Compositions comprising high concentration of biologically active molecules and processes for preparing the same
Inventors
Draghia-Akli, Ruxandra • Hebel, Henry • Cai, Ying
Assignees
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Publication Number
US-11453855-B2
Publication Date
2022-09-27
Expiration Date
Abstract
Large scale processes for producing high purity samples of biologically active molecules of interest from bacterial cells are disclosed. The methods comprise the steps of producing a lysate solution by contacting a cell suspension of said plurality of cells with lysis solution; neutralizing said lysate solution with a neutralizing solution to produce a dispersion that comprises neutralized lysate solution and debris; filtering the dispersion through at least one filter; performing ion exchange separation on said neutralized lysate solution to produce an ion exchange eluate; and performing hydrophobic interaction separation on said ion exchange eluate to produce a hydrophobic interaction solution. Further, provided are compositions comprising large scale amounts of plasmid DNA produced by the disclosed large scale processes.
Core Innovation
The invention is directed to a large scale process for producing high purity samples of at least one biologically active molecule of interest from a plurality of bacterial cells. The process produces a lysate solution by contacting a cell suspension of the plurality of bacterial cells with a lysis solution, then neutralizes the lysate solution with a neutralizing solution to produce a dispersion comprising neutralized lysate solution and debris. The dispersion is filtered through at least one filter to separate debris from the neutralized lysate solution.
After debris separation, the neutralized lysate solution is subjected to ion exchange separation to produce an ion exchange eluate. The ion exchange eluate is then processed by hydrophobic interaction separation to produce a hydrophobic interaction solution. This sequential combination of ion exchange separation followed by hydrophobic interaction separation is used as part of the large scale purification workflow.
In the process, large scale transitions between successive steps are characterized as substantially continuous. One described arrangement includes substantially continuous transitions occurring from the contacting step through the passing of the filtered solution step, while also specifying an approach that uses a bubble column mixer for neutralizing and includes downstream steps of passing through an ion-exchange column and through a hydrophobic interaction column or a hydrophobic interaction membrane, followed by ultrafiltration to form a high purity sample.
Claims Coverage
Two independent claims are provided. Each claim specifies a large scale sequential purification workflow that includes lysing, neutralizing, filtering, ion exchange separation, hydrophobic interaction separation, and in one claim ultrafiltration, with one claim additionally requiring substantially continuous transitions between steps.
Large scale lysate preparation and neutralization to obtain a neutralized lysate dispersion
Producing a lysate solution by contacting a cell suspension of said plurality of bacterial cells with lysis solution; neutralizing said lysate solution with a neutralizing solution to produce a dispersion comprising neutralized lysate solution and debris.
Debris removal by filtration to form a clarified neutralized lysate
Filtering the dispersion through at least one filter to separate debris from the neutralized lysate solution.
Sequential ion exchange separation followed by hydrophobic interaction separation
Performing ion exchange separation on said neutralized lysate solution to produce an ion exchange eluate; and performing hydrophobic interaction separation on said ion exchange eluate to produce a hydrophobic interaction solution.
Substantially continuous transitions between process steps in a large scale workflow
Wherein each transition from one step to a subsequent step in the large scale process from the contacting step to the passing the filtered solution step occur substantially continuously.
Bubble column mixer neutralization, dual-stage filtering, and downstream ion exchange/hydrophobic interaction with ultrafiltration
Neutralizing the lysate solution by mixing a neutralization solution into the lysate solution with a bubble column mixer to form a neutralized mixture; filtering the neutralized mixture through a primary filter and a secondary filter to form a filtered solution; passing the filtered solution through an ion-exchange column to form a ion-exchange solution; passing the ion-exchange solution through a hydrophobic interaction column or a hydrophobic interaction membrane to form a hydrophobic interaction solution; and ultrafiltration of the hydrophobic interaction solution to form a high purity sample of at least one biologically active molecules of interest.
The independent claim set centers on a large scale purification process that sequentially combines lysate generation and neutralization, filtration to remove debris, ion exchange separation, and hydrophobic interaction separation, with one independent claim further requiring substantially continuous transitions and a specific sequence of mixer, staged filtration, column/membrane formats, and ultrafiltration to obtain a high purity sample.
Stated Advantages
Produces high purity samples of at least one biologically active molecule of interest from a plurality of bacterial cells.
Enables large scale processing with substantially continuous transitions between successive steps in the process.
Documented Applications
No documented applications found
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