Purification of CRM 197 from bacteria
Inventors
Blattner, Frederick R. • Frisch, David • Landry, Charles • Brandon, John
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11174284-B2
Publication Date
2021-11-16
Expiration Date
Abstract
The present invention relates to the use of redox agents for purification of the CRM 197 variant of diphtheria toxin. The invention further relates to multi-step purification of CRM 197 from bacterial fermentates.
Core Innovation
The invention relates to purifying CRM 197 obtained from bacterial cell culture by contacting a bacterial cell lysate comprising CRM 197 or a fermentation medium comprising CRM 197 with a redox agent and optionally a solubilizing agent, followed by chromatographic purification. The method includes mixed mode cation exchange chromatography using media having a ligand comprising an aromatic group substituted with a primary amine and a carboxylic acid group, under conditions in which CRM 197 binds and is eluted to generate a first eluate comprising CRM 197. Subsequent steps contact the first eluate with an anion exchange chromatography media to generate a second eluate comprising CRM 197, and then contact the second eluate with a hydroxyapatite chromatography media under conditions in which CRM 197 binds and is eluted.
A central aspect is mitigating redox-driven degradation of recombinant CRM 197 and maintaining intramolecular disulfide stability during purification. The partial content describes use of an oxidized redox agent, including glutathione disulfide/cystine and additional oxidized species such as cystamine and diphenyl disulfide, as well as other redox agents, and optional use of a solubilizing agent such as L-arginine with the bacterial lysate or fermentation medium. The approach is described as supporting reduced proteolysis products and improved maintenance of disulfide-related structural integrity.
The purification scheme is further characterized by multi-step chromatography while restricting chromatographic scope between the three principal steps. The method proceeds from the mixed mode cation exchange step to an anion exchange step and then to hydroxyapatite, with the limitation that no additional chromatographic purifications occur between the steps. The partial content also notes process optimizations such as reducing the number of steps and using alternate resin order to eliminate certain intermediate hydrophobic interaction chromatography or related intermediate steps.
Claims Coverage
The partial content provides one independent claim that covers a purification method using three sequential chromatographic stages with redox assistance and optional solubilizing agent, while explicitly limiting that no additional chromatographic purifications occur between the stages.
Redox-assisted purification with three chromatography stages without interposed chromatographic purifications
A method for purifying CRM 197 obtained from a bacterial cell culture by contacting a bacterial cell lysate comprising CRM 197 or a fermentation medium comprising CRM 197 with a redox agent and optionally a solubilizing agent, then performing mixed mode cation exchange chromatography using a specified aromatic/primary amine/carboxylic acid ligand media to bind and elute CRM 197, contacting the first eluate with an anion exchange chromatography media to generate a second eluate comprising CRM 197, and contacting the second eluate with hydroxyapatite chromatography media to bind and elute CRM 197, wherein no additional chromatographic purifications steps occur between the three stages.
Across the provided independent claim, the principal coverage is the combination of redox and optional solubilizing assistance with a defined three-stage purification sequence together with an explicit restriction that no additional chromatographic purifications occur between the stages.
Stated Advantages
Reduced proteolysis products during CRM 197 purification when a redox agent is used.
Maintenance of intramolecular disulfide stability for recombinant CRM 197.
Improved dimer-to-monomer conversion behavior after heat treatment of a stored CRM 197 formulation.
Process optimization by reducing the number of purification steps.
Elimination of certain intermediate steps via alternate resin order.
Documented Applications
Vaccine conjugation applications for CRM 197, as indicated by conjugate-related products and vaccines including Menveo, Prevnar, Menjugate, Meningitec, Vaxem-Hib, and HibTITER.
Interested in licensing this patent?