l-Asparaginase and HCP quantification by SWATH LC-MS/MS for new and improved purification step in Erwinia...

Erwinase® or Erwinaze® are the proprietary names for the L-asparaginase enzyme derived from Erwinia chrysanthemi.L-asparaginase is an integral part of the treatment of Acute Lymphoblastic Leukaemia (ALL) in children and adolescents. E. chrysanthemiL-asparaginase was first developed in the early 1970s at Porton Down and is currently manufactured by Porton Biopharma Ltd. One of the early purification steps during E. chrysanthemiL-asparaginase manufacture, involves use of batch cation exchange carboxymethyl resin, and alternatives to this older technology are currently under investigation using mass spectrometry to understand the impact of resin changes on the impurity profile. In this study, a novel SWATH library was developed for E. chrysanthemi proteome and used to evaluate this potential process change on product yield and host cell protein (HCP) profile and clearance. An ELISA assay is currently used as a quality control release test for quantifying HCPs at the Drug Substance (DS) stage, but these early extract samples are too crude for interference-free analysis by ELISA. Given that ELISA assay could not be used in the assessment of new resin options, SWATH LC-MS/MS analysis proved to be pivotal in selecting a resin for further scale-up and implementation. The data quantified that L-asparaginase from the new process step was 2.28-fold higher in concentration than in legacy-process samples. The new step, using a modern ion exchanger, was at least equivalent and in some cases outperformed the legacy resin step in terms of HCP clearance for 78.2% of total HCPs (528 of 675 total proteins).