1) The document discusses challenges in formulating biotherapeutics like proteins and how commonly used excipients like gelatin and human serum albumin have issues related to purity, consistency, and potential risk of transmitting diseases. 2) It describes how recombinant DNA technology has been used to produce recombinant versions of gelatin and human serum albumin as excipients that are highly pure, consistent, and avoid risks from animal or human sources. 3) Recombinant human albumin called Recombumin has been commercially developed and clinically tested as a safer alternative to use in biotherapeutic formulations compared to traditional excipients from animal or human sources.

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Compression: Using
Excipients as binders
Recombinant Proteins
As Excipients
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2. Recombinant
Proteins as
Excipients
By S Berezenko, Research Director, Delta Biotechnology
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 to use them in the
preparation of a stable drug formulation that has the
desired shelf life and bioavailability. There are signifi-
cant differences between the functions of excipients
in small molecule pharma and biotherapeutic formu-
lations. In the former, the role of an excipient can be
to aid tablet formation by, for example, affecting
compressibility, act as a lubricant, disintegrant, filler
or glidant. The aforementioned functions of excipi-
ents in small molecule pharma are not what is
required for biotherapeutics (proteins, peptides and
vaccines). For biotherapeutics, the end point of a
stable, safe formulation with the desired bioavailabil-
ity, 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 inter-
faces; additionally, pH and buffer components can
inactivate these molecules. Biotherapeutics also
have more mechanisms of decomposition on top of
the usual drug degradation pathways such as
oxidation, racemisation and hydrolysis: these include
disulphide exchange, beta elimination, aggregation
and deamidation. Whilst there is no typical formula-
tion for biotherapeutics, there are some generalities
that can be considered.
Biotherapeutics formulation constraints
The pH of the formulation has two significant constraints, the obvious
one is that the pH has to be within a range in which the protein is sta-
ble 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 stabilise the
protein. Some commonly used excipients are in Table 1 and include
amino acids, sugars, polyols and polymers.
Table 1 Commonly used excipients for biotherapeutics
Sugars Trehalose Amino Acids Histidine
Mannose Aspartic acid
Sucrose Alanine
Dextrose Glutamic acid
Polyols Sorbitol Polymers Polysorbate
Mannitol Albumin
Glycerol Gelatin
The above 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 denatura-
tion at the air-liquid interface; to reduce this problem, 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 sorbitan and fatty acid side chain, Polysorbate 20, 40 and 80
respectively, having laurate, palmitate and oleate 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 protein under going denaturation at this
interface.
For many proteins, the combinations of pH, detergents and low
molecular weight excipients may still not make an ideal formulation.
One aspect of this is that biotherapeutics are very 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; in particular two proteins 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. In
comparison with expensive biotherapeutics (which have been pre-
pared via cell culture, cell separation and downstream processing)
these two proteins are relatively cheap and available commercially in
large quantities. Gelatin is derived from collagen extracted from the
hides and bones of cows or pigs. HSA is the most abundant protein
in blood plasma and is fractionated or purified from donated blood.
However, this simple approach of adding animal- and human-derived
proteins to formulations is now being challenged.
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3. Perceived problems with animal- and
human-derived proteins
There are two problems, firstly these proteins are heterogeneous and
relatively impure. Gelatin is a heterogeneous mixture of polypeptides
and this raises issues with lot to lot consistency. HSA has a pharma-
copoeial purity requirement of only ≥96%, (USP), the rest of the pro-
tein 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.
The second issue is the drive to remove all animal and human derived
products from pharmaceuticals, caused by the advent of “mad cow”
disease (Bovine Spongiform Encephalopathy) and the prion disease
in humans, variant Creutzfeldt-Jakob Disease (vCJD), 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 (European Commission, 2001) or blood (Aguzzi and Glatzel,
2004) and the HSA derived from it. Although there is no evidence that
this can happen it has pushed formulation scientists to think twice
about using these proteins as excipients. Also, given the very high
purity of recombinant DNA derived biotherapeutics it seems some-
what illogical to adulterate them with such impure excipients.
