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and the program can be terminated if a complete match is found. The most difficult algorithm to construct will be that corresponding to (2). The majority of proton resonances in oligosaccharide NMR spectra resonate between 3.5-4.0ppm and crosspeaks are often poorly resolved. Nevertheless, the search procedure is less demanding than that for proteins since the number of resonances is much reduced, and all of the spin coupling constants with the exception of those involving exocyclic protons are known. In this regard, crosspeaks belonging to each monosaccharide residue generate characteristic multiplicity patterns. Other NMR methods may be useful for this stage of the analysis. For example, multiple step relayed correlation spectroscopy (RELAY) or homonuclear Hartmann-Hahn spectroscopy (HOHAHA)7 might offer significant advantages in terms of reduced crosspeak overlap. The I-D counterparts of these experiments, recorded by selective excitation of the resolved anomeric proton of each monosaccharide residue, might significantly reduce the complexity of the algorithm. A n entirely analogous algorithm to that required for (2) will be used at (3). However, the analysis of NOESY spectra of oligosaccharides is much simpler than for COSY, since the only connectivities containing sequence information are those from the anomeric proton resonance of one residue to a second proton on an adjacent, glycosidically linked residue. The relevant crosspeaks are thus generally fully resolved. Acknowledgements The author is grateful to the Wellcome Trust for financial support. Rqfkrencrs 1 . Calvo, F. 0. & Ryan, R. J., Biochemistry, 24 (1985) 1953. 2. Takasaki, S., Mizuochi, T. & Kobata. A., Methods in Enzymol., 83 (1982) 263. 3. Likhosherstov, L. M . , Novika, 0. S., Piskarev, V. E., Trusikhina, E. A,, Derevitskaya, V. A . & Kochetkov, N . K., Curbohydr. Res., 178 (1988) 155. 4. Vliegenthart. J . F. G., Dorland, L. & van Halbeek, H., A h . Curb. Chrm. Biochem., 41 (1983) 209. 5 . Homans, S. W., Dwek, R. A. & Rademacher, T. W., Biochemistry, 26 (1987) 6571. 6. Homans, S. W.. Ferguson, M . A. J., Dwek, R. A,, Rademacher. T. W., Anand, R. & Williams. A. F.. Nnture, 333 (1988) 269. 7. Homans, S. W.. A Dictioncrry of’ Concepts in N M R . Clarendon Press, Oxford, 1989 (and Refs. therein).

lmmunoassays for Protein Contaminants Richard S. Williams Celltech Limited, 216 Bath Road, Slough, Berkshire SLI 4EN. U K

Introduction There are limitations as to how far one can utilise general biochemical methods, such as electrophoresis and liquid chromatography, for purity analysis of

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macromolecules obtained from biotechnological processes. There comes a point when one has to stop and ask ‘what are the process steps likely to introduce protein contaminants and what is their nature?’. Once the potential contaminants have been identified one can seek to analyse for them with methods specifically designed for that purpose. Origins of protein contaminants A great diversity exists in the types of contaminants that may be present in biotechnology-derived products but this short paper will briefly review only those of a proteinaceous nature. Protein contaminants are unavoidable in today’s biotechnology processes as the cells used to produce the product of interest will also secrete many other proteins and peptides in addition to the contamination contributed by intracellular proteins released following cell death and lysis. Although in recent years there has been a reduction in the use of extraneous proteins added to cell culture media (particularly in the use of foetal calf serum) many media preparations still contain a variety of protein components to achieve optimal cell growth and product yields. These are obviously a further potential source of protein contamination. Apart from the fermentation process, protein contaminants may also be introduced further downstream. If the purification of the product utilities an affinity process involving proteinaceous ligands (e.g. antibodies, Protein A, Protein G or lectins) then these ligands have the potential for leaching off the matrix support and contaminating the product. Complexity of analysis As described above, the types of protein contaminants may vary from well defined (e.g. bovine serum albumin, transferrin) to those of a more complex and ill defined nature (e.g. foetal calf serum, host cell proteins). Although ligands from affinity processes are well defined their analysis as contaminants is complicated by the fact that they are present specifically to interact with the product; hence interference in analytical procedures is problematical. This will be discussed briefly later using Protein A as an example. A further level of complexity is introduced if the assay is to be used for in-process testing as opposed to final product testing only. In the latter case one has a consistent protein profile, single buffer system and narrow range of analyte concentration, whereas in the former situation the assay has to accommodate enormous variability in all of these parameters. Immunoassay approach During the purification process, generally, one has used the most appropriate resolution techniques to purify product from contaminants. I t is therefore unlikely that their use at an analytical level would be totally effective. One must then turn to more specific approaches such as immunoassay, a technique which relies on the unique specificity of antibody-antigen interactions.’.2 In addition to the specificity, immunoassays have a high throughput and can measure very low analyte concentrations at the lower end of a wide dynamic range. Very few other

