Abstract
A significant barrier to insulin is affordability. In this manuscript we describe improvements to key steps in the insulin production process in Pichia pastoris that reduce cost and time. The strategy for recovery and processing of human insulin precursor has been streamlined to two steps from bioreactor to the transpeptidation reaction. In the first step the insulin precursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostak™ module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the protein is applied at very high loadings on a cation exchange resin and eluted in a mixture of water and ethanol to obtain a concentrated insulin precursor, suitable for use directly in the transpeptidation reaction. Overall the yield from insulin precursor to human insulin was 51% and consisted of three purification chromatography steps. In addition we describe a method for recovery of the excess of H-Thr(tBu)-OtBu from the transpeptidation reaction mixture, one of the more costly reagents in the process, along with its successful reuse.
Highlights
Insulin is a relatively low-priced drug the chronic nature of Diabetes means the cost for insulin treatment is high, and together with an increasing number of patients, this financial burden challenges healthcare systems worldwide
The methylotrophic yeast Pichia pastoris has shown a number of attractive characteristics for heterologous protein production in virtue of its capacity of reaching high cell densities and expressing high amounts of recombinant proteins under control of strong and tightly regulated promoters [5]. Using this system we have previously described a simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase for secretory insulin precursor (IP) production resulting in a ~2 fold enhancement of IP production compared to the highest values reported, significantly increasing the efficiency of insulin manufacture [6]
This was relatively unexpected as the use of N-terminal extension of the insulin precursor (EEAEAEAEPK) has been shown to increase the Kex2 endoprotease efficiency and the insertion of Glu before the N-terminal Glu to inhibit cleavage by Dipeptidyl aminopeptidase A (DPAPA) [10]
Summary
Insulin is a relatively low-priced drug the chronic nature of Diabetes means the cost for insulin treatment is high, and together with an increasing number of patients, this financial burden challenges healthcare systems worldwide. Two major pathways for large-scale production of recombinant human insulin are currently used [2]. One route uses E. coli as an expression host, where the overexpressed insulin precursor (IP) forms inclusion bodies requiring solubilisation and oxidative refolding. The second route uses yeast-based expression systems (mainly Saccharomyces cerevisiae) where the IP is directly secreted in the culture supernatant in its correctly folded conformation. Half of the world insulin supply derives from Saccharomyces cerevisiae [3]. The standard recovery and purification process of human insulin produced in Saccharomyces cerevisiae can include up to fifteen steps [4]
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