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Effective DirectCompression: UsingExcipients as binders

Recombinant ProteinsAs Excipients

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RecombinantProteins asExcipientsBy S Berezenko, Research Director, Delta Biotechnology

Formulation requirements – small molecules versus biotherapeutics

The term excipient is defined as a raw material thatis 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 thepreparation of a stable drug formulation that has thedesired shelf life and bioavailability. There are signifi-cant differences between the functions of excipientsin small molecule pharma and biotherapeutic formu-lations. In the former, the role of an excipient can beto aid tablet formation by, for example, affectingcompressibility, act as a lubricant, disintegrant, filleror glidant. The aforementioned functions of excipi-ents in small molecule pharma are not what isrequired for biotherapeutics (proteins, peptides andvaccines). For biotherapeutics, the end point of astable, safe formulation with the desired bioavailabil-ity, is still a necessity but the challenges offered bythe formulation of proteins are different.

Proteins are often sensitive to heat, denaturationfrom liquid shear or denaturation at air-liquid inter-faces; additionally, pH and buffer components caninactivate these molecules. Biotherapeutics alsohave more mechanisms of decomposition on top ofthe usual drug degradation pathways such as oxidation, racemisation and hydrolysis: these includedisulphide exchange, beta elimination, aggregationand deamidation. Whilst there is no typical formula-tion for biotherapeutics, there are some generalitiesthat can be considered.

Biotherapeutics formulation constraints

The pH of the formulation has two significant constraints, the obviousone 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 willresult in the patient suffering injection site pain during administrationof 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 theprotein. Some commonly used excipients are in Table 1 and includeamino 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 aprotein as well as stabilising the product in solution. One specificproblem 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-ionicdetergent 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 amphipathicdetergent molecules gather at the air liquid interface, with thehydrophobic moiety in the air and the hydrophilic tail in the aqueousenvironment, thus preventing protein under going denaturation at thisinterface.

For many proteins, the combinations of pH, detergents and lowmolecular weight excipients may still not make an ideal formulation.One aspect of this is that biotherapeutics are very often required invery small therapeutic doses and can be denatured by surfaceadsorption to glass containers or container closures such as butylsepta. This has resulted in many formulations requiring a bulkingagent; in particular two proteins have been used extensively, notablygelatin and human serum albumin (HSA). These proteins can beadded in large excess over the active protein and thus reduce the risk of protein denaturation by surface adsorption. Incomparison 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 inlarge quantities. Gelatin is derived from collagen extracted from thehides and bones of cows or pigs. HSA is the most abundant proteinin blood plasma and is fractionated or purified from donated blood. However, this simple approach of adding animal- and human-derivedproteins to formulations is now being challenged.

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Perceived problems with animal- and human-derived proteins

There are two problems, firstly these proteins are heterogeneous andrelatively impure. Gelatin is a heterogeneous mixture of polypeptidesand 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 plasmaproteins that remain from the purification, additionally these otherproteins are denatured during the pasteurisation process that HSAfinal product undergoes.

The second issue is the drive to remove all animal and human derivedproducts from pharmaceuticals, caused by the advent of “mad cow”disease (Bovine Spongiform Encephalopathy) and the prion diseasein humans, variant Creutzfeldt-Jakob Disease (vCJD), which has beenlinked to eating BSE infected products. This has raised the questionas to whether prions or other viral diseases could be transmitted viagelatin (European Commission, 2001) or blood (Aguzzi and Glatzel,2004) and the HSA derived from it. Although there is no evidence thatthis can happen it has pushed formulation scientists to think twiceabout using these proteins as excipients. Also, given the very highpurity 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 andremove the protein from the product. Factor VIII (AntihemophilicFactor) from Bayer HealthCare, USA is now a third generation prod-uct. It began as a plasma derived product, and was thenmanufactured using recombinant DNA technology, but still using HSAas an excipient. Now it is manufactured using the same recombinantDNA technology, but is formulated with sucrose thus avoiding theaddition of protein excipients (Kogenate® FS). A similar example hasbeen the removal of HSA from a formulation of recombinant humaninterferon-α-2 (Ruiz et al 2003).

A new approach - Recombinant DNA technologyexcipients

An alternative solution to the costly and time consuming search for anew 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 recombinantgelatin and recombinant human albumin (Recombumin®) for use asexcipients (Dodsworth et al, 1996 and Tarelli et al, 1998).

Recombinant human gelatins (FibroGen, South San Francisco,California) are engineered from specific segments of human collagengenes. They are expressed in the methylotrophic yeast Pichia pas-toris and manufactured avoiding the use of animal or human-derivedmaterials. FibroGen’s proprietary technology describes the productionof discrete, reproducible batches of gelatin fragments with specificmolecular weights, providing customers with the ability to select aproduct optimised for specific applications. FibroGen has also per-formed a clinical safety study of recombinant human gelatin, findingthe study material safe and well tolerated.

The other recombinant excipient, Recombumin®, is further ahead indevelopment than the recombinant gelatin and is available as a commercial product. Recombumin® is manufactured by DeltaBiotechnology Ltd, (Nottingham, England, a subsidiary of AventisPharma). Recombumin® is derived from the yeast Saccharomycescerevisiae and is manufactured to cGMP using a process that is com-pletely free from the use of animal or human derived products. Theproduct is structurally identical HSA but significantly purer (Figure 1).

The characterisation of the recombinant albumin molecule has beentaken to the level of x-ray crystallography studies using crystalsgrown under zero gravity on the NASA Space Shuttle (He and Carter,1992) and laboratory studies have crystallised Recombumin® in thepresence of ligands (Curry et al1998) (Figure 2).

Recombumin® is an ultra-highpurity product, with residualyeast content of less than 0.15ppm. A comparative clinicaltrial using Recombumin® andHSA was performed.Recombumin® was well toler-ated in both an i.m. repeatdose study (5 x 65mg) evaluated in 500 subjects (250 rHA, 250 HSA),as well as in an escalating dose i.v. study administering a maximumof 50g and a cumulative dose of 80g. Currently, Recombumin® isbeing 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 formulationproteins 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