pharma-supplementsSPRING 2004available by subscription onlyNOWEffective DirectCompression: UsingExcipients as bindersRecombinant ProteinsAs ExcipientsLatest Technology& New Products
pharma-supplementsNOW SPRING 2004Biotherapeutics formulation constraintsThe 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 stableand 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 orisotonicity. Thereafter, if the protein is not stable under theseconditions, 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 biotherapeuticsSugars Trehalose Amino Acids HistidineMannoseAspartic acidRecombinantProteins asExcipientsBy S Berezenko, Research Director, Delta BiotechnologyFormulation requirements – small moleculesversus biotherapeuticsThe term excipient is defined as a raw material thatis purposely added to a pharmaceutical; it is aninactive material that can perform a number offunctions but the ultimate aim is to use them in thepreparation of a stable drug formulation that has thedesired shelf life and bioavailability. There are significantdifferences between the functions of excipientsin small molecule pharma and biotherapeutic formulations.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 excipientsin small molecule pharma are not what isrequired for biotherapeutics (proteins, peptides andvaccines). For biotherapeutics, the end point of astable, safe formulation with the desired bioavailability,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 interfaces;additionally, pH and buffer components caninactivate these molecules. Biotherapeutics alsohave more mechanisms of decomposition on top ofthe usual drug degradation pathways such asoxidation, racemisation and hydrolysis: these includedisulphide exchange, beta elimination, aggregationand deamidation. Whilst there is no typical formulationfor biotherapeutics, there are some generalitiesthat can be considered.SucroseDextroseAlanineGlutamic acidPolyols Sorbitol Polymers PolysorbateMannitolGlycerolAlbuminGelatinThe 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 denaturationat 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. Thisfamily of detergents is based on a polyoxyethylene backbone witha sorbitan and fatty acid side chain, Polysorbate 20, 40 and 80respectively, 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 reducethe risk of protein denaturation by surface adsorption. Incomparison with expensive biotherapeutics (which have been preparedvia 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.
pharma-supplementsNOW SPRING 2004Perceived problems with animal- andhuman-derived proteinsThere 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 pharmacopoeialpurity requirement of only ≥96%, (USP), the rest of the proteinpresent 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 somewhatillogical to adulterate them with such impure excipients.One approach, taken to avoid the issues surrounding the use ofprotein 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 product.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 technologyexcipientsAn alternative solution to the costly and time consuming search for anew formulation has emerged from the same source as the biotherapeutics,namely recombinant DNA technology. Using genetically modifiedyeast 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 pastorisand 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 performeda clinical safety study of recombinant human gelatin, findingthe study material safe and well tolerated.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 toleratedin both an i.m. repeatFigure 2dose 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 devicesas an alternative to HSA inin vitro fertilisation reagentsin the manufacturing process for avaccine as a stabiliser replacing HSAIn conclusion, the advent of recombinant excipients offers theopportunity to use a potentially safer, more consistent and purerprotein as an excipient rather than the current sources of formulationproteins being used now.ReferencesAguzzi, A and Glatzel, M. (2004) The Lancet 363 9407 411-412Curry, S., Mandelkow, H., Brick, P. and Franks, N. (1998)Nature Structural Biology 5, 827-835Dodsworth, N., Harris, R., Denton, K., Woodrow, J., Wood,P.C and Quirk, A (1996) Biotechnol. Appl. Biochem. 24 171-176European Commission (2001), The safety with regard to TSE risksof gelatine derived from ruminant bones or hides from cattle,sheep or goatsHe, M.X. and Carter, D.C. (1992) Nature 358 209-215Ruiz, L., Reyes, N., Duany, L., Franco, A,. Aroche, K. and Rando,E.H. (2003) Int. J. Pharmaceutics 264 57-72Tarelli E, Mire-Sluis A, Tivnann HA et al (1998)Biologicals 26 331-346The other recombinant excipient, Recombumin ® , is further ahead indevelopment than the recombinant gelatin and is available as acommercial 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 completelyfree from the use of animal or human derived products. Theproduct is structurally identical HSA but significantly purer (Figure 1).Figure 1