Proteinbased 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 are typically supplemented with bovine serum/foetal calf serum (2-25 per cent), 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

Pharmaceutical biotechnology: concepts and applications Gary Walsh © 2007 John Wiley & Sons, Ltd ISBN 978 0 470 01244 4 (HB) 978 0 470 01245 1 (PB)

Table 7.1 The range and medical significance of potential impurities present in biopharmaceutical products destined for parenteral administration

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 that, 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

endonuleases to the cell homogenate to degrade the large quantity of DNA liberated upon 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 good manufacturing practice (GMP), however, should minimize contamination from such sources.

The clinical significance of protein-based impurities relates to (a) their potential biological activities and (b) their antigenicity. Whereas some contaminants may display no undesirable biological activity, others may exhibit activities deleterious to either the product itself (e.g. proteases that could modify/degrade the product) or the recipient patient (e.g. the presence of contaminating toxins).

Their inherent immunogenicity also renders likely and 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 biopharmaceu-ticals). Although the product itself is likely to be non-immunogenic (usually 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. Although 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 (e.g. see Table 6.5).

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