Roman Necina

Short monolithic columns as stationary phases for biochromatography.

Monolithic supports represent a novel type of stationary phases for liquid and gas chromatography, for capillary electrochromatography, and as supports for bioconversion and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, monolithic supports are cast as continuous homogeneous phases. They represent an approach that provides high rates of mass transfer at lower pressure drops as well as high efficiencies even at elevated flow rates. Therefore, much faster separations are possible and the productivity of chromatographic processes can be increased by at least one order of magnitude as compared to traditional chromatographic columns packed with porous particles. Besides the speed, the nature of the pores allows easy access even in the case of large molecules, which make monolithic supports a method of choice for the separation of nanoparticles like pDNA and viruses. Finally, for the optimal purification of larger biomolecules, the chromatographic column needs to be short. This enhances the speed of the separation process and reduces backpressure, unspecific binding, product degradation and minor changes in the structure of the biomolecule, without sacrificing resolution. Short Monolithic Columns (SMC) were engineered to combine both features and have the potential of becoming the method of choice for the purification of larger biomolecules and nanopartides on the semi-preparative scale.

Affinity chromatography of human blood coagulation factor VIII on monoliths with peptides from a combinatorial library

FVIII is a very complex molecule of great therapeutic significance. It is purified by a sequence of chromatographic steps including immunoaffinity chromatography. A peptide affinity chromatography method has been developed using peptides derived from a combinatorial library. Spot technology using cellulose sheets has been applied for this purpose. The dual positional scanning strategy was used for identification of the amino acids in random positions. Approximately 5000 possible candidates found in the first screening round were reduced to a panel of 36. Six candidates have been selected empirically. Five peptides seem to be directed against the light chain of FVIII, one peptide seems to be directed against the heavy chain. The peptides have been immobilized on conventional beaded material and CIM polymethacrylate monoliths. Much better performance with respect to capacity and selectivity has been observed with the monolithic material. Exposure of the ligand and its ensuing accessibility are responsible for these properties.

Continuous matrix-assisted refolding of proteins.

A refolding reactor was developed for continuous matrix-assisted refolding of proteins. The reactor was composed of an annular chromatography system and an ultrafiltration system to recycle aggregated proteins produced during the refolding reaction. The feed solution containing the denatured protein was continuously fed to the rotating bed perfused with buffer promoting folding of the protein. As the protein passed through the column, it was separated from chaotropic and reducing agents and the refolding process took place. Native proteins and aggregates could be continuously separated due to different molecular size. The exit stream containing aggregates was collected, concentrated by ultrafiltration and recycled to the feed solution. The high concentrations of chaotropic and reducing agents in the feed solution enabled dissociation of the recycled aggregates and consequently were fed again to the refolding reactor. When the initial feed mixture of denatured protein is used up, only buffer-containing chaotropic agents and recycled aggregates are fully converted to native protein. This process resulted in a stoichiometric conversion from the denatured protein to its correctly folded native state. The system was tested with bovine alpha-lactalbumin as model protein. Superdex 75 PrepGrade was used as size-exclusion medium. The yield of 30% active monomer in the batch process was improved to 41% at a recycling rate of 65%. Assuming that the aggregates can be redissolved and recycled into the feed stream in a quantitative manner, a refolding yield close to 100% is possible. The method can be also applied to other chromatographic principles suited for the separation of aggregates.

Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density.

A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content.

Peptide affinity chromatography of human clotting factor VIII: Screening of the vWF-binding domain

The region of von Willebrand factor, which is involved in the complex formation with factor VIII, was used to generate a panel of octapeptides. A peptide ladder was generated from the von Willebrand factor region aa40 to aa100 and was synthesized on cellulose membranes by spot technology. Four peptides with affinity for factor VIII were identified by incubation with plasma derived factor VIII and recombinant factor VIII. The peptides denoted as 010 (LCPPGMVRHE), 011 (RCPCFHQGK), 014 (CFHQGKEYA) and 015 (RDRKWNCTDHVC) were further characterized by real-time interaction analysis and small scale affinity chromatography. Biotinylated peptides were used for blotting assays. These experiments showed that the peptides are directed against the light chain of FVIII. We consider these peptides as valuable tools for in situ labeling and also as ligands suitable for affinity chromatography.

Determination of immune complexes by high-performanced gel chromatography (positive cooperativity of antibody-antigen reaction)

Positive cooperativity of antibody-antigen complex formation using human Cu/Zn superoxide dismutase and a murine monoclonal antibody as model is demonstrated by high-performance gel chromatography using TSK G3000sw and TSK G4000sw columns. Three different antibody-antigen complexes named Type I, II and III composed from two antigen molecules+two antibody molecules, two antigen molecules+one antibody molecule and one antigen molecule+one antibody molecule could be separated. The degree and ratio of complexes present in solution depends mainly on the ratios of antibody and antigen.

Large Scale CGMP Manufacturing of pDNA for Gene Therapy

The therapeutic fields for pDNA presently are in cardiovascular diseases with genes for expression of angiopoietin 4, DEL-1, Erythropoietin, monocyte contracting protein-1 (MCPI), nitric oxidase and VEGF. Therapeutic doses required are often 1 mg to 10 mg of pDNA. In oncology genes for CEA-B 7.1, IL2, IL12, p58 are under clinical investigations in dosages ranging from 10 microgram to 10 mg of pDNA. In gene repair cystic fibrosis, factor VIII, factor IX and interferon alpha are targets with therapeutic doses of 10 microgram to 100 microgram of pDNA. As anti-infectiva for vaccination, Hepatitis B infections, HIV infections, malaria, and tuberculosis are in the focus with therapeutic doses of 10 microgram to 100 microgram of pDNA.