- Email: [email protected]
Resin optimization for affinity chromatography media
Abstracts / Journal of Biotechnology 136S (2008) S198–S200
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References
III4-O-005
Hanson, B.J., Boon, A., Lim, A., Webb, A., Ooi, E.E., Webby, R., 2006. Passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza H5 hemagglutinin in mice. Respir. Res. 7, 126. Simmons, C.P., Bernasconi, N.L., Suguitan Jr., A.L., Mills, K., Ward, J.M., Chau, N.V.V., Hien, T.T., Sallusto, F., Ha, D.Q., Farrar, J., de Jong, M.D., Lanzavecchia, A., Subbarao, K., 2007. Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza. PLoS Med. 4, e178.
Bio-prospecting for novel polysaccharides in microalgae using novel glycan microarrays William G.T. Willats ∗ , Iben Sørensen The University of Copenhagen, Copenhagen, Denmark E-mail address: [email protected] (W.G.T. Willats).
doi:10.1016/j.jbiotec.2008.07.417 III4-O-004 Bio-prospecting across the plant kingdom for industrially relevant cell wall polysaccharides using novel glycan microarrays Iben Sørensen ∗ , William Willats The University of Copenhagen, Copenhagen, Denmark E-mail address: [email protected] (I. Sørensen). Plant cell walls are one of the most abundant sources of biomass on earth and provide a wealth of materials ranging from unrefined bulk products such as timber, to highly defined oligosaccharides with specific pharmacological activities. However, the vast majority of plant cell walls have not been analyzed and it is likely that some contain polysaccharides with valuable industrial or nutraceuticals properties. We have recently developed a technique that enables the occurrence of cell wall glycans to be systematically and rapidly mapped throughout plants. The technique (comprehensive microarray polymer profiling, or CoMPP) combines the high-throughput capacity of microarrays, with the specificity of monoclonal antibodies (mAbs) and carbohydrate binding modules (CBMs) (Moller et al., 2007; Sorensen et al., in press). CoMPP provide a global snap-shot of cell wall composition and we have used CoMPP to undertake a large scale survey of cell walls across the plant kingdom from the charophyte green microalgae to angiosperms. This survey has revealed an extraordinary diversity of cell wall compositions and, for example, lead to the discovery of mixed linkage (1 → 3), (1 → 4)--d-glucan (MLG) in the horsetail Equisetum arvense. The discovery of a novel and abundant source of MLG is significant because MLG has well-documented health benefits in relation to controlled glycemic index and reducing the risk of coronary heart disease (Lazaridou and Biliaderis, 2007). The results we have obtained so far indicate that further wide scale surveying of cell walls in phylogenetically diverse plant species may well yield novel plant products and will contribute to our understanding of cell wall evolution.
Algae are a vast but largely unexploited bio-resource. Many algal cells contain high level of starch, oils and proteins and are surrounded by a wall constructed of complex polysaccharides (Metting, 1996). All these components have commercial applications but relatively few of the several thousand known species of algae have been investigated in detail or commercially developed. We have initiated a project aimed at surveying a large number of diverse microalgae for polymers with application as novel functional food ingredients. To do this we have used a recently developed high-throughput technique for polysaccharide profiling. The technique, comprehensive microarray polymer profiling (or CoMPP), combines the high throughput capacity of microarrays with the specificity of monoclonal antibodies and carbohydrate binding modules (Moller et al., 2007). Using CoMPP we have carried out an initial survey of charophyte microalgae and discovered that several species contain high levels of pectins and mixed linkage linked glucans. It is anticipated that further large scale surveying of microalgae may well identify species that contain novel polymers with functional food ingredient and/or nutraceutical applications.
References Metting, F.B., 1996. Biodiversity and application of microalgae. J. Ind. Microbiol. 17, 477–489. Moller, I., Sørensen, I., Bernal, A.J., Blaukopf, C., Lee, K., Øbro, J., Pettolino, F., Roberts, A., Mikkelsen, J.D., Knox, J.P., Bacic, A., Willats, W.G.T., 2007. High-throughput mapping of cell-wall polymers within and between plants using novel microarrays. Plant J. 50, 1118–1128.
