Exploring the Unique Selectivity of Hydrophobic Cation Exchanger Nuvia cPrime for the Removal of a Major Process Impurity: A Case Study with IgM.

Background: Mixed-mode chromatography is becoming an important tool for downstream process purification, as it provides the selectivity and robustness unmatched by conventional singlemode chromatographic methods. The joint action of multiple functionalities present on the ligands of mixed-mode chromatography matrices effectively enhances the separation of target molecules from impurities.

Material And Methods: Using Nuvia cPrime as an example, we elucidate the separation principles of hydrophobic cation exchange mixed-mode chromatography and its difference from traditional strong cation exchangers.

Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction.

The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA.

Production of plasmid DNA for pharmaceutical use.

The concept of curing diseases at the genetic level was already introduced in the 1970s, but only the evolution of molecular biology and tools for genetic manipulation brought the idea into labs and clinics during the last 16 years. Viral and non-viral vectors and delivery systems were developed to transfer therapeutic genes into the target cells. In the case of non-viral approaches plasmid DNA has become a very promising gene delivery vector because it can easily be genetically manipulated and produced by cultivation of plasmid harbouring Escherichia coli and subsequent downstream processing, thus making production easy in comparison to other gene delivery vectors.

Reverse micellar extraction systems for the purification of pharmaceutical grade plasmid DNA.

Plasmid DNA as an active pharmaceutical ingredient (API) is gaining more and more importance. For the production of multigram quantities of this substance robust and scalable processes comprising several purification steps have to be designed. One main challenge is the initial separation of plasmid DNA and RNA in such a purification scheme.

Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use.

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein.

Immobilisation of a repressor protein for binding of plasmid DNA.

The use of plasmid DNA in gene therapy and genetic vaccination has increased the need for scalable and sustainable production processes. One key challenge for bioprocess engineering is the separation of plasmid DNA from structurally related impurities. Affinity purification procedures allow a highly selective capturing of the target molecule.

Production of supercoiled multimeric plasmid DNA for biopharmaceutical application.

Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance. Non-viral vectors like plasmid DNA are currently investigated in various clinical trials. Supercoiled multimeric plasmids are of particular interest for pharmaceutical purpose because they contain multiple copies of a therapeutic gene and can therefore be more efficient vectors.

Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer.

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation.

Intratumoral low-volume jet-injection for efficient nonviral gene transfer.

Jet-injection has become an applicable technology among other established nonviral delivery systems, such as particle bombardment or in vivo electroporation. The low-volume jet injector employed in this study uses compressed air to inject solutions of 1.5-10 microL containing naked DNA into the desired tissue.