Integrated process for the production of therapeutics (human albumin, immunoglobulins, clotting factor VIII and clotting factor IX) from human plasma
Inventors
VADDE, NEELIMA • Samaddar, Mitali • NUVULA, ASHOK • CHAKRABORTY, ZINIA • DUGGINENI, SWAPNA SAGAR • Komath, Uma Devi
Assignees
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Publication Number
US-9663553-B2
Publication Date
2017-05-30
Expiration Date
Abstract
The invention is an integrated process for the isolation and purification of plasma derived clotting Factor VIII, clotting Factor IX, albumin and immunoglobulin without the use of ethanol precipitation. The integrated process comprises of sequential chromatography steps of gel filtration, anion exchange and cation exchange chromatographies, followed by viral inactivation and removal steps. The present invention has the advantage of being a mild process that does not denature or aggregate the proteins and provides high yields of several therapeutic grade plasma proteins from a given volume of plasma.
Core Innovation
The invention describes an integrated process for the isolation and purification of plasma derived clotting Factor VIII, clotting Factor IX, albumin and immunoglobulin (IgG) without the use of ethanol precipitation. Plasma is first subjected to gel filtration chromatography to obtain three fractions, Fraction I, Fraction II and Fraction III, which serve as the shared starting step for routing different plasma proteins into different downstream purification paths.
After gel filtration, Fraction I and Fraction III are further subjected to chromatographic separations using anion and cation exchange resins. The process includes viral inactivation and recovery of isolated and purified clotting Factor VIII from Fraction I and clotting Factor IX, albumin and immunoglobulin (IgG) from Fraction III, while maintaining an integrated sequence based on chromatography and viral inactivation.
The documented process elements further emphasize virus inactivation and removal integrated with chromatography-based purification to maintain purified proteins while handling multiple targets in parallel fractions. Fraction I and/or Fraction III are processed through additional chromatographic steps, including exchange and resin binding modes, followed by viral inactivation and removal steps such as solvent-detergent treatment, low-pH treatment, nanofiltration, freeze-drying, and heat treatment, with further concentration and diafiltration in later steps.
Claims Coverage
The document provides one independent claim covering an integrated, ethanol-precipitation-free workflow with gel filtration followed by further anion and cation exchange separations and viral inactivation, with protein-specific recovery routing from Fraction I versus Fraction III. Dependent claims add further inventive features that refine the chromatography mode used and specify additional viral inactivation and processing steps for Factor VIII, Factor IX, albumin, and IgG.
Ethanol-precipitation-free integrated gel filtration into three fractions
A process for the isolation and purification of plasma derived clotting Factor VIII, clotting Factor IX, albumin and immunoglobulin (IgG) without the use of ethanol precipitation, comprising subjecting plasma to gel filtration chromatography to obtain three fractions (Fraction I, Fraction II and Fraction III).
Exchange resin separations of Fraction I and Fraction III
Further subjecting Fraction I and Fraction III to further chromatographic separations using anion and cation exchange resins.
Fraction-directed viral inactivation and protein recovery
Subjecting to viral inactivation and recovering isolated and purified clotting Factor VIII from Fraction I, and clotting Factor IX, albumin and immunoglobulin (IgG) from Fraction III.
Factor VIII recovery from Fraction I using cation exchange
Isolated and purified clotting Factor VIII is obtained from Fraction I by further chromatographic separation using a cation exchange column.
Factor IX bound fraction and IgG/albumin unbound recovery from Fraction III
Factor IX, albumin, and IgG are obtained by fractionating Fraction III on an anion exchange column to produce a bound Fraction IX fraction and recover IgG and albumin from an unbound fraction.
Factor IX viral inactivation followed by affinity or metal chelate binding
Factor IX is obtained from a bound fraction by performing solvent-detergent viral inactivation, followed by affinity or metal chelate chromatography to bind Factor IX, and then employing gel filtration chromatography and nanofiltration for viral removal.
Albumin purification from unbound fraction using anion then cation exchange with low-pH viral inactivation and heat treatment
Albumin is obtained from an unbound fraction containing albumin and IgG using anion-then-cation exchange chromatography, followed by viral inactivation steps using caprylate at low pH and incubation, then heat treatment, filtration, further concentration, addition of formulation excipients, and pasteurization.
IgG purification from unbound fraction using anion exchange, low-pH and S/D inactivation, and cation exchange with nanofiltration and diafiltration
IgG is obtained from an unbound fraction using anion-exchange chromatography, low-pH treatment followed by solvent-detergent viral inactivation, cation-exchange chromatography to collect IgG eluate, nanofiltration with diafiltration concentration, and final formulation with excipients.
Across the independent claim and its dependents, the core coverage is an integrated, ethanol-precipitation-free process built on gel filtration into Fraction I/II/III, followed by anion/cation exchange-based fraction separations, then viral inactivation with fraction-directed recovery. Dependent features further specify cation-versus-anion exchange roles, bound versus unbound fraction recovery for Factor IX versus IgG/albumin, and additional binding modes for Factor IX, with expanded viral inactivation and downstream concentration and processing for Factor VIII, Factor IX, albumin, and IgG.
Stated Advantages
The process isolates and purifies plasma derived clotting Factor VIII, clotting Factor IX, albumin and immunoglobulin (IgG) without the use of ethanol precipitation.
The integrated chromatography-based workflow together with viral inactivation and recovery yields isolated and purified proteins from defined fractions.
Documented quality control indicates impurity reduction and protein integrity measures, including aggregate and IgG/functional and impurity-related measures.
Documented Applications
Integrated purification of therapeutic-grade human albumin, IgG, Factor VIII, and Factor IX from plasma using an all-chromatography fractionation scheme without ethanol precipitation.
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