BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY - PART 4 pps

conditions of low pH or elevated temperature (it is stable for over 10 h at 608C). It also displays excellent solubility characteristics. It is postulated that albumin stabilizers exert their stabilizing influences by both direct and indirect means. Certainly, it helps decrease the level of surface adsorption of the active biopharmaceutical to the internal walls of final product containers. It also could act as an alternative target, e.g. for traces of proteases or other agents that could be deleterious to the product. It may also functi on to directly stabilize the native conformation of many proteins. It has been shown to be an effective cryoprotectant for several biopharmaceuticals (e.g. IL-2, tPA and various interferon preparations), helping to minimize potentially detrimental effects of the freeze-drying process on the product. However, the use of HSA is now discouraged, due to the possibility of accidental transmission of blood-borne pathogens. The use of recombinant HSA would overcome such fears. . Various amino acids are also used as stabilizing agents for some biopharmaceutical products (Table 3.24). Glycine is most often employed and it (as well as other amino acids) has been found to help stabilize various interferon preparations, as well as erythropoietin, factor VIII, THE DRUG MANUFACTURING PROCESS 151 Table 3.22. Some major excipient groups that may be added to protein-based biopharmaceuticals in order to stabilize the biological activity of the finished product Serum albumin Various individual amino acids Various carbohydrates Alcohols and polyols Surfactants Table 3.23. Various biopharmaceutical preparations for which human serum albumin (HSA) has been described as a potential stabilizer a- and b-Interferons Tissue plasminogen activator g-Interferon Tumour necrosis factor Interleukin-2 Monoclonal antibody preparations Urokinase g-Globulin preparations Erythropoietin Hepatitis B surface antigen Table 3.24. Amino acids, carbohydrates and polyols that have found most application as stabilizers for some biopharmaceutical preparations Amino acids Carbohydrates Polyols Glycine Glucose Glycerol Alanine Sucrose Mannitol Lysine Trehalose Sorbitol Threonine Maltose Polyethylene glycol urokinase and arginase. Amino acids are generally added to final product at concentrations of 0.5–5%. They appear to exert their stabilizing influence by various means, including reducing surface adsorption of product, inhibiting aggregate form ation, as well as directly stabilizing the conformation of some proteins, particularly against heat denaturation. The exact molecular mechanisms by which such effects are achieved remain to be elucidated. . Several polyols (i.e. molecules displaying multiple hydroxyl groups) have found application as polypeptide-stabilizing agents. Polyols include substances such as glycerol, mannitol, sorbitol and polyethylene glycol, as well as inositol (Table 3.24 and Figure 3.26). A subset of polyols are the carbohydrates, which are listed separately (and thus somewhat artificially) from polyols in Table 3.24. Various polyols have been found to directly stabilize proteins in solution, while carbohydrates in particular are also often added to biopharmaceutical products prior to freeze-drying, in order to provide physical bulk to the freeze-dried cake. . Surfactants are well-known protei n denatura nts. However, when sufficiently dilute, some surfactants (e.g. polysorbate) exert a stabilizing influence on some protein types. Proteins display a tendency to aggregate at interfaces (air–liquid or liquid–liquid), a process which often promotes their denaturation. Addition of surfactant reduces the surfac e tension of aqueous solutions and often increases the solubility of proteins dissolved therein. This helps to reduce the rate of protein denaturation at interfaces. Polysorbate, for example, is included in some g-globulin preparations and in the therapeutic monoclonal antibody, OKT-3 (Chapter 10). 152 BIOPHARMACEUTICALS Figure 3.26. Structure of some polyols sometimes used to stabilize proteins Final product fill An overview of a typical final product filling process is presented in Figure 3.27. The bulk final product firstly undergoes QC testing to ensure its compliance with bulk product specifications. While implementation of GMP during manufacturing will ensure that the product carries a low microbial load, it will not be ster ile at this stage. The product is then passed through a (sterilizing) 0.22 mm filter, Figure 3.28. The sterile product is housed (temporarily) in a sterile product-holding tank, from where it is aseptically filled into pre-sterilized final product containers (usually glass vials). The filling pro cess normally employs highly automated liquid filling systems. All items of equipment, pipework, etc. with which the sterilized product comes into direct contact must obviously themselves be sterile. Most such equipment items may be sterilized by autoclaving, and be aseptically assembled prior to the filling