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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 elicit 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 addition 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 pharmacokinetic 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 downstream procedures for protein-based biopharmaceuticals include both gel-filtration 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, partial denaturation and limited proteolysis can also alter the shape and surface charge of proteins, facilitating their
THE DRUG MANUFACTURING PROCESS 161
Table 3.26. Methods used to characterize (protein-based) finished product biopharmaceuticals. An overview of most of these methods is presented over the next several sections of this chapter
Non-denaturing gel electrophoresis Denaturing (SDS) gel electrophoresis 2-D electrophoresis Capillary electrophoresis Peptide mapping
HPLC (mainly reverse phase—HPLC)
Amino acid analysis
Circular dichromism studies
Bioassays and immunological assays
removal from the product by ion-exchange or other techniques, such as hydrophobic interaction chromatography.
The range of chromatographic techniques now available, along with improvements in the resolution achievable using such techniques, renders possible the routine production of protein biopharmaceuticals that are in excess of 97-99% pure. This level of purity represents the typical industry standard with regard to biopharmaceutical production.
A number of different techniques may be used to characterize protein-based biopharmaceu-tical products, and to detect any protein-based impurities that may be present in that product (Table 3.26). Analyses for non-protein-based contaminants are described in subsequent sections.