Industrially scalable process for recovering biologically active recombinant carrier proteins

An industrially scalable method for preparation of Cross Reactive Material 197 (CRM197), the method comprising the steps of:

• a) transformation of Escherichia coli with a gene coding for CRM197 using a plasmid vector,
• b) culturing the transformed Escherichia coli in suitable culture medium under suitable conditions,
• c) isolation and purification of inclusion bodies,
• d) denaturation and solubilization of inclusion bodies at high pH value ranging from 9 to 14,
• e) followed by pH adjustment within a range of 6 to 8.5, preferably at 8 of the solubilized protein using in-line static mixer for a period of 0.1 to 200 msec, to produce refolded protein,
• f) intermediate purification of the refolded protein using ion exchange chromatography to obtain >90% pure native CRM197 and
• g) the semi purified CRM197 obtained in step (f) is further purified by one or more chromatographic separation using anion exchange chromatography, hydrophobic interaction chromatography, metal & dye affinity chromatography, affinity chromatography, multimodal chromatography, hydroxyapatite chromatography and size exclusion chromatography to obtain CRM197.

A process for preparation of recombinant carrier proteins selected from tetanus toxoid (TT), diphtheria toxoid (DT), Cross Reactive Material 197 (CRM197), Haemophilus influenzae protein D, Neisseria outer membrane protein, Pertussis toxin (PT), Pertactin (PRN) and Filamentous Hemagglutinin (FHA) from a prokaryotic expression system, wherein protein is expressed as inclusion bodies which comprises:

• a. transformation of bacterial cells with a desired gene coding for carrier proteins using a plasmid vector,
• b. culturing the transformed bacterial cells in chemically defined media supplemented with glucose as carbon source wherein pH is maintained at 5-9 and at temperature of 20-40° C.;
• c. lysing bacterial cells by mechanical means above 800-1200 bars thereby producing a lysate containing inclusion bodies and cellular components;
• d. clarifying the cell lysate by separating solids from the solution;
• e. isolation and purification of inclusion bodies (IBs) by using buffers selected from carbonate, bicarbonate, Tris, borate, Glycine and NaOH to remove cellular contaminants to form a pellet of purified IBs;
• f. denaturation and solubilisation of (IBs) at a high pH value ranging from 9 to 14 using at least one buffer selected from carbonate, bicarbonate, Tris, borate, Glycine and NaOH buffer;
• g. rapid pH adjustment within a range of 6 to 8.5, preferably at 8 of solubilized carrier proteins using acid containing redox systems and inline static mixer for a period of 0.1 to 200 msec, to produce refolded protein,
• h. intermediate purification of the refolded protein using ion exchange chromatography to obtain >90% pure native carrier protein and
• i. the semi purified protein obtained in step (h) is further purified by one or more chromatographic separations using anion exchange chromatography, hydrophobic interaction chromatography, metal & dye affinity chromatography, affinity chromatography, multimodal chromatography, hydroxyapatite chromatography and size exclusion chromatography to obtain to obtain carrier protein.