operation (which is undertaken under Grade A laminar flow conditions). The final product containers must also be pre-sterilized. This may be achieved by autoclaving, or passage through special equipment which subjects the vials to a hot WFI rinse, followed by sterilizing dry heat and UV treatment. If the product can be filled into plastic-based containers, alternative ‘blow–fill–seal’ systems may be used, Figure 3.29; as its name suggests, such equipment first moulds plastic into the final product container (the moulding conditions ensure container sterility), followed immediately by THE DRUG MANUFACTURING PROCESS 153 Figure 3.27. Final product filling. The final bulk product (after addition of excipients and final product QC testing) is filter sterilized by passing through a 0.22 mm filter. The sterile product is aseptically filled into (pre-sterilized) final product containers under grade A laminar flow conditions. Much of the filling operation uses highly automated filling equipment. After filling, the product container is either sealed (by an automated aseptic sealing system) or freeze-dried first, followed by sealing 154 BIOPHARMACEUTICALS Figure 3.28. Photographic representation of a range of filter types and their stainless steel housing. Most filters used on an industrial scale are of a pleated cartridge design which facilitates housing of maximum filter area within a compact space (a). These are generally housed in stainless steel housing units (b). Some process operations, however, still make use of flat (disc) filters, which are housed in a tripod-based stainless steel housing (c). Photos courtesy of Pall Life Sciences, Ireland automated filling of sterile product into the container and its subsequent sealing. In this way operator intervention in the fillin g process is minimized. Freeze-drying Freeze-drying (lyophilization) refers to the removal of solvent directly from a solution while in the frozen state. Removal of water directly from (frozen) biopharmaceutical products via THE DRUG MANUFACTURING PROCESS 155 Figure 3.29. Photographic representation of a blow–fill–seal machine, which can be particularly useful in the aseptic filling of liquid products (refer to text for details). While used fairly extensively in facilities manufacturing some traditional parenteral products, this system has not yet found application in biopharmaceutical manufacture. This is due mainly to the fact that many biopharmaceutical preparations are sold not in liquid, but in freeze-dried format. Also, some proteins display a tendancy to adsorb onto plastic surfaces. Photo courtesy of Rommelag a.g., Switzerland lyophilization yields a powdered product, usually displaying a water content of the order of 3%. In general, removal of the solvent water from such products greatly reduces the likelihood of chemical/biological-mediated inactivation of the biopharmaceutical. Freeze-dried biopharma- ceutical products usually exhibit longer shelf-lives than products sold in solution. Freeze drying- is also recognized by the regula tory authorities as being a safe and acceptable method of preserving many parenteral products. Freeze-drying is a relatively gentle way of removing water from proteins in solution. However, this process can promote the inactivation of some protein types and specific excipients (cryoprotectants) are usually added to the product in order to minimize such inactivation. Commonly used cryoprotectants include carbohydrates, such as glucose and sucrose; proteins, such as human serum albumin; and amino acids, such as lysine, arginine or glutamic acid. Alcohols/polyols have also found some application as cryoprotectants. The freeze-drying process is initiated by the freezing of the bioph armaceutical product in its final product containers. As the temperature is decreased, ice crystals begin to form and grow. This results in an effective concentration of all the solutes present in the remaining liquid phase, including the protein and all added excipients, e.g. the concentration of salts may increase to levels as high as 3 M. Increased solute concentration alone can accelerate chemical reactions damaging to the protein product. In addition, such concentration effectively brings individual protein molecules into more intimate contact with each other, which can prompt protein– protein interactions and, hence, aggregation. As the temperature drops still lower, some of the solutes present may also crystallize, thus being effectively removed from the solution. In some cases, individual buffer constituents can crystallize out of solution at different temperatures. This will dramatically alter the pH values of the remaining solution and, in this way, can lead to protein inactivation. As the temperature is further lowered, the viscosity of the unfrozen solution increases dramatically until molecular mobility effectively ceases. This unfrozen solution will contain the protein, as well as some excipients and (at most) 50% water. As molecular mobility has 156 BIOPHARMACEUTICALS THE DRUG MANUFACTURING PROCESS 157 Figure 