A simplified bioprocess for human alpha-fetoprotein production from inclusion bodies.
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.
Leong
Susanna S J
SS
Centre for Biomolecular Engineering, Division of Chemical Engineering, The University of Queensland, St. Lucia, Queensland 4072, Australia.
Middelberg
Anton P J
AP
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D052617:Solid Phase Microextraction / Q000379:methods*; D000509:alpha-Fetoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D000509:alpha-Fetoproteins