Sub/supercritical fluid chromatography purification of biologics.
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Date
2020
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Abstract
Peptide and protein drugs are highly versatile with numerous therapeutic properties such as anti-cancer,
anti-diabetic, anti-hypertensive, and anti-microbial; which are therefore ideal candidates for the
development of next-generation drugs. This is exemplified using these drugs for the treatment of diseases
such as diabetes. Diabetes is one of the most prevalent non-communicable diseases worldwide. The rapid
increase in the number of diabetic patients globally places a burden on current insulin manufacturers. The
traditional reversed-phase high-performance liquid chromatography (RP-HPLC) purification methods of
insulin and peptides are problematic, tedious with long run times of approximately 50 minutes, low yields
employ harsh solvents such as acetonitrile, which has a negative impact on the environment. There is a
need for a greener process for the purification of insulin and peptides. Sub/supercritical fluid
chromatography (SFC) can provide the solution since it utilises greener mobile phases such as carbon
dioxide (CO2) and methanol, which can be recycled. However, there is a paucity of knowledge regarding
the SFC purification of human insulin and peptides. Therefore, this research study aimed to provide an
efficient, innovative approach for the biosynthesis of human insulin and the SFC purification of
biosynthesised human insulin, as well as the extension into the SFC purification of peptides. The
background of these topics is presented in Chapter One.
Chapter two (manuscript one) presents the development of a novel and more efficient method of human
insulin biosynthesis in Escherichia coli (E. coli). Several of the conventional steps were eliminated. The
crude biosynthesised protein sequence was verified using protein sequencing, which had a 100%
similarity to the human insulin sequence. The biological activity of the biosynthesised human insulin was
tested in vitro using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The
biosynthesis of human insulin was conducted on a laboratory-scale basis; future studies should investigate
scaling up of this method.
Chapter three (manuscript two) was based on the SFC purification of the commercially available standard
sample of human insulin and a crude biosynthesised sample of human insulin. The SFC purified standard
the biosynthesised human insulin samples were detected and quantified using liquid chromatographymass spectrometry (LC-MS) and protein sequencing techniques. SFC columns, i.e., silica, 2’ ethyl
pyridine, diol-HILIC, and the pentafluoro phenyl (PFP), were evaluated to determine the ideal column.
The PFP column gave the best results since it displayed good peak shapes, resolution, retention factors,
retention times, and the least relative standard deviation in comparison to the other columns. Therefore,
the aforementioned column was selected for further analysis using the biosynthesised human insulin,
whereby a column efficiency test was conducted on a semi-preparative scale, yielding 84% recovery.
Subsequently, the biological activities of the SFC purified standard sample of human insulin and
biosynthesised version were tested in vitro using a MTT assay. The results indicated that the biological
activities of the standard and biosynthesised human insulin derivatives were retained subsequent to SFC
purification. The biological activities were highly significant, with a p-value of < 0.0001.
From chapter three, band broadening and phase separation peaks were experienced during SFC
purification of the commercially available standard sample of human insulin and biosynthesised human
insulin. Therefore, in Chapter four (manuscript three), a SFC purification method was developed to purify
peptides at an analytical scale. A tetrapeptide [insulin β chain peptide (15-18)], octapeptide [angiotensin
II], nonapeptide [insulin β chain peptide (15-23)] were purified using four SFC columns, i.e., PFP, diolHILIC, silica, and 2’ ethyl pyridine. Subsequently, the 2’ ethyl pyridine column was selected for further
analysis based on the reproducibility, peak shapes, efficient separations and retention factors. The three
peptides were monitored using LC-MS analysis. The successful peptide recoveries ranged from 80-102%.
Chapter five pertains to the summary and conclusion drawn from the study and reflects on possible future
endeavours. The present study was successful in providing a more affordable and innovative approach for
the biosynthesis of human insulin. The work also successfully developed a rapid, greener, and more
efficient method of SFC purifying biosynthesised human insulin and peptides as opposed to conventional
HPLC purification methods. As far as we are aware, this study is the first of its kind to purify
biosynthesised human insulin and this combination of peptides using SFC purification techniques. Future
research studies can focus on the SFC purification of larger protein molecules and consider the use of
custom columns and or other modifiers for the improvement of the isolation of other highly sought after
biologics within the pharmaceutical industry.
Description
Doctoral Degree. University of KwaZulu-Natal, Durban.