one in particular,
have highlighted these issues with respect to both human and
animal derived blood proteins, including HSA. A recent
FDA/PDA meeting (September, 2004) included a presentation
by WM Egan, Acting Director, Office of Vaccines, FDA,
entitled ‘Bovine-derived Products Used in the Manufacture
and Formulation of Vaccines: Current Policies and Issues
for the Future’. In that presentation, a suggestion was
made regarding the use of recombinant albumin:
consider ‘replacement of human serum albumin (HSA) with
recombinant albumin’.
Sugars Trehalose Amino Acids Histidine
Mannose Aspartic acid
Sucrose Alanine
Dextrose Glutamic acid
Polyols Sorbitol Polymers Polysorbate
Mannitol Albumin
Glycerol Gelatin
Protein Trade name
Erythropoietin EpogenTM
and ProcritTM
(Amgen Inc)
Interferon alfa-2b Intron ATM
(Schering Corporation)
Antihaemophilic factor (FVIII) BioclateTM
(Baxter Healthcare Corp)
Tissue necrosis factor alpha-1a BeromunTM
(Boehringer Ingelheim Int. GmbH)
Interferon beta-1a AvonexTM
(Biogen BV)
Table 1: Commonly Used Excipients for Biotherapeutics
Table 2: A Selection of Therapeutic Products Containing HSA as an Excipient
EuropeanBioPharmaceuticalReviewSummer‘05issue.©SamedanLtd.2006

3. The UK Government recently notified up to 4,000 patients in
the UK that they may be at increased risk of carrying variant
Creutzfeldt-Jakob disease (vCJD) (8). This is a consequence
of those patients having received blood products purified
from donor pools that have subsequently been found to
have contained blood from a donor that has gone on to
develop vCJD. Whilst there has never been a proven case of
transmission of vCJD from processed plasma derived
products, such as FVIII, IgG or HSA, there have been
reports of likely transmissions from blood transfusions in the
UK (7,9).
With respect to gelatin and HSA, all the above issues can
be avoided in two ways: by development of protein-
free formulations or by the use of recombinant versions of
these proteins.
PROTEIN-FREE FORMULATIONS
One approach taken to avoid the above issues has been to
develop new formulations and remove the excipient protein
from the product. Factor VIII (antihaemophilic factor) from
Bayer HealthCare, US, is now a third generation product.
Factor VIII was originally a plasma derived product. It was
then manufactured using recombinant DNA technology, but
still using HSA as an excipient. Now it is manufactured
using the same recombinant DNA technology, but is
formulated with sucrose, thus avoiding the addition of protein
excipients (Kogenate®
FS). A similar example has been the
removal of HSA from a formulation of recombinant human
interferon-α-2 (10).
However, this approach is not always straightforward, as
was exemplified by recent issues with the reformulation
of recombinant human erythropoietin (epoetin-alpha)
distributed outside the US (Eprex®
). Removal of HSA from
the formulation and the introduction of polysorbate 80
resulted in a substantial increase in the incidence of pure red
cell aplasia (PRCA). Investigations have proposed that
organic compounds that were leached from rubber stoppers
through the action of polysorbate 80 could be the cause of
the PRCA. These leachates were not present when the
rubber stoppers were replaced by Teflon®
coated stoppers;
nor were the leachates present in the product formulated
with human serum albumin or in other epoetin products with
different formulations. The authors suggest that the
leachates were the critical contributory factor in the
increased incidence of antibody-mediated PRCA attributed
to Eprex®
(11).
RECOMBINANT PROTEIN EXCIPIENTS
The basic driving forces for developing recombinant
versions of gelatin and HSA are as mentioned previously; the
potential for prion and viral contamination of these
excipients and the fact that gelatin and HSA are
heterogeneous protein preparations and relatively impure.
Gelatin is a heterogeneous mixture of polypeptides, whilst
HSA has a pharmacopoeial purity requirement of only ≥96%,
(USP), the rest of the protein present being a mixture of
polymers of HSA and other plasma proteins that remain
from the purification. Additionally, these other proteins
are denatured during the pasteurisation process that HSA
final product undergoes. Given the very high purity
of recombinant DNA derived biotherapeutics, it seems
somewhat illogical to adulterate them with ‘relatively poorly
defined’ excipients.
AN ANIMAL AND HUMAN FREE EXCIPIENT APPROACH –
RECOMBINANT DNA TECHNOLOGY EXCIPIENTS
An alternative solution to the costly and time-consuming search
for a new formulation for a product containing a protein
excipient has emerged from the same source as the
biotherapeutics, namely recombinant DNA technology. Using
genetically modified yeast, it has been possible to express
and purify recombinant gelatin (3) and recombinant human
albumin (Recombumin®
, Delta Biotechnology) for use as
excipients (12,13).