doi:10.1016/j.jbiotec.2008.07.419 III4-P-003 Resin optimization for affinity chromatography media Nanying Bian 1,∗ , Neil Soice 1 , Chen Wang 1 , K.S. Cheng 1 , Senthil Ramaswamy 1 , Yuan Wong 1 , Kami Beyzavi 2 1 2
References Lazaridou, A., Biliaderis, C.G., 2007. Molecular aspects of cereal -glucan functionality: physical properties, technological applications and physiological effects. Cereal Sci. 46, 101–118. Moller, I., Sørensen, I., Bernal, A.J., Blaukopf, C., Lee, K., Øbro, J., Pettolino, F., Roberts, A., Mikkelsen, J.D., Knox, J.P., Bacic, A., Willats, W.G.T., 2007. High-throughput mapping of cell-wall polymers within and between plants using novel microarrays. Plant J. 50, 1118–1128. Sørensen, I., Pettolino, F.A., Wilson, S.M., Doblin, M.S., Johansen, B., Bacic, A., Willats, W.G.T. Mixed linkage (1 → 3),(1 → 4)--d-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls. Plant J., in press.
doi:10.1016/j.jbiotec.2008.07.418
Millipore Corporation, Bedford, MA, USA Millipore Corporation, Conssett, UK
E-mail address: Nanying [email protected] (N. Bian). Protein A affinity chromatography has been the workhorse in downstream processing of monoclonal antibody (Follman and Fahrner, 2004; Hamilton, 1987; Wlad et al., 2001). With the advances in expression technology, fermentation with higher titer is becoming common practice. This demands increased capacity and productivity in the affinity chromatography step (Low et al., 2007). One of the common base matrices for affinity chromatography media is controlled pore glass (CPG). As a relatively mature material, controlled pore glass provides rigidity, uniform pore size, and superior flow property. In this study, we examined parameters of CPG base matrix and identified key factors that influence the dynamic and static binding capacity of polyclonal and monoclonal antibodies. Pressure-flow property of the optimized media has also been studied to ensure optimum productivity for mAb processing. Optimal pore size and particle size of CPG for antibody capturing is identified. This work provides guidance for base matrix optimiza-
S200
Abstracts / Journal of Biotechnology 136S (2008) S198–S200
tion of Protein A affinity media. The result is a new media with ultra high IgG binding capacity and productivity. References Follman, D., Fahrner, R.L., 2004. Factorial screening of antibody purification processes using three chromatographic steps without Protein A. J. Chromatogr. A 1024, 79–85. Hamilton, R.G., 1987. The Human IgG Subclasses. Calbiochem-Novabiochem Corporation, Texas, pp. 1–75. Low, D., O’Leary, R., Pujar, N.S., 2007. Future of antibody purification. J. Chromatogr. B 848, 48–63. Wlad, H., Ballagi, A., Bouakaz, L., Gu, Z., Janson, J.C., 2001. Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli. Protein Expr. Purif. 22 (2), 325–329.
doi:10.1016/j.jbiotec.2008.07.420 III4-P-006 The study of conjugation condition of enrofloxacin artificial antigens Xiaowei Fu ∗ , Mingzhou Zhang, Huajun Hu, Jun Liu College of Life Science, China Jiliang University, Hangzhou 310018, China The immunogen EN-BSA (Enrofloxacin-bovine serum albumin) and the coating antigen EN-OVA (Enrofloxacin-ovalbumin) were
synthesized by methods of N-hydroxysuccinimide ester (NHS), identified by the FPLC and the UV absorption method. Two main factors involved in the conjugation, that is, temperature and pH , were discussed and optimized. The results showed that when the temperature and pH were keeping at 4 ◦ C and between 7.2 and 7.5 respectively, EN-BSA and EN-OVA were successfully synthesized. References Adamezyk, M., Buko, A., Chen, Y.Y., et al., 1994. Characterization of protein–hapten conjugates. 1. Matrix-assisted laserdesorption ionization mass spectrometry of immuno BSA-hapten conjugates and comparison with other characterization methods. Bioconjugate Chem. 5 (6), 631–638. Chan, E.C.Y., Ho, P.C., 2002. Preparation and characterization of immunogens for antibody production against metanephrine and normetanephrine. Immunol. Methods 266, 143–154. The European agency for the Evaluation of Medicinal products, Veterinary Medicines and information Technology unit. Committee for veterinary medicinal products clenbuterol hydrochloride summary report (1). EMEA/MRL/030/95-FINAL. Erlanger, T.B.F., 1980. The preparation of antigenic hapten-carrier conjugates: a survey. Methods in Enzymology, vol. 70, pp. 85–103. Zhao, S.Q., Sun, Y.M., Le, X.Y., Huang, X.M., 2002. Research progress of artificial antigen synthesis of pesticides IJl Pesticide, 41 (3), 9–11.
doi:10.1016/j.jbiotec.2008.07.421
S199
References
III4-O-005
Hanson, B.J., Boon, A., Lim, A., Webb, A., Ooi, E.E., Webby, R., 2006. Passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza H5 hemagglutinin in mice. Respir. Res. 7, 126. Simmons, C.P., Bernasconi, N.L., Suguitan Jr., A.L., Mills, K., Ward, J.M., Chau, N.V.V., Hien, T.T., Sallusto, F., Ha, D.Q., Farrar, J., de Jong, M.D., Lanzavecchia, A., Subbarao, K., 2007. Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza. PLoS Med. 4, e178.