3.30. Photographic representation of (a) lab-scale, (b) pilot-scale and (c) industrial-scale freeze driers. Refer to text for details. Photo courtesy of Virtis, USA effectively stopped, chemical reactivity also all but ceases. The consistency of this ‘solution’ is that of glass, and the temperature at which this is attained is called the glass transition temperature, Tg’. For most protein solutions, Tg’ values reside between 7408C and 7608C. The primary aim of the init ial stages of the freeze-drying process is to decrease the product temperature below that of its Tg’ value as quickly as possible. The next phase of the freeze-drying process entails the application of a vacuum to the system. When the vacuum is established, the temperature is increased, usually to temperatures slightly in excess of 08C. This promotes sublimation of the crystalline water, leaving behind a powdered cake of dried material. Once satisfactory drying has been achieved, the product container is sealed. The drying chamber of industrial-scale freeze-dryers usually opens into a clean room (Figure 3.27). This facilitates direct transfer of the product-containing vials into the chamber. Immediately prior to filling, rubber stoppers are usually partially inserted into the mouth of each vial in such a way as not to hinder the outward flow of water vapour during the freeze- drying process. The drying chamber normally contains several rows of shelves, each of which can accommodate several thousand vial s (Figure 3.30). These shelves are wired to allow their electrical heating and cooling and their upward or downward movement. After the freeze-drying cycle is complete (which can take 3 days or more), the shelves are then moved upwards. As each shelf moves up, the partially-inserted rubber seals are inserted fully into the vial mouth as they come in contact with the base plate of the shelf immediately above them. After product recovery, the empty chamber is closed and is then heat-sterilized (using its own chamber-heating mechanism). The freeze-drier is then ready to accept its next load. Labelling and packing After the product has been filled (and sealed) in its final product container, it is immediately placed under quarantine. QC personnel then remove representative samples of the product and carry out tests to ensure conformance to final product specification. The most important specifications will relate to product potency, sterility and final volume fill, as well as the absence of endotoxin or other potentially toxic substances. Detection and quantification of excipients added will also be undertaken. Only after QC personnel are satisfied that the product meets these specifications will it be labelled and packed. These operations are highly automated. Labelling, in particular, deserves special attention. Mislabelling of product remains one of the most common reasons for product recall. This can occur relatively easily, particularly if the facility manufactures several different products, or even a single product at several different strengths. Information presented on a label should normally include: . name and strength/potency of the product; . specific batch number of the product; . date of manufacture and expiry date; . storage conditions required. Additional information often presented includes the name of the manufacturer, a list of excipients included and a brief summary of the correct mode of product usage. When a batch of product is labelled and packed, and QC personnel are satisfied that labelling and packing are completed to specification, the QC manager will write and sign a Certificate of Analysis. This details the pre-defined product specifications and confirms conformance of the 158 BIOPHARMACEUTICALS actual batch of product in question to these specifications. At this point, the product, along with its Certificate of Analysis, may be shipped to the customer. ANALYSIS OF THE FINAL PRODUCT All pharmaceutical finished products undergo rigorous QC testing, in order to confirm their conformance to pre-determined specifications. Potency testing is of obvious importance, ensuring that the drug will be efficacious when administered to the patient. A prominent aspect of safety testing entails analysis of product for the presence of various potential contaminants. The range and complexity of analytical testing undertaken for reco mbinant biopharmaceu- ticals far outweighs those undertaken with regard to ‘traditional’ pharmaceuticals manufactured by organic synthesis. Not only are proteins (or additional likely biopharmaceuticals, such as nucleic acids; Chapter 11) much larger and more structurally complex than traditional low molecular mass drugs, their production in biological systems renders the range of potential contaminants far broader (Table 3.25). Recent advances in analytical techniques renders practical the routine analysis of complex biopharmaceutical products. An overview of the range of finished-product tests of recombinant protein biopharmaceuticals is outlined below. Explanation of the theoretical basis underpinning