Russell H. Robins

High-performance tryptic mapping of recombinant bovine somatotropin☆

Experiments are described that have lead to the development of a highly reproducible tryptic map of recombinant DNA derived bovine somatotropin (rbSt). Tryptic digestion of rbSt at 37 degrees C results in the formation of a precipitate. Preliminary characterization of the precipitate suggests that its formation is due to the association of intermediate tryptic fragments. An examination of the temperature dependence of the digestion has revealed that precipitate formation is inhibited when digestion is performed at 10 degrees C or less. The combination of a 5-mg sample, the use of highly purified trypsin, and digestion at 5 degrees C generate a tryptic map that exhibits an average 1.3% RSD (0.5-3.6%) for all anticipated fragments. Validation studies demonstrate that while the peak response precision is rugged to daily variation of operators or chromatographic systems, the fragment retention is not. This dictates that peaks be assigned by qualitative pattern recognition. Assay ruggedness in the peak response domain allows for the implementation of quantitative methods for the comparison of rbSt reference standard and sample tryptic maps. The assay is linear for all anticipated fragments within 50-150% of the operating range. Specificity is established by assay of pituitary somatotropins from other species and rbSt analogs produced by site-specific mutagenesis. The data demonstrate that all single amino acid substitutions examined are identified by using the technique. Assay sensitivity is validated for selected tryptic fragments through analysis of reference standard digests spiked with known amounts of rbSt analog digests. The data indicate that potential impurities of 3.2, 2.0, and 4.5% can be quantitated with statistical confidence in the tryptic fragments T1, T10, and T23 + 25, respectively.

Unequivocal location of sites ofN-oxidation using natural abundance long-range1H,15N GHNMQC two-dimensional NMR

A method is described for unequivocally establishing the site of N‐oxidation which relies on the relatively large downfield shift of aliphatic nitrogen resonances following oxidation. The technique described is based on 15N at natural abundance and does not require labelling. 15N chemical shifts are established on the basis of long‐range 1H–15N heteronuclear coupling pathways detected via two‐dimensional NMR using the GHNMQC pulse sequence. The oxidized nitrogen in the two piperazine‐containing systems studied was shifted downfield by approximately +68 ppm; the opposite, non‐oxidized nitrogen in the piperazine ring was shifted upfield by an average of ‐6 ppm (15N chemical shifts are referenced to liquid ammonia with a chemical shift of 0 ppm). N‐oxidation perturbations in 15N chemical shifts were first parameterized using an oxazolidinone antibiotic (eperezolid) and its N‐oxide as a model system. After the 15N shift due to N‐oxidation was determined, the method was used to establish unequivocally the site of N‐oxidation in PNU‐101387, a piperazine‐containing anxiolytic agent. Conventional spectroscopic methods were equivocal and could not reliably establish the site of N‐oxidation.

EFFECTS OF N-OXIDATION ON THE 15N CHEMICAL SHIFTS IN THE STRYCHNOS ALKALOIDS STRYCHNINE AND BRUCINE

The effects of N‐oxidation on the 15N chemical shifts of the Strychnos alkaloids strychnine and brucine are discussed. The 15N shifts were determined using the inverse‐detected, long‐range GHMBC experiment at natural abundance. Following N‐oxidation, the N‐19 resonance shifted downfield from 35.0 ppm in strychnine to 136.3 ppm in the N‐19‐oxide. The N‐19 resonance of brucine shifted downfield from 35.9 to 135.5 ppm in the N‐19‐oxide. Small upfield shifts were observed for the N‐9 resonances of both N‐oxides. The 1H and 13C shifts of both N‐oxides were assigned using inverse‐detected 2D NMR methods to ensure proper assignments of the long‐range 1H– 15N couplings. Copyright

Gradient high performance liquid chromatographic assay for degradation products of adinazolam mesylate in a sustained release tablet formulation

