Purification of monoclonal antibodies by simulated moving-bed chromatography.

A simulated moving bed (SMB) system has been developed for the biospecific purification of monoclonal antibodies. Adsorption and desorption of the desired product is performed under different conditions. To increase the purity and yield of the antibodies, two purge steps have to be introduced. The steady-state performance of the SMB system was modelled by solving the governing differential equations using a linear driving force approximation. The model parameters were determined independently in batch experiments. They were used to determine the operating conditions of the SMB system for the purification of monoclonal antibodies from cell culture supernatant. The antibodies could be isolated with a yield of > or = 90. SDS gel electrophoresis of the feed and product stream showed that more than 99% of the contaminating proteins were removed in a single step by SMB chromatography.

Perfusible and non-perfusible supports with monoclonal antibodies for bioaffinity chromatography of Escherichia coli penicillin amidase within its pH stability range.

Several monoclonal antibodies (mABs) have been prepared and immobilized for the biospecific isolation of penicillin amidase (PA) from Escherichia coli (EC 3.5.1.11), an enzyme without S-S bridges and a pH stability range of 4-9. During the immobilization the fluorescence emission maxima of the mABs were found to change from 336 nm to ca. 350 nm. Only one of these mABs was found to be suitable for preparative bioaffinity chromatography of PA within the pH stability range. This mAB was immobilized on different spherical supports (Eupergit C 250 L and Sepharose) and one perfusible support (Knauer Quick Disc) and used for analytical and preparative bioaffinity chromatography. Under isocratic conditions the plate height for the perfusible biospecific adsorbent was found to be an order of magnitude lower than for the other supports. The different forms of this proteolytically processed bacterial enzyme could not be separated, however, by the biospecific adsorbents. At the mAB density used in the adsorbents (10-30 microM), less than 30% of the theoretical binding capacity of the immobilized mABs could be used to adsorb the enzyme.

[Lab-On-A- Chip--systems for biomedical research and diagnosis]. / Lab-On-A-Chip--Systeme für Die Biomedizinische Forschung und Diagnostik.

In today's biomedical research and diagnosis, a number of substances and agents have to be checked. Frequently, plastic micro titer plates are used for this purpose as large-area test platforms. For the first time, plastic micro titer plates with 96 identical microfluidic labon-a-chip structures for simultaneous capillary electrophoresis (CE) have now been produced using microtechnical fabrication methods. Such structures are suited for e.g. the separation of biomolecules. In completely sealed microfluidic channel systems, smallest sample volumes can be processed, separated, mixed with other substances, or detected. Due to the small channel dimensions, these microfluidic systems are characterized by very small sample volumes needed.

Two-dimensional electrochromatography/capillary electrophoresis on a microchip.

A two-dimensional separation system on a microfabricated device was demonstrated using open-channel electrochromatography as the first dimension and capillary electrophoresis as the second dimension. The first dimension was operated under isocratic conditions, and the effluent from the first dimension was repetitively injected into the second dimension every few seconds. A 25-cm separation channel with spiral geometry for open-channel electrochromatography was chemically modified with octadecylsilane and coupled to a 1.2-cm straight separation channel for capillary electrophoresis. Fluorescently labeled products from tryptic digests of beta-casein were analyzed in 13 min with this system.

Integrated microchip-device for the digestion, separation and postcolumn labeling of proteins and peptides.

A microchip device was demonstrated that integrated enzymatic reactions, electrophoretic separation of the reactants from the products and post-separation labeling of proteins and peptides prior to detection. A tryptic digestion of oxidized insulin B-chain was performed in 15 min under stopped flow conditions in a heated channel, and the separation was completed in 1 min. Localized thermal control of the reaction channel was achieved using a resistive heating element. The separated reaction products were then labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and detected by laser-induced fluorescence. A second reaction at elevated temperatures was also demonstrated for the on-chip reduction of disulfide bridges using insulin as a model protein. This device represents one of the highest levels, to date, of monolithic integration of chemical processes on a microchip.