these analytical methodologies is not undertaken, as this would considerably broaden the scope of the text. Appropriate references are provided in Further Reading at the end of the chapter for the interested reader. Protein-based contaminants Most of the chromatographic steps undertaken during downstream processing are specifically included to separate the protein of interest from additional contaminant proteins. This task is not an insubstantial one, particularly if the recombinant protein is expressed intracellularly. In addition to protein impurities emanating directly from the source material, other proteins may be introduced during upstream or downstream processing. For example, animal cell culture media is typically supplemented with bovine serum/fetal calf serum (2–25%), or with a defined cocktail of various regulatory proteins required to maintain and stimulate growth of these cells. Downstream processing of intracellular microbial proteins often requires the addition of endonucleases to the cell homogenate to degrade the large quantity of DNA liberated upon THE DRUG MANUFACTURING PROCESS 159 Table 3.25. The range and medical significance of potential impurities present in biopharmaceutical products destined for parenteral administration. Reproduced by permission of John Wiley & Sons Ltd from Walsh & Headon (1994) Impurity Medical consequence Microorganisms Potential establishment of a severe microbial infection — septicaemia Viral particles Potential establishment of a severe viral infection Pyrogenic substances Fever response which, in serious cases, culminates in death DNA Significance is unclear — could bring about an immunological response Contaminating proteins Immunological reactions. Potential adverse effects if the contaminant exhibits an unwanted biological activity cellular disruption (DNA promotes increased solution viscosity, rendering processing difficult; viscosity, being a function of the DNA’s molecular mass, is reduced upon nuclease treatment). Minor amounts of protein could also potentially enter the product stream from additional sources, e.g. protein shed from production personnel. Implementation of GMP should, however, minimize contamination from such sources. The clinical significance of protein-based impurities relates to: (a) their potential biological activities; and (b) their antigenicity. While some contaminants may display no undesirable biological activity, others may exhibit activities deleterious to either the product itself (e.g. proteases which could modify/degrade the product) or the recipient patient (e.g. the presence of contaminating toxins). Their inherent immunogenicity also renders likely an immunological reaction against protein- based impurities upon product administration to the recipient patient. This is particularly true in the case of products produced in microbial or other recombinant systems (i.e. most biopharmaceuticals). While the product itself is likely to be non-immunogenic (being coded for by a human gene), contaminant proteins will be endogenous to the host cell, and hence foreign to the human body. Administration of the product can e licit an immune response against the contaminant. This is particularly likely if a requirement exists for ongoing, repeat product administration (e.g. administration of recombinant insulin). Immunological activation of this type could also potentially (and more seriously) have a sensitizing effect on the recipient against the actual protein product. In add ition to distinct gene products, modified forms of the protein of interest are also considered impurities, rendering desirable their removal from the product stream. While some such modified forms may be innocuous, others may not. Modified product ‘impurities’ may compromise the product in a number of ways, e.g: . biologically inactive forms of the product will reduce overall product potency; . some modified product forms remain biologically active but exhibit modified pharmacoki- netic characteristics (i.e. timing and duration of drug action); . modified product forms may be immunogenic. Altered forms of the protein of interest can be generated in a number of ways by covalent and non-covalent modifications (see e.g. Table 3.20). Removal of altered forms of the protein of interest from the product stream Modification of any protein will generally alter some aspect of its physicochemical characteristics. This facilitates removal of the modified form by standard chromatographic techniques during downstream processing. Most downst ream procedures for protein-based biopharmaceuticals include both gel-fi ltration and ion-exchange steps. Aggregated forms of the product will be effectively removed by gel-filtration (because they now exhibit a molecular mass greater by several orders of magnitude than the native product). This technique will equally efficiently remove extensively proteolysed forms of the product. Glycoprotein variants whose carbohydrate moieties have been extensively degraded will also likely be removed by gel- filtration (or ion-exchange) chromatography. Deamidation and oxidation will generate product variants with altered surface charge characteristics, often rendering their removal by ion- exchange relatively straightforward. Incorrect disulphide bond formation, parti al denaturation and limited proteolysis can also alter the shape and surface charge of proteins, facilitating their 160 BIOPHARMACEUTICALS [...]