Progress 10/01/05 to 09/30/06
Outputs
Continuing from the research of last year, this project focused on the second and the third specific aims of improvement of cryptophycin production process and scale-up cryptophycin production with micro-algae. During our studies on the biosynthesis of cryptophycin, it was discovered that both Nostoc sp. GSV 224 and ATCC 53879 released a relatively large amount of cryptophycin into their aqueous media. Because the organic compounds recovered from media were the active component without too many impurities, it is highly desirable to develop a convenient, easy to scale-up method to collect cryptophycin from a large quantity of aqueous media. More importantly, a continuous production system might be developed by replacing the old media with fresh media and then separating cryptophycin from that media. Methylene chloride was first used to extract both Nostoc sp. GSV 224 and ATCC 53879 media. Although the active components could be extracted, it is difficult for the organic
layer to fully separate from the aqueous layer. A third layer formed between the upper and lower layers and it took more than 12 hours to obtain a reasonably clear organic layer. A series tests were designed to compare various organic solvents for this extraction purpose. Ethyl acetate was chosen to replace methylene chloride. The organic layer, which is lighter than water in this case, separated better from the media. According to our previous experiments, the best media for Nostoc sp. GSV 224 is BG-11 while Nostoc sp. ATCC 53879 grows best in A3M7. Two sets of experiments were designed to compare the amount of cryptophycin in media at various growth stages for extraction. Table 1 and Figure 1 summarized the result obtained from these experiments. Table 1: Cryptophycin Concentration in GSV 224 and ATCC 53879 Media Algae Media Day 10 Day 20 Day 30 ATCC 53879 A3M7 < 0.5 mg/L 1.9 mg/L 5.6 mg/L GSV 224 Normal BG-11 < 0.5 mg/L 1.1 mg/L 2.0 mg/L Figure 1: Accumulation of Cryptophycin in
Media at Different Growth Stages All studies on the biosynthesis of cryptophycin have been focusing on axenic algal cultures by far. A non-axenic culture, however, would be highly desirable in order to produce cryptophycin cost-effectively. A direct comparison of these two cultures was implemented and the result was summarized in Table 2. Table 2: Comparison of ATCC 53879 in Axenic and Non-axenic Cultures Experiment Code (20 L culture) Dry Cells (g) Active Fraction (mg) Yield (mg/L) 4060401 Non-axenic 11.80 27.6 1.4 4060402 Axenic 11.52 60 (combined yield) 1.5 4060403 Axenic 16.87 0401261 Axenic 9.60 29.3 1.5 It is obvious that axenic and non-axenic cultures produced the active components in similar yield. This result is significant because non-axenic culture can dramatically reduce cost in large scale production. A pilot-scale experiment using Nostoc sp. GSV 224 was implemented using an 800 L photo-bioreactor. Relatively less amount of biomass (0.13 g/L dry cells) was harvested on
day 30. The purified fraction was at 0.1 mg/L.
Impacts
The goal of this project is to evaluate the potential use of microalgae to produce cryptophycin cost-effectively. Since this project started in October of 2003, we have improved the separation process of the bioactive components from the crude extract of algae so that this procedure can be scale-up easily in commercial production. By comparing the growth and cryptophycin production yields of different blue-green algae, we have selected GSV 224, which produced at least twice as much of cryptophycin as other strains did, for optimal biosynthesis of cryptophycin. Studies on the lifecycle of GSV 224 resulted in early harvest, cutting the cultivation period by half. We have successfully harvested almost pure cryptophycin from algal media. This new development makes the continuous production a very attractive system. Specifically designed experiments were implemented to compare the production yields of axenic and non-axenic cultures; the latter can dramatically reduce
production costs. Our research has helped UH Office of Technology Transfer and Economic Development secure a $380,000 investment from one of the largest pharmaceutical companies for an exclusive option to test the Cryptophycin Technology. Cyanotech, a local microalgae producer, is working closely with us to explore the feasibility of producing cryptophycin in its facility. Developing high value products that require advanced technology in production will improve the competitiveness of local microalgae producers. Cryptophycin, a high-valued anticancer drug, is a promising product for Hawaii microalgae industry.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/06
Outputs
