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The use of either a polyampholyte buffer or a simple buffer system for the high-performance cation-exchange chromatofocusing of monoclonal antibodies is demonstrated for the case where the pH gradient is produced entirely inside the column and with no external mixing of buffers. The simple buffer system used was composed of two buffering species, one which becomes adsorbed onto the column packing and one which does not adsorb, together with an adsorbed ion that does not participate in acid-base equilibrium. The method which employs the simple buffer system is capable of producing a gradual pH gradient in the neutral to acidic pH range that can be adjusted by proper selection of the starting and ending pH values for the gradient as well as the buffering species concentration, pKa, and molecular size. By using this approach, variants of representative monoclonal antibodies with isoelectric points of 7.0 or less were separated with high resolution so that the approach can serve as a complementary alternative to isoelectric focusing for characterizing a monoclonal antibody based on differences in the isoelectric points of the variants present. Because the simple buffer system used eliminates the use of polyampholytes, the method is suitable for antibody heterogeneity analysis coupled with mass spectrometry. The method can also be used at the preparative scale to collect highly purified isoelectric variants of an antibody for further study. To illustrate this, a single isoelectric point variant of a monoclonal antibody was collected and used for a stability study under forced deamidation conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Recombinant human glucocerebrosidase (imiglucerase, Cerezyme) is used in enzyme replacement therapy for Gaucher disease. Complex oligosaccharides present on Chinese hamster ovary cell-expressed glucocerebrosidase (GCase) are enzymatically remodeled into a mannose core, facilitating mannose receptor-mediated uptake into macrophages. Alternative expression systems could be used to produce GCase containing larger oligomannose structures, offering the possibility of an improvement in targeting to macrophages. A secondary advantage of these expression systems would be to eliminate the need for carbohydrate remodeling. Here, multiple expression systems were used to produce GCase containing primarily terminal oligomannose, from Man2 to Man9. GCase from these multiple expression systems was compared to Cerezyme with respect to affinity for mannose receptor and serum mannose-binding lectin (MBL), macrophage uptake, and intracellular half-life. In vivo studies comparing clearance and targeting of Cerezyme and the Man9 form of GCase were carried out in a Gaucher mouse model (D409V/null). Mannose receptor binding, macrophage uptake, and in vivo targeting were similar for all forms of GCase. Increased MBL binding was observed for all forms of GCase having larger mannose structures than those of Cerezyme, which could influence pharmacokinetic behavior. These studies demonstrate that although alternative cell expression systems are effective for producing oligomannose-terminated glucocerebrosidase, there is no biochemical or pharmacological advantage in producing GCase with an increased number of mannose residues. The display of alternative carbohydrate structures on GCase expressed in these systems also runs the risk of undesirable consequences, such as an increase in MBL binding or a possible increase in immunogenicity due to the presentation of non-mammalian glycans.

Recombinant antibodies and their derivatives are increasingly being used as therapeutic agents. Clinical applications of antibodies often require large amounts of highly purified molecules, sometimes for multiple treatments. The development of very efficient expression systems is essential to the full exploitation of the antibody technology. Production of recombinant protein in the milk of transgenic dairy animals is currently being tested as an alternative to plasma fractionation for the manufacture of a number of blood factors (human antithrombin, human alpha-1-antitrypsin, human serum albumin, factor IX). The ability to routinely yield mg/ml levels of antibodies and the scale-up flexibility make transgenic production an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. The following review examines the potential of transgenic expression for the production of recombinant therapeutic antibodies.