Industrially scalable process for recovering biologically active recombinant carrier proteins
Inventors
Goel, Akshay • Joglekar, Tushar • Tiwari, Krishnanand • Mishra, Yogesh • Mantena, Narender Dev • Datla, Mahima
Assignees
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Publication Number
US-10927149-B2
Publication Date
2021-02-23
Expiration Date
Abstract
The present invention relates to a preparation method which is performed by expressing the recombinant carrier proteins in Escherichia coli and purification thereof. More particular, the invention relates to industrially scalable process for the recovery of recombinant carrier proteins.
Core Innovation
The invention relates to an industrially scalable method for preparation of 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). The method includes transformation of Escherichia coli with a desired gene coding for the carrier protein using a plasmid vector and culturing the transformed Escherichia coli in suitable culture medium. The expressed protein is isolated and purified as inclusion bodies.
The invention performs denaturation and solubilization of the inclusion bodies at high pH ranging from 9 to 14, followed by pH adjustment within a range of 6 to 8.5, preferably at 8, of the solubilized protein. The pH adjustment is carried out using an in-line static mixer for a period of 0.1 to 200 msec to produce a refolded protein. The approach is further characterized by process variants that include acid containing redox systems in combination with the rapid in-line static mixer pH adjustment.
After refolding, the refolded protein is purified by intermediate purification using ion exchange chromatography to obtain greater than 90% pure native carrier protein. The semi purified protein is then further purified by one or more chromatographic separation methods selected from anion exchange chromatography, hydrophobic interaction chromatography, metal & dye affinity chromatography, affinity chromatography, multimodal chromatography, hydroxyapatite chromatography and size exclusion chromatography to obtain the carrier protein.
Claims Coverage
The document contains three independent claims directed to industrially scalable processes for producing specified prokaryotic recombinant carrier proteins, including general carrier-protein embodiments, a CRM197-specific embodiment, and a parameterized prokaryotic expression/inclusion-body/refolding/purification process with defined media and mechanical lysis constraints.
Industrially scalable carrier protein preparation from E. coli inclusion bodies with high-pH solubilization and in-line static mixer refolding
The method comprises transformation of Escherichia coli with a desired gene coding for selected carrier proteins using a plasmid vector; culturing; isolation and purification of inclusion bodies; denaturation and solubilization of inclusion bodies at high pH value ranging from 9 to 14; pH adjustment within a range of 6 to 8.5, preferably at 8, using in-line static mixer for a period of 0.1 to 200 msec to produce a refolded protein; intermediate purification using ion exchange chromatography to obtain >90% pure native carrier protein; and further purification 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.
Industrially scalable CRM197 preparation from E. coli inclusion bodies with high-pH solubilization and in-line static mixer refolding
An industrially scalable method for preparation of Cross Reactive Material 197 (CRM197) includes transformation of Escherichia coli with a gene coding for CRM197 using a plasmid vector; culturing; isolation and purification of inclusion bodies; denaturation and solubilization of inclusion bodies at high pH value ranging from 9 to 14; pH adjustment within a range of 6 to 8.5, preferably at 8, using in-line static mixer for a period of 0.1 to 200 msec to produce refolded protein; intermediate purification of the refolded protein using ion exchange chromatography to obtain >90% pure native CRM197; and further purification of the semi purified CRM197 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.
Recombinant carrier protein process from prokaryotic expression system with defined media, high-pH inclusion-body solubilization, acid containing redox inline static mixer refolding, and multi-step chromatography
A process for preparation of recombinant carrier proteins selected from TT, DT, CRM197, Haemophilus influenzae protein D, Neisseria outer membrane protein, PT, PRN and FHA from a prokaryotic expression system comprises expressing the protein as inclusion bodies; transformation with a desired gene using a plasmid vector; culturing in chemically defined media supplemented with glucose as carbon source wherein pH is maintained at 5-9 and at temperature of 20-40°C; lysing bacterial cells by mechanical means above 800-1200 bars to produce a lysate containing inclusion bodies and cellular components; clarifying by separating solids from solution; isolating and purifying inclusion bodies using buffers selected from carbonate, bicarbonate, Tris, borate, Glycine and NaOH to form a pellet of purified IBs; denaturation and solubilisation of the IBs at high pH value ranging from 9 to 14 using at least one buffer selected from carbonate, bicarbonate, Tris, borate, Glycine and NaOH; 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; intermediate purification using ion exchange chromatography to obtain >90% pure native carrier protein; and further purification using 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 carrier protein.
Overall, the independent claims cover industrially scalable production of TT, DT, CRM197, Haemophilus influenzae protein D, Neisseria outer membrane protein, PT, PRN and FHA, including CRM197 specifically, by expressing proteins as inclusion bodies, solubilizing at high pH 9-14, rapidly refolding by pH adjustment to 6-8.5 using an in-line static mixer for 0.1-200 msec, optionally with acid containing redox systems, and performing ion exchange capture to obtain >90% pure native protein followed by additional chromatographic polishing using multiple specified chromatographic types.
Stated Advantages
Provides intermediate purification yielding >90% pure native carrier protein.
Enables industrially scalable preparation of the stated carrier proteins.
Produces refolded protein by rapid pH adjustment using an in-line static mixer.
Provides native carrier protein purification by combining ion exchange chromatography with one or more additional chromatographic separation methods.
Documented Applications
Preparation of 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) for industrial-scale manufacturing.
Preparation of Cross Reactive Material 197 (CRM197) in an industrially scalable method.
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