A gradient high performance liquid chromatographic method was developed to determine degradation products of adinazolam mesylate in a sustained release tablet formulation. Sample preparations were chromatographed on a YMC-Basic column using a formate buffer/acetonitrile gradient with absorbance detection at 254 nm. Adinazolam mesylate was found to degrade at high relative humidity and temperature to form a major product, the 6-aminoquinoline analog, plus numerous other compounds. Five of these compounds were identified and their structures indicate that the solid-state degradation of adinazolam, in the presence of sufficient moisture, involves not only a hydrolytic mechanism, but also an oxidative mechanism. Potential process impurities were resolved from the drug and degradation products. Recovery was near 100% over the 0.5 to 10% range for the major degradate (6-aminoquinoline) and over the 0.5 to 1% range for the other analytes. The method was applied to tablet samples stressed at high relative humidity and temperature. The relative standard deviation of the assay for the 6-aminoquinoline was less than 2% and less than 13% for the minor components. Calculated mass balances (sum of adinazolam plus degradation products in the degraded tablet divided by the same sum in the undegraded tablet) were less than 100% and were dependent on the extent of degradation in the tablet. The average mass balance result obtained for samples that were an average of 9.5% degraded was 95.0 ± 1.5%. It is possible that the decrease in mass balance with increase in percent degradation may be explained by the formation of many components at trace levels due to degradation by various permutations of hydrolytic and oxidative reaction pathways.

The 3′-Keto–Diol Equilibrium of Trospectomycin Sulfate Bulk Drug and Freeze-Dried Formulation: Solid-State Carbon-13 Cross-Polarization Magic Angle Spinning (CP/MAS) and High-Resolution Carbon-13 Nuclear Magnetic Resonance (NMR) Spectroscopy Studies

Understanding how moisture interacts with a drug or formulation is a critical component of product development. This study demonstrates how water affects the 3′-gem-diol ↔ 3′-keto equilibrium in trospectomycin sulfate bulk drug and freeze-dried formulation, as probed by solid-state carbon-13 cross-polarization magic angle spinning (CP/MAS) and high-resolution nuclear magnetic resonance (NMR) spectroscopy. Drying the bulk drug or formulation to low water levels dehydrates trospectomycin sulfate from the diol to the keto form. Carbon-13 CP/MAS NMR spectroscopy measures the keto drug concentration in solid samples directly. The bulk drug, which contains approximately 16% water, is more than 90% in the 3′-diol form. Oven drying to <3% water converts approximately 75% of the drug to the 3′-keto form. The drug is formulated as a freeze-dried, sterile powder that can contain up to 12% water depending on the freeze-drying conditions. These studies show that the 3′-keto concentration rises uniformly (up to 75%) with decreasing residual water in the freeze-dried cake. The keto–diol equilibrium was also studied in solution by high-resolution carbon-13 NMR experiments, and it was found that raising the temperature or using dimethyl sulfoxide (DMSO) as a solvent also dehydrates the drug. For example, in aqueous solution at 25°C, nearly all (>95%) of the drug is in the 3′-diol form. After equilibration at 60°C, however, the 3′-keto content increases to 7%, and in d6-DMSO solvent at 25°C the drug is mostly (60%) in the 3′-keto form.

N-ε-Acetylation Can Occur at Lysine Residues 157, 167, 171, And 180 of Recombinant Bovine Somatotropin

Publisher Summary It was found that a significant fraction of the p 7 rbSt arises from acetylation of certain lysine residues. This chapter discusses the isolation and structural elucidation of the acetylated pI 7 bands in rbSt. Acetylation of lysine is a well known post-translational modification mediated by an acetyltransferase enzyme using acetyl-CoA as a substrate. It has been demonstrated with the histones, where it is believed to regulate the binding of these very basic proteins to the negatively charged ribose backbone of the nucleic acids. The acetylation of lysines on rbSt is a post-translational modification carried out by the host E. coli organism. Acetylation of lysines makes them unrecognizable to trypsin and in each case result in the removal of one positive charge in the parent protein, thus lowering the pi. The data indicate that at least four of the pi 7 species are produced by acetylation of lysines 157, 167, 171, and 180. Another species is formed by the base catalyzed rearrangement of asparagine 99 to isoaspartic acid.