Comparison of batch and perfusion culture in combination with pilot-scale expanded bed purification for the production of soluble recombinant beta-secretase.

beta-Secretase is one of the prime targets for therapeutic intervention of Alzheimer's disease. For the development of a secretase inhibitor a steady supply of large quantities of a homogeneous and active recombinant beta-secretase is a prerequisite. Therefore various culture modes were investigated using HEK-293 cells stably transfected with soluble recombinant beta-secretase. The coupling of the Fc part of human IgG1 to the ectodomain of beta-secretase (residues 1-460) allowed a fast purification of the protein with rProtA expanded bed chromatography. Batch cultures of 5 to 50 L working volume run for 7 days showed reproducible cell growth and product yields of 3 mg/L purified protein. A 20 L perfusion culture was operated for 21 days, reaching a cell density of 30 x 10(6) cells/mL at a dilution rate of 2/d. The total product yield of the perfusion culture was 1.4 g of purified protein. The effect of different perfusion rates on cell growth, protein yield, and quality was investigated and compared to the results obtained in batch cultures. Protein quality was consistent as analyzed on 1D SDS-PAGE, and the final product contained both the mature and the pro form of beta-secretase. Although the cell specific protein expression was slightly reduced in perfusion culture, a substantial increase in specific activity of over 75% was achieved. Some of the increase in activity can be explained by an increase in the percentage of the mature form of the recombinant protein.

Rapid structural characterisation of a murine monoclonal IgA alpha chain: heterogeneity in the oligosaccharide structures at a specific site in samples produced in different bioreactor systems.

A murine hybridoma cell line which secretes a monoclonal IgA antibody, directed against the LPS antigen of Vibrio cholerae, was grown in either a continuous stirred tank reactor, a fluidised bed reactor or a hollow fibre reactor. Different methods were used for the structural characterisation of the IgA alpha chains. A classical approach consisted of Edman sequencing and mass determination of peptides separated by reversed phase HPLC. Alternatively, peptides and glycopeptides from a tryptic digest of each alpha chain were identified directly by MALDI-TOF mass spectrometry. A detailed analysis of the oligosaccharide structures at an unique site on the alpha chain was made by labelling the oligosaccharides released by N-glycosidase F with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone. After separation by HPLC, mass measurements were made using matrix-assisted laser desorption time of flight mass spectrometry before and after digestion with specific exoglycosidases. The primary structure of the alpha chain of IgA was not affected by different cell culture conditions; in contrast, significant variations could be detected in the pattern of N-linked oligosaccharide structures, most prominently in the degree of sialylation. The efficiency of the analytical techniques in providing quality control of the identity, integrity and consistency of the glycoprotein is shown and discussed.

Thiol-disulfide redox buffers maintain a structure of immunoglobulin A that is essential for optimal in vitro binding to secretory component.

We have shown that human secretory component (SC) binds in vitro to different samples of human and murine dimeric immunoglobulin A (IgA). The binding ratio in the IgA/SC complex is 1:1. IgA which is stably bound to SC is separated from unreacted IgA by anion exchange chromatography. A part of IgA/SC complexes formed in vitro is unstable to this elution; the proportion varies between different samples of IgA; it increases following prolonged incubation of IgA at 37 degrees C. Incubation of IgA with glutathione/glutathione disulfide (GSH/GSSG) redox buffers increases the proportion able to form a stable complex with SC to approximately 90%. The presence of bound SC is not essential for this process but does allow it to occur at a lower GSH/GSSG concentration. The stable IgA/SC complex consists of a structure with a disulfide bond between IgA and SC apparently in equilibrium with a structure in which this bond is absent. The proportion bound covalently is similar for different samples of IgA and is insensitive to incubation with GSH/GSSG. It is significantly greater for secretory IgA (sIgA) and for IgA and SC incubated together with a starting mixture of cysteine/cystine. Monoclonal, antigen-specific IgA, all of which is optimally bound to SC in essentially the same way as in native sIgA, can be isolated in high yield. Our results support a mechanism for optimal binding of IgA to SC, that can occur both in vitro and in vivo, in which a thiol disulfide interchange occurs between a free IgA thiol and a sensitive SC disulfide following the initial non-covalent interaction.

Chitin biosynthesis enhancement by the endochitinase inhibitor allosamidin.

Membrane preparations of Artemia salina synthetize radiolabelled chitin from UDP-[U-14C]GlcNAc at a low rate (Horst, M.N. (1981) J. Biol. Chem. 256, 1412-1419). We now report that, when the specific endochitinase inhibitor allosamidin is present in addition to the established activators trypsin and GlcNAc, incorporation of [U-14C]GlcNAc into chitin is increased up to 58-fold over the basic synthesis rate. Thus, a greatly enhanced apparent chitin synthase activity is observed in membranes from an arthropod species when simultaneous degradation of chitin is inhibited.