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`Biotechnology
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`Recombinant Human Albumin: Applications
`as a Biopharmaceutical Excipient
`In addition to batch-to-batch consistency, the use of recombinant human
`albumin in biopharmaceutical formulation provides many of the recognised
`benefits of using human serum albumin as an excipient, whilst avoiding
`the risks of transmitting viral and prion contaminants.
`
`By David Mead, Dermot Pearson and Maree Devine at Novozymes Delta Ltd
`
`Dr David Mead is Director of Intellectual Property and Business Development at Novozymes Delta Ltd, based in Nottingham
`(UK). His first degree was in Microbiology from the University of Kent, followed by a PhD from UMIST (Manchester, UK) in
`plasmid-host interactions in yeast. He initially worked as a Research Scientist in Glaxo’s Biotechnology Group, followed by
`a post-doc back in academia (University of Manchester) managing a project between chemistry and molecular biology on
`superoxide dismutase. Dr Mead has had a number of roles within Novozymes Delta Ltd, including Manager of Fermentation
`with responsibility for the development of commercial and scaleable fermentation processes integrated with molecular
`biology and downstream purification, including technology transfer, both internally and externally. He was also responsible
`for setting up and managing the Technical Support function for Recombumin® manufacturing, before taking responsibility
`for the company’s intellectual property and business development.
`
`Dermot Pearson has worked for Novozymes Delta Ltd since 1987, following completion of a PhD in Fermentation
`Technology at Dublin City University (Dublin, Ireland). Prior to that, he was a graduate in Biochemistry from University
`College, Dublin. While with Novozymes Delta Ltd, he has performed a number of scientific and management roles,
`from R&D Process Development through QC and Operations Management to his current role as Director of Commercial
`Operations. In this role, he is responsible for Marketing and Business Development, promoting products and services
`and negotiating technology licence and product supply agreements with customers worldwide; in this capacity, he is
`also responsible for Novozymes Delta Ltd’s Regulatory Affairs function.
`
`Maree Devine is Commercial Operations Manager at Novozymes Delta Ltd. She has a degree in Parasitology from the
`University of Glasgow (Scotland) and, since completing her PhD at Nottingham University, has held positions in both research
`and sales and marketing. Prior to joining Novozymes Delta Ltd, she was a Technical Product Specialist with EMD Biosciences,
`and then joined ThermoFisher (UK) as a Product Merchandising Manager, Life Sciences, where she maintained, developed
`and marketed the product portfolio. In her current role as Commercial Operations Manager, Dr Devine is responsible for sales
`and marketing co-ordination, customer liaison, event management, market research and marketing campaign development.
`
`Human serum albumin (HSA) is one of the most
`widely used proteins in the pharmaceutical industry.
`Synthesised in the liver, this non-glycosylated 66kD
`molecule is well characterised and occurs naturally in
`the body as a plasma protein at concentrations of 42-
`45mg/ml (1). HSA regulates the colloidal osmotic
`pressure of blood and buffers acid-base changes; it is
`also responsible for the transportation of a range of
`substances, which have the potential to be toxic in the
`unbound state, but are non-toxic when bound to
`albumin (1). Traditionally used as a therapeutic agent,
`HSA’s primary function is the restoration and
`maintenance of blood volume in situations such as
`surgery and blood loss, traumatic shock, plasma
`exchange and the treatment of burns. Exhibiting a lack
`of toxicity and immunogenicity, HSA has also been
`used as a manufacturing excipient for numerous
`pharmaceutical and biological products – for example,
`as a stabiliser in vaccines and therapeutic protein drugs,
`
`in coatings for medical devices, and as a component in
`drug delivery systems and imaging reagents such as
`those used for X-rays (2).
`
`HARNESSING HSA PROPERTIES
`HSA’s various in vivo functions and physical properties
`have been exploited in a number of biopharmaceutical
`applications – for example, as an excipient:
`
`N Its amphiphilic properties make it suitable as
`an additive to inhibit adsorption of the active
`protein to the container via competitive
`adsorption mechanisms
`N Its surface active character enables it to fulfil the
`role of a surfactant, thereby preventing protein
`aggregation
`N In some instances, it stabilises the conformational
`structure of the active molecule to maintain its
`bioactivity throughout the product shelf-life
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`HSA also has a high glass transition temperature, which
`in combination with its amphiphilic nature makes it an
`ideal vehicle for cryoprotection.
`
`Native HSA demonstrates remarkable stability, with an in
`vivo half-life of 15-20 days; this is attributable in part to
`the presence of 17 disulphide linkages in the protein. In
`vitro, the molecule’s stability is increased and it remains in
`solution at room temperature helping to sustain the shelf-
`life of the final biopharmaceutical product. During
`manufacture, HSA can withstand heating to 60oC for 10
`hours to facilitate viral inactivation.
