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 biopharma-ceuticals include both gel-filtration and ion-exchange steps (Chapter 6). 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 also 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 disulfide bond formation, partial denaturation and limited proteolysis can also alter the shape and surface charge of proteins, facilitating their 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 biop-harmaceuticals which are in excess of 97-99 per cent 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 biopharmaceutical products, and to detect any protein-based impurities that may be present in that product (Table 7.2). Analysis for non-protein-based contaminant is described in subsequent sections.

7.3.1 Product potency

Any biopharmaceutical must obviously conform to final product potency specifications. Such specifications are usually expressed in terms of 'units of activity' per vial of product (or per thera-

Table 7.2 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

Two-dimensional electrophoresis

Capillary electrophoresis

Peptide mapping

HPLC (mainly RP-HPLC)

Isoelectric focusing

Mass spectrometry

Amino acid analysis

N-terminal sequencing

Circular dichroism studies

Bioassays and immunological assays peutic dose, or per milligram of product). A number of different approaches may be undertaken to determine product potency. Each exhibits certain advantages and disadvantages.

Bioassays represent the most relevant potency-determining assay, as they directly assess the biological activity of the biopharmaceutical. Bioassay involves applying a known quantity of the substance to be assayed to a biological system that responds in some way to this applied stimulus. The response is measured quantitatively, allowing an activity value to be assigned to the substance being assayed.

All bioassays are comparative in nature, requiring parallel assay of a 'standard' preparation against which the sample will be compared. Internationally accepted standard preparations of most biopharmaceuticals are available from organizations such as the World Health Organization (WHO) or the United States Pharmacopeia.

An example of a straightforward bioassay is the traditional assay method for antibiotics. This usually entailed measuring the zone of inhibition of microbial growth around an antibiotic-containing disc, placed on an agar plate seeded with the test microbe. Bioassays for modern biop-harmaceuticals are generally more complex. The biological system used can be whole animals, specific organs or tissue types, or individual mammalian cells in culture.

Bioassays of related substances can be quite similar in design. Specific growth factors, for example, stimulate the accelerated growth of specific animal cell lines. Relevant bioassays can be undertaken by incubation of the growth-factor-containing sample with a culture of the relevant sensitive cells and radiolabelled nucleotide precursors. After an appropriate time period, the level of radioactivity incorporated into the DNA of the cells is measured. This is a measure of the bio-activity of the growth factor.

The most popular bioassay of EPO involves a mouse-based bioassay (EPO stimulates red blood cell production, making it useful in the treatment of certain forms of anaemia; Chapter 10). Basically, the EPO-containing sample is administered to mice along with radioactive iron (57Fe). Subsequent measurement of the rate of incorporation of radioactivity into proliferating red blood cells is undertaken. (The greater the stimulation of red blood cell proliferation, the more iron taken up for haemoglobin synthesis.)

One of the most popular bioassay for interferons is termed the 'cytopathic effect inhibition assay'. This assay is based upon the ability of many interferons to render animal cells resistant to viral attack. It entails incubation of the interferon preparation with cells sensitive to destruction by a specific virus. That virus is then subsequently added, and the percentage of cells that survive thereafter is proportional to the levels of interferon present in the assay sample. Viable cells can assimilate certain dyes, such as neutral red. Addition of the dye followed by spectrophotometric quantitation of the amount of dye assimilated can thus be used to quantitate percentage cell survival. This type of assay can be scaled down to run in a single well of a microtitre plate. This facilitates automated assay of large numbers of samples with relative ease.

Although bioassays directly assess product potency (i.e. activity), they suffer from a number of drawbacks, including:

• Lack of precision. The complex nature of any biological system, be it an entire animal or individual cell, often results in the responses observed being influenced by factors such as metabolic status of individual cells, or (in the case of whole animals) subclinical infections, stress levels induced by human handling, etc.

• Time. Most bioassays take days, and in some cases week, to run. This can render routine bio-assays difficult, and impractical to undertake as a quick QC potency test during downstream processing.

• Cost. Most bioassay systems, in particular those involving whole animals, are extremely expensive to undertake.

Because of such difficulties alternative assays have been investigated, and sometimes are used in conjunction with, or instead of, bioassays. The most popular alternative assay system is the immunoassay.

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with

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