Profiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography–Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-µm Particles

Abstract

In recent years protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficult to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kspi range, accommodates the use of capillary columns in excess of one meter packed with sub-2 μm particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (λ 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at the higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.

Thiyl Radical Reactions in the Chemical Degradation of Pharmaceutical Proteins

“…An important difference between the results of both growth hormones is the formation of an ether cross-link in mGH vs. a vinylether cross-link in hGH. This may be rationalized by sequence differences between hGH and mGH [96], as we also observed significant differences in photooxidation between hGH and rat growth hormone (rGH) [97]. We believe that the ether cross-link is ultimately generated via reduction of a vinyl ether.…”

Proteolysis and Oxidation of Therapeutic Proteins After Intradermal or Subcutaneous Administration

“…A specific improvement for the resolution and detection of these modifications is the use of very long columns (!1m), coupled to mass spectrometry detection. 164,165…”