Downstream processing of recombinant human insulin and its analogues production from E. coli inclusion bodies

Yin Yin Siew,Wei Zhang

doi:10.1186/s40643-021-00419-w

Abstract

The Global Diabetes Compact was launched by the World Health Organization in April 2021 with one of its important goals to increase the accessibility and affordability of life-saving medicine—insulin. The rising prevalence of diabetes worldwide is bound to escalate the demand for recombinant insulin therapeutics, and currently, the majority of recombinant insulin therapeutics are produced from E. coli inclusion bodies. Here, a comprehensive review of downstream processing of recombinant human insulin/analogue production from E. coli inclusion bodies is presented. All the critical aspects of downstream processing, starting from proinsulin recovery from inclusion bodies, inclusion body washing, inclusion body solubilization and oxidative sulfitolysis, cyanogen bromide cleavage, buffer exchange, purification by chromatography, pH precipitation and zinc crystallization methods, proinsulin refolding, enzymatic cleavage, and formulation, are explained in this review. Pertinent examples are summarized and the practical aspects of integrating every procedure into a multimodal purification scheme are critically discussed. In the face of increasing global demand for insulin product, there is a pressing need to develop a more efficient and economical production process. The information presented would be insightful to all the manufacturers and stakeholders for the production of human insulins, insulin analogues or biosimilars, as they strive to make further progresses in therapeutic recombinant insulin development and production.

Highlights

Diabetes is a global epidemic (WHO 2021)
This will be beneficial in prolonging the effective lifetime of reversed-phase chromatography (RP) stationary phase as it is prone to fouling as a result of irreversible binding or low solubility of contaminants (Kroeff et al 1989)
Insulin is a scarce commodity, with global demands projected to rise due to increasing prevalence of diabetes

Summary

Introduction

Diabetes is a global epidemic (WHO 2021). In April 2021, the World Health Organisation has launched the Global Diabetes Compact to hasten action to tackle diabetes worldwide. The downstream process to purify recombinant human insulin/analogues from E. coli inclusion bodies is complex and involves multiple steps, and the specifics of the processes established by different companies are proprietary. All the critical aspects of downstream processing of recombinant human insulin/analogues produced in E. coli inclusion bodies will be summarized and discussed. Inclusion body washing is critical in recombinant insulin purification, without which numerous impurities will persist and may interfere with the following steps, such as sulfitolysis, renaturation, and enzymatic digestion (Min et al 2011) This could lead to a reduction in purification yield. A chaotropic agent to extract the bacterial outer membrane components

M 4 M NA information not available

M urea 8 M urea 6 M GdnHCl 8 M urea and 6 M

C8 C8 C8

M GdnHCl or 4 M urea

Findings

Conclusions