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The Fourier-transform infrared (FT-IR) spectroscopic approach has been employed to understand the recombinant human G-CSF (rhG-CSF) protein accumulation, secondary structure, and thermal stability in Escherichia coli grown under a temperature shift strategy (37 and 28°C) in various media formulations. The choline + sodium pyruvate (37°C) and sodium pyruvate (28°C) formulations have shown the highest inclusion body (IB) accumulation of 0.41 and 0.46 mg/mL, respectively. Furthermore, insights on the structure of the rhG-CSF within IBs and intact cells have been investigated through secondary structure analysis. Thermal stability experiments were also carried out to explain the pattern of the second derivative structure of rhG-CSF. The studies showed that choline + sodium pyruvate formulation has preserved the protein secondary structure even at 82°C. Overall, the FT-IR spectroscopic technique can also be adopted to accelerate the characterization of other recombinant therapeutic proteins of E. coli origin.

Recombinant proteins are frequently hampered by aggregation during the refolding and purification process. A simple and rapid method for in vitro refolding and purification of recombinant human parathyroid hormone (rhPTH 1–34) expressed in Escherichia coli with protein folding size exclusion chromatography (PF-SEC) was developed in the present work. Discrete effects of potential solution additives such as urea, polypolyethylene glycol, proline, and maltose on the refolding with simultaneous purification of rhPTH were investigated. The results of individual additives indicated that both maltose and proline had remarkable influences on the efficiency of refolding with a recovery yield of 65 and 66 % respectively. Further, the synergistic effect of these additives on refolding was also explored. These results demonstrate that the additive combinations are more effective for inhibiting protein aggregation during purification of rhPTH in terms of recovery yield, purity, and specific activity. The maltose and proline combination system achieved the highest renatured rhPTH having a recovery yield of 78 %, a purity of ≥99 %, and a specific activity of 3.31 × 103 cAMP pM/cell respectively, when compared to the classical dilution method yield (41 %) and purity (97 %). In addition, the role of maltose and proline in a combined system on protein aggregation and refolding has been explained. The molecular docking (in silico) scores of maltose (−10.91) and proline (−9.0) support the in vitro results.

This work, for the first time reports the optimization of rhG-CSF in Escherichia coli using novel media supplements. The rhG-CSF production was optimized using Plackett–Burman (P–B) and Central composite design (CCD) operated in an integrated process. Three novel media supplements (sodium pyruvate, methylglucoside and choline) along with two conventional essential elements (sodium citrate and magnesium sulphate) were screened with a P–B design. An integrated application of CCD represented a statistically significant response (R2 = 98.56 % and P < 0.0001) for the factors under the study, that suggested a quadratic model for the prediction of rhG-CSF. The CCD results reveal the highest rhG-CSF production of 31.88 µg/mL with optimized components comprising 1.75 g/L of sodium pyruvate, 1.5 g/L of methylglucoside, 2.215 g/L of sodium citrate, 1.7 g/L of choline and 0.036 g/L of magnesium sulphate.

Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0 M) and arginine (0.6 M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6 × 108 IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF.