A simplified bioprocess for human alpha‐fetoprotein production from inclusion bodies

Abstract

A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies.

Expression and purification of Canis interferon α in Escherichia coli using different tags

“…This result was corresponding with other researches [44]. Leoing found that the amount of protein recovered in a native form was highly dependent on the ratio of the denatured protein and in his study, 50-fold dilution of target protein was found to be most favorable to increase protein solubility [23]. Hwang pointed out that high refolding yield (>70%) could be easily achieved at a refolding protein concentration of 30µg/mL [21].…”

“…Additionally, contaminants, especially hydrophobic proteins and lipids, are expected to bind to target proteins after refolding [21]. This highlights the importance of product purification before the critical refolding step [23]. Thus, the sediment was dissolved in 8 M urea.…”

A chromatography-focused bioprocess that eliminates soluble aggregation for bioactive production of a new antimicrobial peptide candidate

“…hBDs are cysteine-rich and disulfide bonds have been suggested to be important for tertiary structure stabilisation of ␤-defensins [35][36][37]. Therefore, optimising the refolding buffer with respect to redox environment is considered important to enhance oxido-shuffling cysteines and hence promote correct formation of disulfide bonds [38,39]. The effect of varying GSH:GSSG ratio in the refolding buffer on the antimicrobial activity of hBD25 was investigated.…”

A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies

“…Evidence that PMP was retained on the retentate side of the filter was provided by SDS-PAGE analyses which indicated roughly the same PMP concentration in all retentate samples collected from the different wash experiments (data not shown). Consequently, urea/phos8/EDTA was excluded as an optimal wash solution, because the inclusion bodies were partly dissolved and also because the risk of carbamylation of the product protein with use of urea (Leong and Middelberg, 2007).…”

“…IBs, cell debris, host cell proteins (HCP), host DNA, and endotoxin. It is known that residual contaminants may negatively affect the renaturation performance (Shire et al, 1984;Maachupalli-Reddy et al, 1997;Thatcher and Hitchcock, 1994;Batas et al, 1999;Leong and Middelberg, 2007).…”