Bio-prospecting for novel polysaccharides in microalgae using novel glycan microarrays William G.T. Willats ∗ , Iben Sørensen The University of Copenhagen, Copenhagen, Denmark E-mail address: [email protected] (W.G.T. Willats).
doi:10.1016/j.jbiotec.2008.07.417 III4-O-004 Bio-prospecting across the plant kingdom for industrially relevant cell wall polysaccharides using novel glycan microarrays Iben Sørensen ∗ , William Willats The University of Copenhagen, Copenhagen, Denmark E-mail address: [email protected] (I. Sørensen). Plant cell walls are one of the most abundant sources of biomass on earth and provide a wealth of materials ranging from unrefined bulk products such as timber, to highly defined oligosaccharides with specific pharmacological activities. However, the vast majority of plant cell walls have not been analyzed and it is likely that some contain polysaccharides with valuable industrial or nutraceuticals properties. We have recently developed a technique that enables the occurrence of cell wall glycans to be systematically and rapidly mapped throughout plants. The technique (comprehensive microarray polymer profiling, or CoMPP) combines the high-throughput capacity of microarrays, with the specificity of monoclonal antibodies (mAbs) and carbohydrate binding modules (CBMs) (Moller et al., 2007; Sorensen et al., in press). CoMPP provide a global snap-shot of cell wall composition and we have used CoMPP to undertake a large scale survey of cell walls across the plant kingdom from the charophyte green microalgae to angiosperms. This survey has revealed an extraordinary diversity of cell wall compositions and, for example, lead to the discovery of mixed linkage (1 → 3), (1 → 4)--d-glucan (MLG) in the horsetail Equisetum arvense. The discovery of a novel and abundant source of MLG is significant because MLG has well-documented health benefits in relation to controlled glycemic index and reducing the risk of coronary heart disease (Lazaridou and Biliaderis, 2007). The results we have obtained so far indicate that further wide scale surveying of cell walls in phylogenetically diverse plant species may well yield novel plant products and will contribute to our understanding of cell wall evolution.
Algae are a vast but largely unexploited bio-resource. Many algal cells contain high level of starch, oils and proteins and are surrounded by a wall constructed of complex polysaccharides (Metting, 1996). All these components have commercial applications but relatively few of the several thousand known species of algae have been investigated in detail or commercially developed. We have initiated a project aimed at surveying a large number of diverse microalgae for polymers with application as novel functional food ingredients. To do this we have used a recently developed high-throughput technique for polysaccharide profiling. The technique, comprehensive microarray polymer profiling (or CoMPP), combines the high throughput capacity of microarrays with the specificity of monoclonal antibodies and carbohydrate binding modules (Moller et al., 2007). Using CoMPP we have carried out an initial survey of charophyte microalgae and discovered that several species contain high levels of pectins and mixed linkage linked glucans. It is anticipated that further large scale surveying of microalgae may well identify species that contain novel polymers with functional food ingredient and/or nutraceutical applications.
References Metting, F.B., 1996. Biodiversity and application of microalgae. J. Ind. Microbiol. 17, 477–489. Moller, I., Sørensen, I., Bernal, A.J., Blaukopf, C., Lee, K., Øbro, J., Pettolino, F., Roberts, A., Mikkelsen, J.D., Knox, J.P., Bacic, A., Willats, W.G.T., 2007. High-throughput mapping of cell-wall polymers within and between plants using novel microarrays. Plant J. 50, 1118–1128.
