Most recombinant biopharmaceuticals are produced in microbial or mammalian cell lines. Thus, although the product is derived from a human gene, all product-unrelated contaminants will be derived from the producer organism. These non-self proteins are likely to be highly immunogenic in humans, rendering their removal from the product stream especially important. Immunoassays may be conveniently used to detect and quantify non-product-related impurities in the final preparation (immunoassays generally may not be used to determine levels of product-related impurities, as antibodies raised against such impurities would almost certainly cross-react with the product itself).
The strategy usually employed to develop such immunoassays is termed the 'blank run approach'. This entails constructing a host cell identical in all respects to the natural producer cell, except that it lacks the gene coding for the desired product. This blank producer cell is then subjected to upstream processing procedures identical to those undertaken with the normal producer cell. Cellular extracts are subsequently subjected to the normal product purification process, but only to a stage immediately prior to the final purification steps. This produces an array of proteins that could co-purify with the final product. These proteins (of which there may be up to 200 as determined by two-dimensional electrophoretic analysis) are used to immunize horses, goats or other suitable animals. Therefore, polyclonal antibody preparations capable of binding specifically to these proteins are produced. Purification of the antibodies allows their incorporation in radioimmunoassay or enzyme-based immunoassay systems, which may subsequently be used to probe the product. Such multi-antigen assay systems will detect the sum total of host-cell-derived impurities present in the product. Immunoassays identifying a single potential contaminant can also be developed.
Immunoassays have found widespread application in detecting and quantifying product impurities. These assays are extremely specific and very sensitive, often detecting target antigen down to parts per million levels. Many immunoassays are available commercially, and companies exist that will rapidly develop tailor-made immunoassay systems for biopharmaceutical analysis.
Application of the analytical techniques discussed thus far focuses upon detection of proteina-ceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses.
Amino acid analysis remains a characterization technique undertaken in many laboratories, in particular if the product is a peptide or small polypeptide (molecular mass <10 kDa.). The strategy is simple. Determine the range and quantity of amino acids present in the product and compare the results obtained with the expected (theoretical) values. The results should be comparable.
The peptide/polypeptide product is usually hydrolysed by incubation with 6 mol l_1 HCl at elevated temperatures (110 °C), under vacuum, for extended periods (12-24 h). The constituent amino acids are separated from each other by ion-exchange chromatography and identified by comparison with standard amino acid preparations. Reaction with ninhydrin allows subsequent quantification of each amino acid present.
Although this technique is relatively straightforward and automated amino acid analysers are commercially available, it is subject to a number of disadvantages that limits its usefulness in bi-opharmaceutical analysis. These include:
• hydrolysis conditions can destroy/modify certain amino acid residues, in particular tryptophan, but also serine, threonine and tyrosine;
• the method is semi-quantitative rather than quantitative;
• sensitivity is at best moderate; low-level contaminants may go undetected (i.e. not significantly alter the amino acid profile obtained), particularly if the product is a high molecular mass protein.
These disadvantages, along with the availability of alternative characterization methodologies, limit application of this technique in biopharmaceutical analysis.
A major concern relating to biopharmaceuticals produced in high-expression recombinant systems is the potential occurrence of point mutations in the product's gene, leading to an altered primary structure (i.e. amino acid sequence). Errors in gene transcription or translation could also have similar consequences. The only procedure guaranteed to detect such alterations is full sequencing of a sample of each batch of the protein, which is a considerable technical challenge. Although partial protein sequencing is normally undertaken (see later), the approach most commonly used to detect alterations in amino acid sequence is peptide (fingerprint) mapping.
Peptide mapping entails exposure of the protein product to a reagent that promotes hydrolysis of peptide bonds at specific points along the protein backbone. This generates a series of peptide fragments. These fragments can be separated from each other by a variety of techniques, including one- or two-dimensional electrophoresis, and RP-HPLC in particular. A standardized sample of the protein product when subjected to this procedure will yield a characteristic peptide fingerprint, or map, with which the peptide maps obtained with each batch of product can subsequently be compared. If the peptides generated are relatively short, then a change in a single amino acid residue is likely to alter the peptide's physico-chemical properties sufficiently to alter its position within the peptide map (Figure 7.4). In
Fragmentation agent (e.g. trypsin)
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