Recombinant human gelatins (FibroGen) are engineered from
specific segments of human collagen genes. They are
expressed in the methylotrophic yeast Pichia pastoris and
manufactured avoiding the use of animal or human derived
materials. FibroGen’s proprietary technology allows the
production of discrete, reproducible batches of gelatin
fragments with specific molecular weights, providing
customers with the ability to select a product optimised for
The UK Government recently notified up to 4,000 patients in the UK that
they may be at increased risk of carrying variant Creutzfeldt-Jakob disease
(vCJD) (8).This is a consequence of those patients having received blood
products purified from donor pools that have subsequently been found to
have contained blood from a donor that has gone on to develop vCJD.Whilst
there has never been a proven case of transmission of vCJD from processed
plasma derived products,such as FVIII,IgG or HSA,there have been reports
of likely transmissions from blood transfusions in the UK (7,9).

4. specific applications (3). FibroGen has also performed a
clinical safety study with recombinant human gelatin and
found it to be safe and well tolerated.
The other recombinant excipient, Recombumin®
, is available
as a commercial product (14). It is derived from the yeast
Saccharomyces cerevisiae and is manufactured to cGMP in an
FDA inspected facility using a process that is completely free
from the use of animal or human derived products. The
product is structurally identical to HSA but significantly purer
(see Figures 1 and 2).
The characterisation of the recombinant albumin molecule has
been taken to the level of x-ray crystallography studies and
laboratory studies have crystallised Recombumin®
in the
presence of ligands (15) (see Figure 3).
In conclusion, the advent of recombinant protein excipients
such as gelatin and albumin offers the opportunity to use
potentially safer, more consistent and purer proteins as
excipients rather than the currently human and animal derived
products. Moreover, recombinant excipients should allow a
simpler replacement alternative
when redeveloping a formulation
rather than developing a protein-
free formulation from scratch. N
The author can be contacted at
steve.berezenko@aventis.com
References
1. Peters T, All about Albumin,
Academic Press, ISBN 0-12
552110-3, 1996
2. Cohn EJ, Chem Rev 28: pp395-417, 1940
3. Olsen D, Yang C, Bodo M, Chang R, Leigh S, Baez J, Carmichael
D, Perälä M, Hämäläinen ER, Jarvinen M and Polarek J,
Advanced Drug Delivery Reviews 55: pp1,547-1,567, 2003
4. Cooperman, L and Michaeli D, J Am Acad Dermatol 10:
pp355-3,656, 1984
5. Sakakaguchi M and Inouye S, Jpn J Infect Dis 53: pp189-
195, 2000
6. European Commission, The safety with regard to TSE risks
of gelatine derived from ruminant bones or hides from
cattle, sheep or goats, 2001
7. Aguzzi A and Glatzel M, The Lancet 363 9407: pp411-
412, 2004
8. Kmietowicz Z, B Med J 329: p702, 2004
9. Pincock S, Br Med J 329: p251, 2004
10. Ruiz L, Reyes N, Duany L, Franco A, Aroche K and Rando
EH, Int J Pharmaceutics 264: pp57-72, 2003
11. Boven K, Knight J, Bader F, Rossert J, Eckardt KU
and Casadevall N, Nephrol Dial Transplant 20 Suppl 3:
iii33-iii40, 2005
12. Dodsworth N, Harris R, Denton K, Woodrow J, Wood PC and
Quirk A, Biotechnol Appl Biochem 24: pp171-176, 199613.
13.Tarelli E, Mire-Sluis A, Tivnann, HA, Tivnann HA, Bolgiano
B, Crane DT, Gee C, Lemercinier X, Athayde, ML, Sutcliffe N
and Corran PH, Biologicals 26: pp331-346, 1998
13. He MX and Carter DC, Nature 358: pp209-215, 1992
14. Bosse D, Praus M, Kiessling P, Nyman L, Andresen C, Waters
J and Schindel F, J Clin Pharmacol 45: pp57-67, 2005
15. Curry S, Mandelkow H, Brick P and Franks N, Nature
Structural Biology 5: pp827-835, 1998
Figure 1: Recombinant Albumin Crystals
Figure 3: Recombumin®
X-Ray Crystal
Structure with Myristate Ligands
Figure 2: Electrospray Mass Spectrometry
of HSA and Recombumin®
Electrospray Mass Spectrometry
Unmodified
monomer
DesAsp-Ala + blocked
free thiol Monomer + blocked
free thiol (cys34 + cys)
Monomer lacking
C-terminal Leu
Recombumin®
HSA
Monomer lacking
N-terminal Asp-Ala
100
%
65,500 66,000 66,500 67,000 67,500