Recombinant granulocyte colony-stimulating factor (filgrastim): Optimization of conjugation conditions with polyethylene glycol

Abstract

In order to create an active pharmaceutical substance of the drug with prolonged action the modification of recombinant human granulocyte colony-stimulating factor GCSF (filgrastim) with polyethylene glycol (PEG, M 21.5 kDa) was conducted. A method for preparation of PEG-filgrastim designed for the development and scaling-up of the technological process of production was described. Modification of proteins with PEG was performed by selective covalent attachment of the molecule alpha-methyl-PEG-propionaldehyde to the alpha-amino group of the N-terminal methionine amino acid residue of the recombinant GCSF. The conditions of the reaction, which provide the desired product yield at least 85% of the total protein, also high protein concentration in the reaction mixture (more than 9 mg/mL) and reduce consumption of PEG in terms of terminal alpha-amino group of the protein was chosen. The data of RP HPLC and MALDI-mass spectrometry showed that the produced drug modified by the N-terminal residue and contains no more than 10% of products with a high degree of modification.

Recombinant granulocyte colony stimulating factor (filgrastim) of prolonged action: Optimization of conditions for extraction and purification from inclusion bodies

“…Under these conditions, the concentration of the purified recombinant protein sample reached a value of 5 mg/mL. The concentration of the purified GCSF failed to meet the optimal conditions for its PEGylation and had to be increased to 12 mg/mL [21]. Therefore, in order to narrow the chromato graphic zone during IOC, different conditions were to be selected for the gradient elution.…”

“…Previously, it was shown that the effective modifi cation of the renatured GCSF occurs at a protein con centration of 12 mg/mL in Na 3 citrate buffer system at pH 6.0, and the preliminary renaturation step pro ceeds at pH 8.0 [21]. To combine all the processes in a single line, the conditions for the primary fraction ation of the recombinant GCSF were optimized after the IB solubilization and renaturation.…”

Site-selective protein conjugation at histidine

“…Prior to purification, the PEGylated IFN variants were treated with sodium triacetoxyborohydride to reduce the aryl ketone to prevent retro-Michael induced PEG deconjugation 64,108. Carbonyl reduction using a mild hydride reagent acts as a stabilisation step and is comparable to the analogous process conducted during reductive amination85,86,109–111 in the manufacture of pegfilgrastim (Neulasta®) 84. It has become evident even with maleimides that it is necessary to conduct a post-conjugation hydrolysis stabilisation step to avoid deconjugation 112–114…”

Production and Optimization of Site-Specific monoPEGylated Uricase Conjugates Using mPEG-Maleimide Through RP–HPLC Methodology

“…It was observed that the yeild of PEGylated uricase was highly dependent upon the molecular weight of the mPEG-mal molecule used. It is indispensible to find the optimum PEGylation reaction conditions which can maintain a high yield of the desired product during subsequent scaling, in order to preserve the biological activity of the enyzme and minimize side effects upon usage in therapeutic applications [19]. The PEGylation reaction can be optimized to maximize the yield of the desired product while limiting production of undesired byproducts.…”