`
`TRADITIONAL HSA MANUFACTURE
`HSA is currently used in greater volumes than any other
`biopharmaceutical solution, with worldwide manufacture
`in the order of hundreds of tonnes annually (3). Since
`1940, it has been produced by fractionation of plasma
`obtained from donors (4). While the safety profile of
`HSA with respect to viral transmission has been excellent,
`the theoretical risk of the transmission of new and known
`infectious agents (such as variant Creutzfeld-Jacob
`disease, HIV, hepatitis and West Nile virus) via the
`continued use of blood- and plasma-derived products is
`ever-present and unlikely to be completely eliminated.
`This has resulted in regulatory authorities worldwide
`creating a myriad of regulations to limit the use of
`plasma-derived materials with the aim of minimising
`transmission risks and necessitating a dedicated drive
`from within the industry to develop substitute products
`and ever-more sophisticated tools for the detection,
`clearance and removal of adventitious agents from serum-
`derived products (5).
`
`Such safety concerns provide the strongest motivations to
`develop recombinant human albumin (rHA) as a suitable
`alternative to HSA, for use as an excipient
`in
`biotherapeutics. As well as avoiding the transmission of
`serum-derived disease agents, other key advantages of using
`rHA over HSA
`include
`increased batch-to-batch
`consistency (which for industrial applications could mean
`the difference between performing several timely and costly
`batch verifications per year or not) and breaking a heavy
`reliance on an increasingly unpredictable supply chain.
`
`DEVELOPING AN rHA
`A number of microbial host/vector systems – including
`K. lactis (6), P. pastoris (7), H. polymorpha (8) and S.
`cerevisiae (baker’s yeast) (9) – have been looked at for the
`production of rHA. However, over the past few years
`particular advances in yeast-based protein expression and
`scale-up have led to the development of an industrial-
`scale manufacturing process that can produce a high
`
`purity, high quality rHA that is animal-free and suitable
`for use as an excipient in biotherapeutics.
`
`The molecular engineering of a series of proprietary S.
`cerevisiae strains to select for various traits, such as
`genetic stability and high copy number, has been
`pioneered by Novozymes Delta Ltd (previously Delta
`Biotechnology Ltd) for the production of rHA
`(Recombumin®, the company’s lead product). Based on
`a proprietary 2-micron plasmid construct, their yeast-
`based expression system is optimised for the production
`of recombinant proteins where glycosylation does not
`naturally occur or can be engineered without loss of
`performance of the active molecule. This proprietary 2-
`micron plasmid construct is in an otherwise plasmid-free
`background, and the high copy number plasmids are
`very stable with an expression cassette consisting of only
`yeast DNA and the cDNA for HSA, removing concerns
`about the use of antibiotic resistance genes of bacterial
`origin. The Novozymes Delta S. cerevisae strains have
`been engineered to be protease-deficient, and can
`generate yields of up to 5g/L, avoiding the use of
`hazardous solvents in the process. Furthermore, like the
`majority of molecules expressed in the system, the
`Recombumin® molecule is secreted which significantly
`aids down-stream processing. The Recombumin®
`production process has been successfully scaled up from
`10L to 8,000L at the company’s cGMP-compliant rHA
`manufacturing facility at Nottingham, UK.
`
`Two physiological phenomena related to the many
`accumulative genetic changes in S.cerevisae had to be
`overcome during the development of a robust industrial-
`scale process for the production of such a high grade
`rHA. The first was a reduction in the critical growth rate,
`µcrit, which is the highest rate at which growth is fully
`aerobic without production of ethanol or acetate. Values
`above µcrit will result in the build-up of unwanted by-
`products. Although a lower µcrit value theoretically results
`in a reduction in bioreactor productivity, this is of little
`economic significance since – at large scale – factors such
`as mass and heat transfer limit the maximum growth
`rate. The decreased µcrit is accommodated by lowering the
`parameter used in the automatic feed control algorithm
`that determines the effective growth rate in the process.
`
`The second phenomenon is a tendency of the organism to
`produce acetate under conditions where there is a slight
`excess in nutrient supply. Ethanol production is readily
`detected by a rise in respiratory quotient (RQ) determined
`by exit gas analysis. Hence, the control algorithm is
`designed to adjust the feed rate automatically. Acetic
`acid cannot be detected by a change in RQ but can
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`Figure 1: Structure
`of rHA with five
`molecules of
`myristate bound
`
`t
`
`in
`identified by changes
`be
`conductivity. This principle was
`used to develop a sub-routine in
`the automatic control procedure to
`adjust the feed rate appropriately.
