Kenneth A. Cohen

Measurement of amino acid compositions of glycoprotein systems by gas-phase hydrolsis and reversed-phase high-performance liquid chromatography

Interest in glycoproteins and their compositions has increased in recent years. Work described in this report illustrates the use of an amino acid analysis protocol involving gas-phase hydrolysis and reversed-phase high-performance liquid chromatography of glycoprotein systems at microgram levels. In other amino acid analysis protocols the problem of losses of amino acids of glycoproteins has been documented. These losses were due to various reactions, referred to as browning or Maillard reactions, which yielded a residue from which amino acids were not recoverable. In our work, three glycoprotein systems are examined: ovalbumin, sICAM-1, and bovine serum albumin--which is naturally unglycosylated, but is spiked with about 30% saccharides. In all three cases, the compositional agreement between the molar ratio of amino acids determined empirically and that predicted is greater than 90%. Thus it is shown that the adverse effects of Maillard-type reactions are avoided, and the presence of carbohydrates causes negligible interferences with amino acid analysis performed under the conditions described herein.

Rapid, sensitive and efficient HPLC assays for HIV-1 proteinase

The proteinase encoded by human immunodeficiency virus type 1 (HIV-1) cleaves peptide substrates of sequences derived from processing sites in HIV-1 gag-pol polypeptide. Based on this cleavage, assays that utilize HPLC to measure activity of HIV-1 proteinase are reported herein. In the assay first described, a baseline separation of unlabeled substrate and products is achieved with a run time of 10 min and UV detection. Enzyme concentrations as low as 1 nM, which is the lowest reported for an assay employing underivatized peptide substrate, are attained. Even more powerful, versatile and sensitive, a second method that takes advantage of a peptide substrate labeled at its N-terminus with the fluorescein derivative is described as well. Because of the fluorescein label, this method offers several superior features, including very fast analysis of substrate and product in less than 3 min and fluorescence detection which provides essentially total freedom from interference. Synthesis of fluorescein-labeled peptide substrate is accomplished by solid-phase peptide synthesis.

High-performance liquid chromatography and photoaffinity crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human immunodeficiency virus type-1

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.

Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: Preparation of heterodimeric enzyme devoid of unprocessed gene product

A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.

Best Practices for Drug Substance Stress and Stability Studies During Early Stage Development. Part III—How to Make Science- and Risk-based Stability Testing Decisions for Drug Substance Batches Produced after Manufacturing Process Changes

During phase 1 and phase 2 drug development (early stage drug development), it is normal to continuously improve a manufacturing process, with changes made to the synthetic pathway, reagents, reaction conditions, crystallization parameters, drying conditions, or manufacturing equipment or scale or site. These manufacturing process changes (“process changes” used thereafter) may or may not affect quality attributes such as impurities, and quality attribute changes may or may not affect drug substance (DS) stability. But a common misconception is that almost all process changes and/or quality attribute changes affect DS stability, and a new (or repeat) stability study is conducted for the DS batch produced after process changes. This misconception is clearly refuted by our many years of DS stability experience. To understand how process changes might affect DS stability, we compiled and analyzed manufacturing processes, quality test results, and stability data for 48 batches from seven drug substances in recent development. Of these 48 batches, the seven first DS clinical batches were used as references against which the other respective 41 batches, which were produced after process changes, were compared for changes in manufacturing processes, quality test results, and stability data. This comparison showed that the chemical and physical stability of 36 (of the 41) batches was not affected by process changes, and the chemical or physical stability of the other 5 batches was affected by residual inorganic impurities, significant amounts of water or residual solvents, or significant changes in DS particle size distribution or surface area. These quality attributes that affect stability are called stability-related quality attributes (SRQAs). A new (or repeat) stability study is warranted only if process changes significantly affect SRQAs. We have established a procedure to systematically assess changes in manufacturing process and quality attributes (particularly impurity profiles), to identify SRQAs (risk assessment), and to make science- and risk-based stability testing decisions on whether and how stability testing for new DS batches should be conducted (risk management).