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May 30, 2012 - The only online database in. Anal. Chem. , 1987, 59 (15), pp 973A–973A. DOI: 10.1021/ac00142a748. Publication Date: August 1987...
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New in vitro assays are needed to replace whole-animal methods and other laborious and imprecise techniques for the testing of recombinant products. photometric monitoring of the time required for lysis of the synthetic clot by plasmin produced by TPA's activation of plasminogen. According to Garnick, this assay was demonstrated to be far more accurate, precise, and stabilityindicating than typical in vivo bioassays. Analytical wish list It is hoped t h a t the future of analytical biotechnology will include other in vitro assays capable of replacing wholeanimal methods and other laborious and imprecise techniques for the testing of recombinant products. One of the first items on Frank Robey's wish list, for example, is the development of in vitro assays for the prediction of immunogenicity. "For example," said Robey, "if a product is not exactly identical to a host protein, the body can recognize t h a t and raise antibodies against it. These antibodies can neutralize the activity of a drug, they can cause its removal from the circulation, or they can induce anaphylactic shock resulting in death. Today, the only way we can determine the immunogenicity of a product is by clinical trials in humans. If we had in vitro assays to replace these trials, it would save everybody a lot of money and a lot of time." Robert Garnick favors the development of cloned receptors to proteins for use in cell-based or biochemical in vitro assays and the use of H P L C assays to replace in vivo bioassays for potency determinations. According to Garnick, it is important to remove as many in vivo bioassays as possible from the repertoire because of the cost, imprecision, and laboriousness of these techniques. Both Robey and Garnick would like to see proteins being profiled by tryptic mapping/mass spectrometry (MS) (2) or tryptic mapping/HPLC/MS. At the present time, H P L C alone is generally used to create tryptic maps after protein digestion. Although H P L C provides a fingerprint pattern of the protein, it doesn't provide information (aside from retention time) t h a t helps in the identification of specific peaks in the map. "Using mass spectrometry," said Robey, "the N-terminus and Cterminus could both be positively identified. I predict t h a t in the next 5-10

years LC/MS will be a standard technique in laboratories doing quality control of recombinant products." Biotechnologists would also like to see better C-terminal protein sequencing techniques to complement the Edm a n degradation, which begins its work at the N-terminus. "Caroboxyterminal sequencing is just as important as amino-terminal sequencing to verify the homogeneity of a protein product," said Robey. "Today's methods for carboxy-terminal sequencing are antiquated, generally requiring enzymatic digestion and kinetics to show the disappearance of one peak and the appearance of another peak as the enzyme eats into the C-terminal portion. That's not very helpful." According to Robey, the tryptic m a p p i n g / H P L C / MS technique mentioned above would be one way to effectively satisfy this need. Robey is also looking forward to the a p p e a r a n c e of a u t o m a t e d carbohyd r a t e compositional and sequence analysis. "We have a tremendous fear," he said, " t h a t these glycosylated proteins are going to be a problem. Considering t h a t in a single hexose you have six different positions a t which you can link another hexose, the number of isomers can explode very quickly as you link on each sugar. As a result, many of the recombinantly produced products may not have the carbohydrate composition and sequence of the host protein, and t h a t could be trouble. If the carbohydrate isn't on there as it's supposed to be in the native protein, then you're dealing with a case where you're affecting the composition, the configuration, the activity, the immunogenicity, and other properties." Other items on biotechnologists' wish lists include • more sensitive nuclear magnetic resonance instruments capable of verifying the purity of proteins at pg/mL levels, • increased commercialization of new forms of M S (e.g., 252 Cf plasma desorption) t h a t are capable of handling higher molecular weight species, • improved microanalysis for contaminants, and • improved automation for all of the labor-intensive techniques currently used in molecular biology, such as plasmid preparations, blotting, and sequencing, in addition to endotoxin, protein content, and electrophoretic assays. Stu Borman

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Suggested reading (1) An interesting and informative article on mammalian cell culture appeared recently: Lewis, R. High Technol. J u n e 1987, pp. 30-37. (2) See "Mass Spectrometry Methods for Protein Sequencing," by K. Biemann, Anal. Chem. 1986,58[13], 1288-1300 A.

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ANALYTICAL CHEMISTRY, VOL. 59, NO. 15, AUGUST 1, 1987 · 973 A