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Figure 3.36. Photo of a modern protein sequencing system. Photo courtesy of Perkin-Elmer Applied Biosystems Ltd, UK
fast and automated determination of up to the first 100 amino acids from the N-terminus of most proteins, and usually requires a sample size of less than 1 mM to do so (Figure 3.36).
Analogous techniques facilitating sequencing from a polypeptide’s C-terminus remain to be satisfactorily developed. The enzyme carboxypeptidase C sequentially removes amino acids from the C-terminus, but often only removes the first few such amino acids. Furthermore, the rate at which it hydrolyses bonds can vary, depending on which amino acids have contributed to bond formation. Chemical approaches based on principles similar to the Edman procedure have been attempted. However, poor yields of derivitized product and the occurrence of side reactions have prevented widespread acceptance of this method.
Analysis of secondary and tertiary structure
Analyses such as peptide mapping, N-terminal sequencing or amino acid analysis yield information relating to a polypeptide’s primary structure, i.e. its amino acid sequence. Such tests yield no information relating to higher-order structures (i.e. secondary and tertiary structure of polypeptides, along with quaternary structure of multi-subunit proteins). While a protein’s 3-D conformation may be studied in great detail by X-ray crystallography or NMR spectroscopy, routine application of such techniques to biopharmaceutical manufacture is impractical from both a technical and economic standpoint. Limited analysis of protein secondary and tertiary structure can, however, be more easily undertaken using spectroscopic methods, particularly far-UV circular dichroism. More recently proton-NMR has also been applied to studying higher orders of protein structure.
Endotoxin and other pyrogenic contaminants
Pyrogens are substances which, when they enter the blood stream, influence hypothalamic regulation of body temperature, usually resulting in fever. Medical control of pyrogen-induced fever proves very difficult, and in severe cases results in patient death.
Pyrogens represent a diverse group of substances, including various chemicals, particulate matter and endotoxin (lipopolysaccharide, LPS — a molecule derived from the outer membrane of Gram-negative bacteria). Such Gram-negative organisms harbour 3-4 million LPS molecules on their surface, representing in the region of 75% of their outer membrane surface area. Gram-negative bacteria clinically significant in human medicine include E. coli, Haemophilus influenzae, Salmonella enterica, Klebsiella pneumoniae, Bordetella pertussis, Pseudomonas aeruginosa, Chylamydia psittaci and Legionella pneumophila.
In many instances the influence of pyrogens on body temperature is indirect, e.g. entry of endotoxin into the bloodstream stimulates the production of interleukin 1 (IL-1; Chapter 5) by macrophages. It is the IL-1 that directly initiates the fever response (hence its alternative name, ‘endogenous pyrogen’).
While entry of any pyrogenic substance into the bloodstream can have serious medical consequences, endotoxin receives most attention because of its ubiquitous nature. It is therefore the pyrogen most likely to contaminate parenteral (bio)pharmaceutical products. Effective implementation of GMP minimizes the likelihood of product contamination by pyrogens, e.g. GMP dictates that chemical reagents used in the manufacture of process buffers be extremely pure. Such raw materials are therefore unlikely to contain chemical contaminants displaying pyrogenic activity. Furthermore, GMP encourages filtration of virtually all parenteral products through a 0.45 mm or 0.22 mm filter at points during processing and prior to filling in final product containers (even if the product can subsequently be sterilized by autoclaving). Filtration ensures removal of all particulate matter from the product. In addition, most final product containers are rendered particle-free immediately prior to filling by an automatic pre-rinse using WFI. As an additional safeguard, the final product will usually be subject to a particulate matter test by QC before final product release. The simplest format for such a test could involve visual inspection of vial contents, although specific particle detecting and counting equipment is more routinely used.
Contamination of the final product with endotoxin is more difficult to control because:
• many recombinant biopharmaceuticals are produced in Gram-negative bacterial systems, thus the product source is also a source of endotoxin;
• despite rigorous implementation of GMP, most biopharmaceutical preparations will be contaminated with low levels of Gram-negative bacteria at some stage of manufacture. These bacteria shed endotoxin into the product stream, which is not removed during subsequent bacterial filtration steps. This is one of many reasons why GMP dictates that the level of bioburden in the product stream should be minimized at all stages of manufacture;