Recombinant proteins, analytical

The MS techniques described previously for characterization of the final recombinant protein product can be applied at all stages during process development. MS might be used upstream to define clone selection, processing format, and purification steps, and downstream to characterize the final product, ascertain lotto-lot reproducibility, determine stability, and define the formulation of biopharmaceutical molecules. Presented here are some examples found either in the literature or from our own experience in which MS has been found to be a useful or necessary tool. Potential limitations of MS methods are discussed, and when appropriate, other analytical methods are mentioned that can be alternatives to MS and are also efficient tools for biopharmaceutical development. 

C. Kaiser, T. Potozki, A. Ellert and R. Luttmann, Applications of PAT - process analytical technology in recombinant protein processes with Escherichia coli, Eng. Life Sci., 8, 132-138 (2008). 

Among these amino acids histidine is the most commonly used one. Attachment of histidine tags to the recombinant proteins polypeptides is the most known development in the field of IMAC. Histidine and other metal affinity tags are widely used for protein purification [26], Adsorbents may be prepared by binding chelators onto the surface and metals to the chelators. Free coordination sites of the metal ions are needed for the analyte to bind to metal ions [25]. 

Capillary electrophoresis has found use in the biotechnology industry for structural analysis of recombinant proteins. The high resolving power of CE for charged analytes makes it a powerful tool for the analysis of tryptic digests. Therefore, many of the techniques given here, such as the determination of thiols, carbohydrates, and amino acids, will be employed for this purpose. 

In addition to analyzing a protein for characteristics dictated by its specific nature, routine analytical assays are performed namely, amino acid analyses and amino- and carboxy-terminal sequencing. If necessary, disulfide assignments are made. A search is ordinarily conducted for oxidations and/or deamidations. In the case of recombinant proteins particular attention is paid to detecting signal sequences or proteolytically degraded species. In contrast, deletions and chemical modifications can be a consequence of proteins prepared by chemical synthesis. Lastly, tests are applied to determine if the protein is correctly folded into its native three dimensional structure. In addition, with recombinant proteins, sensitive immunological procedures are performed to ensure that host cell proteins have not been copurified with the protein of interest. 

In conclusion, studies on isolated cells allow measurements on homogeneous cell populations under well-defined conditions and can aid in the interpretation of metabolic changes seen by NMR in the same or similar cells in the intact animal. In some cases the cells may be a valid system for study in their own right. For example, there have been several NMR studies of commercially important mammalian cell lines which are used in the biotechnology industry for the production of various monoclonal antibodies and recombinant proteins with therapeutic or analytical applications (Mancuso et al., 1990 Gillies et al., 1991). 

This mode of separation, as the name suggests, uses stationary phases with a special affinity for a specific analyte. The affinity ligand immobilized on the stationary phase varies dramatically from peptide, to protein, to oligonucleotide, to monoclonal antibody. In some cases the target molecule is labelled with an affinity tag to simplify the separation. This approach is common in the synthesis of recombinant proteins where the system can be engineered so that the target biomolecule expresses a tag such as polyhistidine. A stationary phase functionalized with aminodiacetic acid and nickel chelate is then used to fish out the required molecule by chelating with the polyhistidine tag. 

The number of residues identified using this method is sequence dependent. Typically 1 to 2 nmol of purified protein will allow sequencing of 3-5 (and sometimes more) cycles fi om the C-terminus. Sequencing of electroblotted samples, where less than 1 nmol of protein is present, has also been successful for 3 or more cycles. Although a few amino acids remain difficult to detect or sequence, our chemistry is especially useful for the verification of the expected C-terminus of recombinant proteins, and has provided information on possible modifications. Here we show specific examples, where the application of this chemistry can be used as an additional analytical tool for protein characterization. 

Minks, C., Huber, R., Moroder, L. and Budisa, N. (2000) Noninvasive tracing of recombinant proteins with fluorophenylalanine-fingers. Analytical Biochemistry, 284, 29-34. 

The presented transcription-translation method is based on T7 RNA polymerase and the micrococcal nuclease-treated reticulocyte lysate as described by Jackson and colleagues,22 and is suitable for a semi-preparative synthesis of recombinant protein. If few reactions on an analytical 10 pi scale are to be carried out the TnT kit from Promega can be recommended. 

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