Compositions comprising high concentration of biologically active molecules and processes for preparing the same

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.

Description

CROSS REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 939,792, filed May 23, 2007, the contents of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to bulk compositions comprising biologically active molecules such as plasmids in high concentrations and processes for producing such compositions.BACKGROUND OF THE INVENTION[0003]There are numerous uses of biologically active molecules produced in and isolated from cells. U.S. Publication Serial No. 20050014245, which is incorporated herein by reference, discloses devices and methods for biomaterial production. Biologically active molecules include proteins and nucleic acid molecules. Production of such molecules in living cells offers numerous advantages over alternative productions methods but isolation and purification issues arise when extracting biologically active molecules from cells. There are various components ...

AI Technical Summary

Benefits of technology

[0015]A further aspect of the present invention comprises large scale processes for producing high purity samples of at least one biologically active molecule of interest from bacterial cells, comprising the steps of: contacting the bacterial cells in a dispersion of cells with lysis solution to form a lysate solution; 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

Problems solved by technology

Production of such molecules in living cells offers numerous advantages over alternative productions methods but isolation and purification issues arise when extracting biologically active molecules from cells.

Cell lysis and the subsequent treatment steps used to prepare a process stream for purification, are the most difficult, complex, and important steps in any plasmid process.

The search for an optimal method, one which is continuous, scaleable, and yielding high quality product that can be formulated at high concentration irrespective of plasmid size, has been an obstacle in getting acceptable processes to commercial capability.

Unfortunately, this method presents significant challenges for large scale preparations (or scaled-up production).

However, this is impractical at large scale, where volumes may be in the range of tens or hundreds of liters.

Common techniques for mixing large volumes of liquid, such as impeller mixing, are problematic because as some cells begin to lyse after initial mixing, they release genomic DNA which dramatically increases solution viscosity.

This increase in viscosity significantly interferes with further mixing.

Another challenge is that excessive incubation at high pH after addition of alkaline lysis solution can lead to permanent denaturation of the plasmid, making it unsuitable for most subsequent uses, especially for therapeutic purposes.

Large amounts of host genomic DNA and endotoxin are present in the flocculent precipitate and if they become mixed with the plasmid they can be difficult to separate from the plasmid during subsequent purification.

There are a variety of existing methods to purify plasmids; however, these methods are not suitable for large scale preparations.

Laboratory scale purification techniques cannot simply be scaled up for the volumes involved in large scale plasmid preparation.

In producing large quantities of plasmid DNA at high concentration, a problem exists in maintaining the plasmid as supercoiled and open circle relaxed form.

Storage conditions generally require high salt, and molecular degradation over time remains a problem even in the presence of salt.

Many existing purification methods rely upon the use of potentially dangerous, toxic, mutagenic or contaminated substances, and / or expensive substances or equipment, which, again, are not desirable for large scale preparations.

Some existing purification methods utilize the use of enzyme to digest protein for eventual elimination and such enzymes are costly for large scale production and can cause a risk of biologic contamination.

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