One approach, taken to avoid the issues surrounding the use of
protein based excipients, has been to develop new formulations and
remove the protein from the product. Factor VIII (Antihemophilic
Factor) from Bayer HealthCare, USA is now a third generation prod-
uct. It began as a plasma derived product, and 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 (Ruiz et al 2003).
A new approach - Recombinant DNA technology
excipients
An alternative solution to the costly and time consuming search for a
new formulation has emerged from the same source as the biothera-
peutics, namely recombinant DNA technology. Using genetically mod-
ified yeast it has been possible to express and purify recombinant
gelatin and recombinant human albumin (Recombumin®) for use as
excipients (Dodsworth et al, 1996 and Tarelli et al, 1998).
Recombinant human gelatins (FibroGen, South San Francisco,
California) are engineered from specific segments of human collagen
genes. They are expressed in the methylotrophic yeast Pichia pas-
toris and manufactured avoiding the use of animal or human-derived
materials. FibroGen’s proprietary technology describes the production
of discrete, reproducible batches of gelatin fragments with specific
molecular weights, providing customers with the ability to select a
product optimised for specific applications. FibroGen has also per-
formed a clinical safety study of recombinant human gelatin, finding
the study material safe and well tolerated.
The other recombinant excipient, Recombumin®, is further ahead in
development than the recombinant gelatin and is available as a
commercial product. Recombumin® is manufactured by Delta
Biotechnology Ltd, (Nottingham, England, a subsidiary of Aventis
Pharma). Recombumin® is derived from the yeast Saccharomyces
cerevisiae and is manufactured to cGMP using a process that is com-
pletely free from the use of animal or human derived products. The
product is structurally identical HSA but significantly purer (Figure 1).
The characterisation of the recombinant albumin molecule has been
taken to the level of x-ray crystallography studies using crystals
grown under zero gravity on the NASA Space Shuttle (He and Carter,
1992) and laboratory studies have crystallised Recombumin® in the
presence of ligands (Curry et al
1998) (Figure 2).
Recombumin® is an ultra-high
purity product, with residual
yeast content of less than 0.15
ppm. A comparative clinical
trial using Recombumin® and
HSA was performed.
Recombumin® was well toler-
ated in both an i.m. repeat
dose study (5 x 65mg) evaluated in 500 subjects (250 rHA, 250 HSA),
as well as in an escalating dose i.v. study administering a maximum
of 50g and a cumulative dose of 80g. Currently, Recombumin® is
being used in a variety of applications including: -
coating medical devices
as an alternative to HSA in
in vitro fertilisation reagents
in the manufacturing process for a
vaccine as a stabiliser replacing HSA
In conclusion, the advent of recombinant excipients offers the
opportunity to use a potentially safer, more consistent and purer
protein as an excipient rather than the current sources of formulation
proteins being used now.
References
Aguzzi, A and Glatzel, M. (2004) The Lancet 363 9407 411-412
Curry, S., Mandelkow, H., Brick, P. and Franks, N. (1998)
Nature Structural Biology 5, 827-835
Dodsworth, N., Harris, R., Denton, K., Woodrow, J., Wood,
P.C and Quirk, A (1996) Biotechnol. Appl. Biochem. 24 171-176
European Commission (2001), The safety with regard to TSE risks
of gelatine derived from ruminant bones or hides from cattle,
sheep or goats
He, M.X. and Carter, D.C. (1992) Nature 358 209-215
Ruiz, L., Reyes, N., Duany, L., Franco, A,. Aroche, K. and Rando,
E.H. (2003) Int. J. Pharmaceutics 264 57-72
Tarelli E, Mire-Sluis A, Tivnann HA et al (1998)
Biologicals 26 331-346
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Figure 1
Figure 2