Continuous process for reducing heterogeneity of therapeutic protein
Inventors
Gadgil, Himanshu • BANERJEE, Abir • Dyaga, Gopal • Pankhania, Ashvin • Londhe, Harshita • Rao, Deepika
Assignees
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Publication Number
US-11618768-B2
Publication Date
2023-04-04
Expiration Date
Abstract
The present disclosure relates to a bio-manufacturing process that enables continuous production of a therapeutic protein with reduced heterogeneity. The bio-manufacturing process disclosed herein utilizes a multi column chromatography system that performs the unit operations of reducing heterogeneity in a therapeutic protein, capturing the therapeutic protein and inactivating viruses, purifying the therapeutic protein, and polishing the purified therapeutic protein. The process disclosed herein can reduce heterogeneity of a therapeutic protein by reducing a proportion of basic isoforms of the therapeutic protein.
Core Innovation
The invention relates to a continuous process for reducing heterogeneity of a therapeutic protein by reducing one or more basic isoforms. The process uses a single multi-column chromatography system that includes first, second, third and fourth columns connected in sequential order in series or in parallel. The first column comprises carboxypeptidase B immobilized on Sepharose, and the flow-through from the first column is fed into the second column.
In the continuous process, a cell culture harvest comprising the therapeutic protein is fed into the first column, thereby reducing heterogeneity of the therapeutic protein. The method captures the therapeutic protein by feeding a flow-through from the first column into the second column. An eluate comprising the therapeutic protein is then fed from the second column into the third column to purify the therapeutic protein, and a flow-through comprising the therapeutic protein from the third column into the fourth column is fed to polish the therapeutic protein.
The disclosed approach is carried out as a continuous workflow using a multi-column chromatography system with sequential column-to-column feeding, including downstream capture, purification and polishing stages. The document further supports the approach with continuous workflow operation and analysis of charge-variant or impurity/charge-isoform outcomes, and it includes support for carboxypeptidase B immobilized on Sepharose resin effectiveness for basic-isoform reduction using residence time and loading density comparisons.
Claims Coverage
The patent includes two independent claims (clm-00001 and clm-00002). Across these claims, the key inventive coverage is a continuous, four-column multi-column chromatography system using a first carboxypeptidase B immobilized-on-Sepharose column to reduce basic isoforms, followed by sequential downstream capture, purification, and polishing through defined flow-through and eluate routing, with optional dependent refinements.
Single continuous multi-column chromatography system with sequentially connected four columns
A continuous process comprises reducing heterogeneity of a therapeutic protein in a single multi-column chromatography system comprising a first, second, third and fourth columns connected in sequential order in series or in parallel.
First carboxypeptidase B immobilized on Sepharose column for basic-isoform reduction
The single multi-column chromatography system includes a first column comprising carboxypeptidase B immobilized on Sepharose, wherein feeding of the therapeutic protein into the first column reduces one or more basic isoforms and thereby reduces heterogeneity.
Flow-through transfer from first to second column
The process feeds a flow-through from the first column into the second column, including continuous capture of the therapeutic protein in the flow-through of the first column.
Sequential eluate and flow-through routing across purification and polishing columns
The process feeds an eluate comprising the therapeutic protein from the second column into the third column to purify the therapeutic protein and feeds a flow-through comprising the therapeutic protein from the third column into the fourth column to polish the therapeutic protein.
Continuous reduction of heterogeneity using cell culture harvest input
Feeding a cell culture harvest comprising the therapeutic protein into the first column thereby reducing heterogeneity of the therapeutic protein.
Collectively, the independent claims center on a continuous four-column multi-column chromatography system in which a first column with carboxypeptidase B immobilized on Sepharose reduces basic isoforms to reduce heterogeneity, and subsequent columns receive defined flow-through and eluate inputs to capture, purify, and polish the therapeutic protein.
Stated Advantages
Reduces heterogeneity of a therapeutic protein.
Reduces one or more basic isoforms of the therapeutic protein.
Enables use of a single multi-column chromatography system in a continuous process.
Documented Applications
Continuous bio-manufacturing process for therapeutic proteins, specifically reducing therapeutic-protein heterogeneity by reducing basic isoforms using a single multi-column chromatography system.
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