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approaches are able to detect 1 part of protein contaminant in the presence of 10' fold excess of product. However, immunoassays do suffer from a number of drawbacks in that there is a need for a reliable supply of appropriate antibodies which may have to be raised specifically, the problems of interference potentially resulting in long development times and the poorer precision of immunological methods compared to some other techniques (e.g. HPLC). Specification and validation When undertaking the development of an assay it is important to understand what the assay is to be used for (e.g. final product testing or in-process analysis) and what performance is expected from the final developed assay in terms of working range, accuracy, precision, specificity and r o b u ~ t n e s s .Stability ~ of test and reference samples and assay reagents are also important issues to be addressed. The two examples, analysis of Protein A and host cell/media proteins, are given below to highlight some of the specific validation issues that may be applicable to different protein contaminants. Protein A Protein A-affinity chromatography is a conventional approach to the purification of many monoclonal a n t i b ~ d i e sHowever, .~ as mentioned previously, there is the potential for the Protein A ligand to leach from the column and contaminate the product. In developing an assay for Protein A there are two major issues; first, what is the acceptable limit of contamination, and secondly, how one overcomes any specific product-ligand interferences. The issue of acceptable limits has to be considered for each product on a case-by-case basis and will be related to risk-benefit analysis of intended use of the product. However, one may expect a level of 1-10 ng Protein A per mg protein product ( i t . 1-10 ppm) to be acceptable. The Protein A antibody interferences observed in the literature and in a number of commercially-available Protein A assay kits presents a significant problem.',' When establishing the approach to take for analysis of Protein A two commercial assays ( R I A and ELISA) and three in-house ELISA formats (competitive and sandwich type) were evaluated. All assays originally showed poor recovery ~ ) into human IgG (approximately 50%) of Protein A (10 ng ~ m - spiked ( I mg cm-'). This interference was attributed to specific interaction with the F, region of the antibody following experiments where the recovery of Protein A from human IgG F, or F(ab'), fragments was measured. However, two of the in-house assays appeared to detect much lower levels of Protein A than the commercial assays; one of these in-house assays was taken for further development, The presence of higher concentrations of human IgG (5 mg c m - 3 ) almost totally suppressed the assay signal for Protein A in the undeveloped assay. However, after addressing such parameters as source of antibodies, buffer components, time and temperatures of incubations and wash cycles much of this interference was overcome. This assay was then able to measure < 2 ng Protein A cm13 in the presence of high concentrations (5 mg cmP3)of human IgG; a significant improvement over

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what has previously been available. It was clear that the assay detected intact Protein A but it is likely that at least a portion of the material leaching from the support matrix may be smaller degradation products. The ability of the assay to detect these degradation products was shown by analysis of a Protein A sample following limited proteolysis with trypsin. I t was found that sample containing no intact Protein A and mostly very low molecular weight material ( 6 2 0 kDa) by SDS-PAGE retained appreciable activity in the ELISA.

