Monoclonal antibodies are used in a wide field of applications also including the use as research tools and for diagnostic and therapeutic purposes.
Since scFv (single-chain variable fragment) and Fab (antigen-binding fragment) as parts of complete antibodies are small and expressible in Escherichia coli and permit a systematic survey and a selection for molecules binding with high affinity, the potential range of applications of these molecules quickly widens. One of the most important limitations of this widening is however the speed of the generation and the analysis of the efficiency of the bond and the effects of antibodies on their target molecules. An acceleration of the antibody production would permit a faster response to health risks caused by a pandemic spread of pathogenic viruses and microorganisms.
The conventional production of antibodies occurs by the culture of animal cells (Nilsang S, Nandakumar K S, Galaev I Y, Rakshit S K, Holmdahl R, Mattiasson B, Kumar A. (2007) Monoclonal antibody production using a new supermacroporous cryogel bioreactor. Biotechnol Prog 23:932-939; Rodrigues M E, Costa A R, Henriques M, Azeredo J, Oliveira R (2010) Technological progresses in monoclonal antibody production systems. Biotechnol Prog 2:332-51). A distinct acceleration and a more economic production of antibodies could be achieved in recent years by the expression thereof in Escherichia coli cells (Humphreys D P (2003) Production of antibodies and antibody fragments in Escherichia coli and a comparison of their functions, uses and modification. Curr Opin Drug Discov Devel 6:188-196). Cell-based methods of the antibody production require however a time and work-intensive cell culture execution. For a bacterial expression, furthermore additional efforts are necessary for cloning the expressible matrices coding for antibodies (Hoogenboom H R (2005) Selecting and screening recombinant antibody libraries. Nat Biotechnol 23:1105-1116).
Meanwhile, the cell-free protein expression is regarded as a promising alternative for overcoming the limitations of cell-based methods. One reason for this is the significant improvement of the productivity, economy and the activity of the synthesized proteins in cell-free prokaryotic systems achieved in recent years (Spirin A S, Baranov V I, Ryabova L A, Ovodov S Y, Alakhov Y B (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242:1162-4; Strey J, Merk H, Stiege W (2004) Verfahren zur präparativen in vitro Proteinbiosynthese. Patent DE 102004032460; Kim T W, Oh I S, Keum J W, Kwon Y C, Byun J Y, Lee K H, Choi C Y, Kim D M (2007) Prolonged cell-free protein synthesis using dual energy sources: combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate. Biotechnol Bioeng 97:1510-1515) and eukaryotic systems (Madin K, Sawasaki T, Ogasawara T, Endo Y (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proc Natl Acad Sci USA 97(2):559-564; Tsuboi T, Takeo S, Iriko H, Jin L, Tsuchimochi M, Matsuda S, Han E, Otsuk, H, Kaneko O, Sattabongkot J, Udomsangpetch R, Sawasaki T, Tori, M, Endo, Y (2008) Wheat germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates. Infection and Immunity 1702-1708; Tarui H, Murata M, Tani I, Imanishi S, Nishikawa S, Hara T (2001) Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment. Appl Microbiol Biotechnol 55:446-453; Kubick S, Schacherl J, Fleischer-Notter H, Royall E, Roberts L O, Stiege W (2003) In vitro Translation in an Insect-Based Cell-Free System. In: Swartz, J. R. (Ed.) Cell-Free Protein Expression. Springer, Berlin Heidelberg New York, 209-217; Mikami S, Masutani M, Sonenberg N, Yokoyama S, Imataka H (2006) An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr Purif 46(2):348-357). Modifications of some of these systems permitted the synthesis of disulfide-bonded proteins including scFv (single-chain antibody) (Ryabova L A, Desplancq D, Spirin A S, Pluckthun A (1997) Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones. Nat Biotechnol 15:79-84; Merk H, Stiege W, Tsumoto K, Kumagai I, Erdmann V A (1999) Cell-free Expression of two Single-Chain Monoclonal Antibodies against Lysozyme: Effect of Domain Arrangement on the Expression. J Biochem 125:328-333). Fab antibody fragments are however more attractive, due to their better binding properties, than scFv. Recently, the cell-free expression of a Fab in a system based on E. coli was shown (Oh I S, Lee J C, Lee M S, Chung J H, Kim D M (2010) Cell-free production of functional antibody fragments. Bioprocess Biosyst Eng 33:127-32).
The drawbacks of the cell-free systems based on E. coli are however a low specific activity of the produced Fab and the necessity of the removal von toxins of the expression system, before the synthesized Fab can be used for a cell-based quality test. E. coli systems meanwhile offer in their simple version (batch system) synthesis performances of up to 1 milligram newly synthesized protein per milliliter reaction solution. The highest value for the yield of functional Fab from a cell-free system is however only 30 μg/ml (Oh I S, Lee J C, Lee M S, Chung J H, Kim D M (2010) Cell-free production of functional antibody fragments. Bioprocess Biosyst Eng 33:127-32). In spite of the high productivity of the cell-free E. coli system, thus only a fraction of the synthesized protein can be used. Furthermore, in order to obtain precise measurement results, the active portion of the synthesized protein must be separated from the inactive portion, which means more efforts for this cleaning step.
Cell-free systems based on E. coli have high activities, which during the reaction will substantially vary the redox potential. This variation reduces the activity of the synthesized antibody fragments. A reduction of the variation of the redox potential was achieved by chemical pre-treatment of the cell lysate, Kim D M, Swartz J R (2004) Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol Bioeng 85:122-9; Oh I S, Kim D M, Kim T W, Park C G, Chloi C Y (2006) Providing an oxidizing environment for the cell-free expression of disulfide-containing proteins by exhausting the reducing activity of Escherichia coli S30 extract. Biotechnol Prog 22:1225-89. This is however disadvantageous for the handling of the system, since another step is required, which furthermore can be automated with additional efforts only.
In order to be able to synthesize in E. coli-based cell-free systems noteworthy concentrations of soluble and active antibody fragment, they are supplemented with chaperones, for instance GroE, DnaK and protein disulfide isomerase, for instance PDI, DsbC (Ryabova L A, Desplancq D, Spirin A S, Pluckthun A (1997) Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones. Nat Biotechnol 15:79-84; Merk H, Stiege W, Tsumoto K, Kumagai I, Erdmann V A (1999) Cell-free Expression of two Single-Chain Monoclonal Antibodies against Lysozyme: Effect of Domain Arrangement on the Expression. J Biochem 125:328-333; Tsumoto K, Nakaoki Y, Ueda Y, Ogasahara K, Yutani K, Watanabe K, Kumagai I (1994) Effect of the order of antibody variable regions on the expression of the single-chain HyHEL10 Fv fragment in E. coli and the thermodynamic analysis of its antigen-binding properties. Biochem Biophys Res Commun 201:546-51; Oh I S, Lee J C, Lee M S, Chung J H, Kim D M (2010) Cell-free production of functional antibody fragments. Bioprocess Biosyst Eng 33:127-32; Kim D M, Swartz J R (2004) Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol Bioeng 85:122-9; Oh I S, Kim D M, Kim T W, Park C G, Chloi C Y (2006) Providing an oxidizing environment for the cell-free expression of disulfide-containing proteins by exhausting the reducing activity of Escherichia coli S30 extract. Biotechnol Prog 22:1225-8).