Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.

PURPOSE: BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. METHODS: SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. RESULTS: Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. CONCLUSION: Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.

Stabilization of chimeric BR96-doxorubicin immunoconjugate.

Chimeric BR96-doxorubicin conjugate (BR96-DOX) is an immunoconjugate designed to specifically target and kill certain tumor cells. The linker between the chimeric BR96 antibody and DOX is an acid-labile hydrazone group which was designed to undergo lysosomal hydrolysis to release DOX in vivo. Stability studies indicated that acid-catalyzed hydrazone hydrolysis was the major degradation route in vitro. Even under optimal conditions of pH and temperature, the stability of BR96-DOX in solution was not acceptable for long-term storage. Lyophilization of BR96-DOX in the presence of added sugars, such as lactose or sucrose, and subsequent storage of the lyophile under refrigeration significantly improved the stability. Therefore lyophilization appears to be a viable approach for achieving long-term stabilization of BR96-DOX.

Chemical and physical stability of chimeric L6, a mouse-human monoclonal antibody.

Chimeric L6 is a mouse-human monoclonal antibody specific for tumor cell-associated antigens. The factors affecting the physical and chemical stability of chimeric L6 were assessed at elevated temperatures (30-60 degrees C) and by multiple freezing and thawing. Three routes of degradation were observed: chemical degradation to smaller molecular weight species, irreversible aggregation, and formation of a reversible dimer. The specific pathway depended on the stress condition applied and the pH, with maximal overall stability to both thermal stress and multiple freezing/thawing observed at about pH 5.5. Other factors including antibody concentration, buffer concentration, NaCl concentration, and agitation had minimal influence on the stability. Commonly used sugars, polyhydric alcohols, and amino acids effectively prevented freeze/thaw-induced aggregation.

Mechanistic investigation of the degradation of sulfamic acid 1,7-heptanediyl ester, an experimental cytotoxic agent, in water and 18oxygen-enriched water.

The hydrolytic degradation of sulfamic acid 1,7-heptanediyl ester was carried out in water and 18O-enriched water at 47 degrees C. The degradation of 1 was also studied at various pH values in the range of 2.5 to 8.0 at constant ionic strength (0.15 M) and temperature (25 degrees C). The hydrolysis was first order and independent of pH with a mean (+/- SD) observed rate constant (kobs) of 2.38 +/- 0.6 X 10(-3) h-1. No significant buffer catalysis was observed. From TLC, HPLC, and mass spectral studies, 1 initially degraded to sulfamic acid 1,7-heptanemonoyl ester and subsequently to 1,7-heptanediol. The site of bond cleavage was assessed by mass spectrometry of the 18O-enriched water reaction mixtures. Exclusive C--O bond fission was observed. Several mechanistic pathways for the degradation of 1 could be postulated. The results from 18O-labeling studies, the pH-rate profile and buffer studies, and kinetic solvent isotope effect (KSIE) studies were consistent with an SN2 mechanism with an early transition state (reactant-like transition state) where no appreciable bond had developed between the incoming nucleophile, water, and the carbon atom of 1. Although an SN1 mechanism was unlikely, based on the need to postulate the formation of a primary carbocation, this mechanism could not be totally ruled out.