`
`HSA VERSUS rHA: SAFETY
`AND TOLERABILITY
`X-Ray crystallography and mass
`spectrometry
`studies
`revealed
`rHA
`is
`that Recombumin®
`structurally identical to HSA (see
`Figure 1)
`and
`significantly
`more homogeneous (10). A Phase I study has been
`conducted comparing the safety, tolerability, and
`pharmacokinetics/pharmacodynamics of rHA with
`HSA (11). Two double-blind, randomised trials were
`performed in healthy volunteers using intravenous (IV)
`and
`intramuscular
`(IM) administration. Thirty
`volunteers participated in the IV trial, each receiving
`increasing doses (10g, 20g and 50g) of either rHA or
`HSA. The IM trial comprised 500 volunteers, each
`receiving 5 repeat doses of 5mg (100 subjects), 15mg
`(100 subjects) or 65mg (300 subjects) of rHA or HSA.
`Both trials recorded all adverse events and were
`conventionally classified; potential allergic responses
`were also monitored. Blood samples were taken in both
`studies to test for IgG or IgE antibodies against the test
`human albumin products and potential impurities.
`
`For the IV study, pharmacokinetic/pharmacodynamic
`assessments were carried out to include measurement of
`serum albumin, colloid osmotic pressure and
`haematocrit pre- and post-infusion. No serious or
`potentially allergic events were noted with either product
`in
`the IV
`study. Furthermore,
`there was no
`immunological response to either product, and dose level
`did not influence the study outcomes. Serum albumin,
`colloid osmotic pressure changes and haematocrit ratio
`were as expected, with no differences between rHA and
`HSA. The study concluded that rHA and HSA exhibited
`similar
`safety,
`tolerability and pharmacokinetic/
`pharmacodynamic profiles, with no evidence of any
`immunological response.
`
`Another study found Recombumin® rHA to be
`equivalent to native HSA in its capacity to protect
`immunological, biological and biochemical properties in
`preparations of thyroid stimulating hormone (TSH),
`interleukin 15 (IL-15) and granulocyte colony-
`stimulating factor (G-CSF). The study recommended
`the use of rHA in the preparation of lyophilised products
`and reference agents (12).
`
`COMMERCIAL VALIDATION
`AND REGULATORY STATUS
`The first and only commercially available recombinant
`human albumin whose use has been approved by the
`FDA and EMEA in the manufacture of biotherapeutics,
`Recombumin® is used in the production of childhood
`vaccines for measles, mumps and rubella {M-M-R® II
`(Merck & Co) and M-M-RVAXPRO® (Sanofi Pasteur
`MSD)} and is supported by a Type V Biologics Master
`File (BMF) with the US FDA.
`
`CONCLUSION
`HSA is a well-characterised protein that is known to have an
`important therapeutic role and has been used previously as
`an excipient for biotherapeutics. Most recently, its use as a
`drug stabiliser has been met with increasing regulatory
`resistance due to the perceived risk of disease transmission.
`To address these concerns and enable the biotherapeutic
`industry to rediscover the benefits of albumin as an excipient,
`recombinant albumin (rHA) has been developed. At
`Novozymes Delta, we have successfully developed a robust
`industrial-scale manufacturing process using a proprietary
`S. cerevisae based expression system that produces
`Recombumin®, a highly consistent and pure animal-, virus-
`and prion-free recombinant human albumin product. Being
`structurally identical to HSA and with a similar safety,
`tolerability and pharmacokinetic/ pharmacodynamic profile,
`Recombumin® is now supplied worldwide for use in the
`manufacture of better biotherapeutics.
`
`The authors can be contacted at DVJM@novozymes.com,
`DPRS@novozymes.com and MDEV@novozymes.com
`
`References
`
`1. Emersen T.E, JR. Unique features of albumin; a brief
`review. Critical Care Medicine. 17; pp690-694, 1989
`2. Peters T, All about Albumin, Academic Press, ISBN
`0-12552110-3, 1996
`3. Matejtschuk R et al, Br J Anaesth2000; 85: 887
`4. Cohn EJ, Chem Rev28: pp395-417, 1940
`5. EMEA, CHMP position statement on Creutzfeldt-Jakob
`disease and plasma-derived and urine derived
`medicinal products, London, 23rd June, 2004
`6. Fleer R. et al, Biotechnology9: p968 1991
`7. Cregg J.M. et al, Mol. Biotechnol. 16: p23 2000
`8. Kang H.A. et al, Biotechnol. Bioeng. 76: p175 2001
`9. Wigley, A. et al, GEN27: p2 2007
`10. Dodsworth N. et al, Biotechnol Appl Biochem.
`24 (Pt 2) pp171-176 1996
`11. Dietrich Bosse, MD et al, J Clin Pharmacol2005;
`45: pp57-67
`12. Tarelli E. et al. Biologicals, 1998 Dec 26(4): 331-346
`
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