Host cell/media contaminants The issues and problems related to the analysis of these contaminants are rather different. The concern over their presence lies in their immunogenicity and potential for containing oncogenic and viral proteins or protein sequences. Protein contaminants from host cells and the media in which they are grown pose special problems for analysis in that there is the potential for thousands of different proteins to be present in widely differing amounts and that these may be expressed at different levels depending, for example, on stage of fermentation cycle and composition of cell culture media. In developing immunoassays for these contaminants it is important to develop an appropriate pool of antigens for immunisation and re-immunisation and to develop the antibody reagent so that it is capable of quantitatively detecting all potential host cell/media contaminants. The preparation of antigens involves the use of the most closely related cell line available as non-expressor. This may be a non-producing parent line of a hybridoma or a recombinant line transfected with a similar plasmid but lacking the structural sequence for the product. It is important at this stage to minimise changes in growth conditions so as to not appreciably affect the antigens produced. Antigens obtained from these cell lines can then be applied to the purification process for the product and pools of antigens may be taken at different stages through the process to be used as immunogens. A judgement will have to be made as to the most appropriate stage(s) to select antigens for immunisation. Having obtained an appropriate antigen pool one must look to the immunisation of animals, addressing species to use, numbers of animals and the development of the antibody reagent. Antibody titres raised following immunisation are conveniently screened using a dot-blot immunoassay approach whereas a qualitative measure of how many of the antigens are detected established by silver staining and Western blotting following two dimensional PAGE. Frequently one finds that much of the initial immunological response is directed against a few particularly immunogenic proteins; the specific removal of these proteins by immunoabsorption is a useful approach to take in preparing a depleted antigen pool for more selective re-immunisation. Finally in preparation of the final antibody reagent one may consider removing dominating antibodies by antigen-affinity chromatography. This approach hopefully would lead to a balanced reagent that will quantitatively detect all antigens present. Validation of this type of assay system may include purification of selected antigens from the

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original pool and monitoring their response factors in the developed assay. Similar responses will give further assurance of the validity of the analytical method. Conclusion In contrast to a panel of biochemical methods looking at general physical and chemical characterisation of the product to evaluate product purity, immunoassays have been effectively used to specifically detect trace amounts of known potential protein contaminants. Acknowledgements The author wishes to acknowledge the work performed by R. Francis in the development of the Protein A ELISA and to S. Flatman and F. Wilcox for their contribution to the preparation of this article. Thanks also to M . Turner for her secretarial assistance.

1 . Chan, D. W., In Immunoassay a Practical Guide, ed. D. Chan & M. Perlstein. Academic Press, 1987, pp. 1-23.

2. Howanitz, P. J., Immunoassay-development and directions in antibody technology. Arch. Pathol. Lab. Med., 112 (1988) 7 7 1 4 . 3. Food and Drug Administration, Guideline for Submitting Samples and Analytical Duta for Methods Validation. Center for Drugs and Biologics, Ofice of Drug Research and Review, Rockville, Maryland, USA, 1987. 4. Vetterlein, D., Monoclonal antibodies: production, purification and technology. Adv. Clin. Chem., 27 (1989) 303-54. 5. Dertzbaugh, M . T., Flickinger, M . C. & Lebherz 111, W. B., An enzyme immunoassay for the detection of staphylococcal protein A in affinity-purified products. J . Immunol. Methods, 83 (1985) 169-77. 6. Lucas, C., Nelson, C. Peterson, M . L., Frie, S., Vetterlein, D., Gregory, T. & Chen, A . B., Enzyme-linked immunosorbent assays (ELISAs) for the determination of contaminants resulting from the immunoaffinity purification of recombinant proteins. J . Immunol. Methods, 113 (1988) 113-22.

Immunoassays for protein contaminants.

I36 SCI Biotrchnoloqy Croup Meeting and the program can be terminated if a complete match is found. The most difficult algorithm to construct will b...
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