Biomedical subjects

A Buchacher

Publications and source records attributed to A Buchacher.

22 records · Page 2Linked to original sources

PubMed1990

Isolation of isoproteins from monoclonal antibodies and recombinant proteins by chromatofocusing.

A fast protein liquid chromatographic method for the preparative separation of the various isoproteins is described. Highly purified human monoclonal antibodies, recombinant human superoxide dismutase and human superoxide dismutase from erythrocytes were used as starting material. The isoproteins were separated by chromatofocusing on Mono P columns. A very narrow pH gradient was applied to achieve complete separation of the isoproteins. The prepurification steps and the pretreatment of the samples to achieve optimum resolution are described in detail. The method is also applicable to extremely basic monoclonal antibodies (pI = 9). The successful separation was checked by isoelectric focusing in immobilized pH gradients (Immobilines). The future of these methods is discussed, because for many different biochemical and biophysical investigations pure and homogeneous isoproteins are necessary.

Antibodies, Monoclonal↗

PubMed1989

Comparison of protein A, protein G and copolymerized hydroxyapatite for the purification of human monoclonal antibodies.

Protein A Superose, protein G Sepharose fast flow and copolymerized hydroxyapatite were used for the purification of human monoclonal antibodies against HIV 1. Both desalted culture supernatant and a prepurified protein solution were used as starting materials. The different runs were compared with respect to yield and recovery of biological activity. The biological activity (specific reactivity) was checked by antigen enzyme-linked immunosorbent assay with recombinant antigen. The human monoclonal antibodies could not be selectively eluted from the hydroxyapatite but elution could be effected from the protein A Superose at pH 4.0 and from protein G at pH 3.0. The eluted immunoglobulin G was distributed over a broad pH range when protein G Superose was used. Biologically active material could be obtained from protein A Superose and protein G Sepharose fast flow.

Antibodies, Monoclonal↗

PubMedDate not supplied

Pilot scale production of a human monoclonal antibody against human immunodeficiency virus HIV-1.

Human monoclonal antibodies against the transmembrane protein gp41 of HIV-1 were isolated and purified on a pilot scale. A purification scheme was established for the production of human monoclonal antibodies on the gram scale. 50 1 of culture supernatant can be treated in one purification cycle. The hybridomas were mass cultured in an airlift fermenter. The culture broth was clarified by microfiltration and chromatographed on CM-Sepharose fast flow and protein A Superose. Scale up of the high performance affinity chromatography from 1 ml protein A Superose up to 40 ml is described. All desalting steps were performed by gel filtration on Sephadex G-25 coarse. The yield of the whole purification procedure is in the range of 50-60%. The purity is higher than 99.9%. DNA and reverse transcriptase could not be detected. The whole method is designed as a basis for scale up to industrial scale. Results from quality control assays have proven the validity of this approach.

Antibodies, Monoclonal↗

PubMedDate not supplied

Continuous removal of protein aggregates by annular chromatography.

The removal of polymeric proteins from their monomers is a frequently encountered separation task, especially in the polishing step of therapeutic proteins. Continuous separation of protein polymers from monomers by annular chromatography using size exclusion chromatography has been studied regarding the resolution, recovery, fouling, and productivity and has been compared to conventional chromatography. An IgG preparation rich in aggregates was used as a model protein mixture. Under conditions that maximized the throughput, the polymers could be separated from the monomers, but baseline separation could not be achieved. Baseline separation was also not possible in batch mode using equivalent conditions, which was also confirmed by computer simulation. For separation of the aggregates from the product the entire available separation space (360 degrees ) was indispensable. Therefore only cyclic, discontinuous regeneration could be carried out. Loading was identified as a critical step, since the concentrated protein solution evaded into the headspace instead of migrating into the gel where viscous fingering often occurs in conventional chromatography. The productivity of annular chromatography was two times higher than that of the conventional batch chromatography, and the buffer consumption was reduced to half the conventional value. These two benefits are especially important for protein separation processes that suffer from low loadability, such as size exclusion chromatography. We have demonstrated that size exclusion can be performed on an industrial scale when it is run continuously with the aid of a pressurized annular chromatograph.

Chromatography, Gel↗