Evaluation of different primary recovery methods forE. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Abstract

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.

Characterisation of a Novel Growth Hormone Variant Comprising a Thioether Link between Cys182 and Cys189

“…The first disulfide bridge lies between Cys53 and Cys165, whereas the second one is near the C terminus of the molecule and connects Cys182 and Cys189 (see Figure 1). The protein is recombinantly expressed in both bacteria [1] and mammalian cells and preparations are therapeutically used for various growth and metabolic disorders. [2,3] Heterogeneity in biopharmaceutical products may arise during both the production process and the shelf life of the product resulting in the presence of various forms of posttranslational modifications or degradation products.…”

Adapted Ultra Scale‐Down Approach for Predicting the Centrifugal Separation Behavior of High Cell Density Cultures

“…Current trends in bioprocessing, particularly in recombinant bacterial and yeast cultures, are leading to higher cell densities as manufacturers strive for greater levels of product expression using recombinant systems. Solids concentrations of up to 30% (w/v) are readily achievable (1). The handling of process streams with significant solids loads presents a unique challenge to the downstream processing engineer, specifically in the selecting of appropriate operating conditions for high-speed centrifuges, needed to harvest the cells from the fermentation liquors.…”

Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli

“…The large number of biopharmaceuticals approaching the end of their patent protection period will encourage manufacturers to seek competitive advantages through bioprocess technologies that have not been extensively exploited at commercial scale [1]. A typical process route for the primary recovery of intracellular protein products involves the use of conventional operations of cell disruption, centrifugation and ultrafiltration [2]. Purification is then frequently achieved by multiple chromatography steps [3].…”