doi:10.1016/j.jbiotec.2008.07.419 III4-P-003 Resin optimization for affinity chromatography media Nanying Bian 1,∗ , Neil Soice 1 , Chen Wang 1 , K.S. Cheng 1 , Senthil Ramaswamy 1 , Yuan Wong 1 , Kami Beyzavi 2 1 2
References Lazaridou, A., Biliaderis, C.G., 2007. Molecular aspects of cereal -glucan functionality: physical properties, technological applications and physiological effects. Cereal Sci. 46, 101–118. Moller, I., Sørensen, I., Bernal, A.J., Blaukopf, C., Lee, K., Øbro, J., Pettolino, F., Roberts, A., Mikkelsen, J.D., Knox, J.P., Bacic, A., Willats, W.G.T., 2007. High-throughput mapping of cell-wall polymers within and between plants using novel microarrays. Plant J. 50, 1118–1128. Sørensen, I., Pettolino, F.A., Wilson, S.M., Doblin, M.S., Johansen, B., Bacic, A., Willats, W.G.T. Mixed linkage (1 → 3),(1 → 4)--d-glucan is not unique to the Poales and is an abundant component of Equisetum arvense cell walls. Plant J., in press.
doi:10.1016/j.jbiotec.2008.07.418
Millipore Corporation, Bedford, MA, USA Millipore Corporation, Conssett, UK
E-mail address: Nanying [email protected] (N. Bian). Protein A affinity chromatography has been the workhorse in downstream processing of monoclonal antibody (Follman and Fahrner, 2004; Hamilton, 1987; Wlad et al., 2001). With the advances in expression technology, fermentation with higher titer is becoming common practice. This demands increased capacity and productivity in the affinity chromatography step (Low et al., 2007). One of the common base matrices for affinity chromatography media is controlled pore glass (CPG). As a relatively mature material, controlled pore glass provides rigidity, uniform pore size, and superior flow property. In this study, we examined parameters of CPG base matrix and identified key factors that influence the dynamic and static binding capacity of polyclonal and monoclonal antibodies. Pressure-flow property of the optimized media has also been studied to ensure optimum productivity for mAb processing. Optimal pore size and particle size of CPG for antibody capturing is identified. This work provides guidance for base matrix optimiza-
S200
Abstracts / Journal of Biotechnology 136S (2008) S198–S200
tion of Protein A affinity media. The result is a new media with ultra high IgG binding capacity and productivity. References Follman, D., Fahrner, R.L., 2004. Factorial screening of antibody purification processes using three chromatographic steps without Protein A. J. Chromatogr. A 1024, 79–85. Hamilton, R.G., 1987. The Human IgG Subclasses. Calbiochem-Novabiochem Corporation, Texas, pp. 1–75. Low, D., O’Leary, R., Pujar, N.S., 2007. Future of antibody purification. J. Chromatogr. B 848, 48–63. Wlad, H., Ballagi, A., Bouakaz, L., Gu, Z., Janson, J.C., 2001. Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli. Protein Expr. Purif. 22 (2), 325–329.
doi:10.1016/j.jbiotec.2008.07.420 III4-P-006 The study of conjugation condition of enrofloxacin artificial antigens Xiaowei Fu ∗ , Mingzhou Zhang, Huajun Hu, Jun Liu College of Life Science, China Jiliang University, Hangzhou 310018, China The immunogen EN-BSA (Enrofloxacin-bovine serum albumin) and the coating antigen EN-OVA (Enrofloxacin-ovalbumin) were
synthesized by methods of N-hydroxysuccinimide ester (NHS), identified by the FPLC and the UV absorption method. Two main factors involved in the conjugation, that is, temperature and pH , were discussed and optimized. The results showed that when the temperature and pH were keeping at 4 ◦ C and between 7.2 and 7.5 respectively, EN-BSA and EN-OVA were successfully synthesized. References Adamezyk, M., Buko, A., Chen, Y.Y., et al., 1994. Characterization of protein–hapten conjugates. 1. Matrix-assisted laserdesorption ionization mass spectrometry of immuno BSA-hapten conjugates and comparison with other characterization methods. Bioconjugate Chem. 5 (6), 631–638. Chan, E.C.Y., Ho, P.C., 2002. Preparation and characterization of immunogens for antibody production against metanephrine and normetanephrine. Immunol. Methods 266, 143–154. The European agency for the Evaluation of Medicinal products, Veterinary Medicines and information Technology unit. Committee for veterinary medicinal products clenbuterol hydrochloride summary report (1). EMEA/MRL/030/95-FINAL. Erlanger, T.B.F., 1980. The preparation of antigenic hapten-carrier conjugates: a survey. Methods in Enzymology, vol. 70, pp. 85–103. Zhao, S.Q., Sun, Y.M., Le, X.Y., Huang, X.M., 2002. Research progress of artificial antigen synthesis of pesticides IJl Pesticide, 41 (3), 9–11.
doi:10.1016/j.jbiotec.2008.07.421