... heated to 908C (promotes 180 BIOPHARMACEUTICALS denaturation, forming single strands) and incubated with the baked filter for several hours at 40 8C Lowering the temperature allows re-annealing of single strands via complementary basepairing to occur Labelled DNA will re-anneal with any complementary DNA strands immobilized on the filter After the filter is washed (to remove non-specifically bound radiolabelled... Biotechnology Wiley, Chichester 186 BIOPHARMACEUTICALS Walsh, G (2002) Proteins: Biochemistry and Biotechnology Wiley, Chichester Whyte, W (2001) Cleanroom Technology Wiley, Chichester Articles Sources of biopharmaceuticals and upstream processing Baneyx, F (1999) Recombinant protein expression in E coli Curr Opin Biotechnol 10, 41 1 42 1 Carrio, M & Villaverde, A (2002) Construction and deconstruction of bacterial... 15–20 17.5 30–35 TNF-a GM-CSF G-CSF EPO TPO IGF-1 52* 22 21 36 60 7.6 EGF NGF Insulin hGH FSH LH 6 26 5.7 22 34 28.5 *Biologically active, trimeric form IFN ¼ interferon; IL ¼ interleukin; TNF ¼ tumour necrosis factor; GM-CSF ¼ granulocyte macrophage colony stimulating factor; G-CSF ¼ granulocyte colony stimulating factor; EPO ¼ erythropoietin; TPO ¼ thrombopoietin; IGF ¼ insulin-like growth factor;... undertaken in 15–30 min, and on-line detection at the end of the column allows automatic detection and quantification of eluting bands THE DRUG MANUFACTURING PROCESS 167 Figure 3.33 Photograph of a typical HPLC system (the Hewlett-Packard HP1100 system) Photo courtesy of Hewlett-Packard GmbH, Germany The speed, sensitivity, high degree of automation and ability to directly quantify protein bands renders this... elute from gel-filtration columns much later than contaminating THE DRUG MANUFACTURING PROCESS 179 Table 3.28 The molecular mass of some polypeptide biopharmaceuticals Many are glycosylated, thereby exhibiting a range of molecular masses due to differential glycosylation Protein Molecular mass (kDa) Protein Molecular mass (kDa) Protein Molecular mass (kDa) IFN-a IFN-b IFN-g IL-2 IL-1 IL-12 20–27 20 20–25... 81– 84 Mann, M et al (2001) Analysis of proteins and proteomes by mass spectrometry Ann Rev Biochem 70, 43 7 47 3 Ullao-Aguirre, A et al (1999) Role of glycosylation in function of follicle-stimulating hormone Endocrinology 11(3), 205–215 Wang, W (2000) Lyophilization and development of solid protein pharmaceuticals International J Pharmaceut 203(1– 2), 1–60 Wei, W (1999) Instability, stabilization and. .. any impurities Batch-to-batch variation can also be assessed by comparison of chromatograms from different product runs Ion-exchange chromatography (both cation and anion) can also be undertaken in HPLC format Although not as extensively employed as RP or SE systems, ion-exchange-based systems are of use in analysing for impurities unrelated to the product, as well as detecting and quantifying deamidated... in great detail by X-ray crystallography or NMR spectroscopy, routine application of such techniques to biopharmaceutical manufacture is impractical from both a technical and economic standpoint Limited analysis of protein secondary and tertiary structure can, however, be more easily undertaken using spectroscopic methods, particularly far-UV circular dichroism More recently proton-NMR has also been... (19 94) Pharmaceutical Press, Wallingford, UK The Rules Governing Medicinal Products in the European Community (a multi-volume work; various publication dates) European Commission, Brussels The United States Pharmacopoeia, 26-NF 21 (2003) United States Pharmacopoeial Convention, USA Venn, R (2000) Principles and Practice of Bioanalysis Taylor and Francis, London Walsh, G & Headon, D (19 94) Protein Biotechnology. .. prompting a false-positive result; sub-clinical infection/poor overall animal health can also lead to false-positive results; use of different rabbit colonies/breeds can yield variable results Another issue of relevance is that certain biopharmaceuticals (e.g cytokines such as 1L-1 and TNF; Chapter 5), themselves, induce a natural pyrogenic response This rules out use of the rabbit-based assay for . a potential stabilizer a- and b-Interferons Tissue plasminogen activator g-Interferon Tumour necrosis factor Interleukin-2 Monoclonal antibody preparations Urokinase g-Globulin preparations Erythropoietin. closed and is then heat-sterilized (using its own chamber-heating mechanism). The freeze-drier is then ready to accept its next load. Labelling and packing After the product has been filled (and. modified form by standard chromatographic techniques during downstream processing. Most downst ream procedures for protein-based biopharmaceuticals include both gel-fi ltration and ion-exchange steps.