Biomedical Engineering Reference
In-Depth Information
sometimes be less than straightforward. Glycoproteins, for example, yield extremely complex spectra
(due to their natural heterogeneity), making the signifi cance of the fi ndings hard to interpret.
7.5 Immunological approaches to detection of contaminants
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 de-
rived 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 fi nal preparation
(immunoassays generally may not be used to determine levels of product-related impurities, as anti-
bodies 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 up-
stream processing procedures identical to those undertaken with the normal producer cell. Cellular
extracts are subsequently subjected to the normal product purifi cation process, but only to a stage
immediately prior to the fi nal purifi cation steps. This produces an array of proteins that could co-
purify with the fi nal 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 ani-
mals. Therefore, polyclonal antibody preparations capable of binding specifi cally to these proteins
are produced. Purifi cation 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 impuri-
ties. These assays are extremely specifi c 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 confi rm 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 specifi cation. Tests performed to verify
the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and
spectrophotometric analyses.
7.5.1 Amino acid analysis
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
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