OUTPUTS: During our studies on the biosynthesis of cryptophycin, it was discovered that both Nostoc sp. GSV 224 and ATCC 53879 released a relatively large amount of cryptophycin into their aqueous media. Because the organic compounds recovered from media were the active component without too many impurities, it is highly desirable to develop a convenient, easy to scale-up method to collect cryptophycin from a large quantity of aqueous media. More importantly, a continuous production system might be developed by replacing the old media with fresh media and then separating cryptophycin from that media. Methylene chloride was first used to extract both Nostoc sp. GSV 224 and ATCC 53879 media. Although the active components could be extracted, it is difficult for the organic layer to fully separate from the aqueous layer. A third layer formed between the upper and lower layers and it took more than 12 hours to obtain a reasonably clear organic layer. A series tests were designed to compare various organic solvents for this extraction purpose. Ethyl acetate was chosen to replace methylene chloride. The organic layer, which is lighter than water in this case, separated better from the media. According to our previous experiments, the best media for Nostoc sp. GSV 224 is BG-11 while Nostoc sp. ATCC 53879 grows best in A3M7. Two sets of experiments were designed to compare the amount of cryptophycin in media at various growth stages for extraction. Table 1 and Figure 1 summarized the result obtained from these experiments. Table 1: Cryptophycin Concentration in GSV 224 and ATCC 53879 Media Algae Media Day 10 Day 20 Day 30 ATCC 53879 A3M7 < 0.5 mg/L 1.9 mg/L 5.6 mg/L GSV 224 Normal BG-11 < 0.5 mg/L 1.1 mg/L 2.0 mg/L Figure 1: Accumulation of Cryptophycin in Media at Different Growth Stages All studies on the biosynthesis of cryptophycin have been focusing on axenic algal cultures by far. A non-axenic culture, however, would be highly desirable in order to produce cryptophycin cost-effectively. A direct comparison of these two cultures was implemented and the result was summarized in Table 2. Table 2: Comparison of ATCC 53879 in Axenic and Non-axenic Cultures Experiment Code (20 L culture) Dry Cells (g) Active Fraction (mg) Yield (mg/L) 4060401 Non-axenic 11.80 27.6 1.4 4060402 Axenic 11.52 60 (combined yield) 1.5 4060403 Axenic 16.87 0401261 Axenic 9.60 29.3 1.5 It is obvious that axenic and non-axenic cultures produced the active components in similar yield. This result is significant because non-axenic culture can dramatically reduce cost in large scale production. A pilot-scale experiment using Nostoc sp. GSV 224 was implemented using an 800 L photo-bioreactor. Relatively less amount of biomass (0.13 g/L dry cells) was harvested on day 30. The purified fraction was at 0.1 mg/L. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Hawaii Aquaculture Industry - in particular Hawaii Blue-green algae aquaculture operators. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The goal of this project is to evaluate the potential use of microalgae to produce cryptophycin cost-effectively. Since this project started in October of 2003, we have improved the separation process of the bioactive components from the crude extract of algae so that this procedure can be scale-up easily in commercial production. By comparing the growth and cryptophycin production yields of different blue-green algae, we have selected GSV 224, which produced at least twice as much of cryptophycin as other strains did, for optimal biosynthesis of cryptophycin. Studies on the lifecycle of GSV 224 resulted in early harvest, cutting the cultivation period by half. We have successfully harvested almost pure cryptophycin from algal media. This new development makes the continuous production a very attractive system. Specifically designed experiments were implemented to compare the production yields of axenic and non-axenic cultures; the latter can dramatically reduce production costs. Our research has helped UH Office of Technology Transfer and Economic Development secure a $380,000 investment from one of the largest pharmaceutical companies for an exclusive option to test the Cryptophycin Technology. Cyanotech, a local microalgae producer, is working closely with us to explore the feasibility of producing cryptophycin in its facility. Developing high value products that require advanced technology in production will improve the competitiveness of local microalgae producers. Cryptophycin, a high-valued anticancer drug, is a promising product for Hawaii microalgae industry.
Publications
- Back, S. and J. Liang. 2008. Production of Cryptophycin from Blue-Green Algae. The J. Young Investigators. 19(4). Online
- Liang, J., R.E. Moore, E.D. Moher, J.E. Munroe, R.S. Al-awar, D.A. Hay, D.L. Varie, T.Y. Zhang, J.A. Aikins, M.J. Martinelli, C. Shih, J.E. Ray, L.L. Gibson, V. Vasudevan, L. Polin, K. White, J. Kushner, C. Simpson, S. Pugh, and T.H. Corbett. 2005. Cryptophycins-309,249 and other cyrptophycin analogs: Preclinical efficacy studies with mouse and human tumors. Invest. New Drugs 23:213-224.
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Progress 10/01/04 to 09/30/05
Outputs
Two strains of blue-green algae ATCC 53789 and GSV 224 were grown under carefully controlled conditions in three media: Normal BG-11, A3M7 and Modified BG-11. Each media had a duplicate to minimize the experimental variations. Table 1 summarizes the production yields of these two strains, as well as the effect of media on cryptophycin production in each strain. Table 1: Comparison of Cryptophycin Production in Different Strains and Media Algae Media Cell Density Isolated Fraction Purified Fraction (g/L) (mg/L) (mg/L) ATCC Normal 0.255 6.1 1.30 53789 BG-11 Modified 0.313 6.1 0.95 BG-11 A3M7 0.324 11.6 1.25 GSV Normal 0.652 13.1 2.55 224 BG-11 Modified 0.377 9.2 1.80 BG-11 A3M7 0.321 7.7 2.40. First of all, GSV 224 produced at least twice as much purified cryptophycin as ATCC 53789 did in all three types of media under identical conditions. Therefore, GSV 224 is clearly a better strain than ATCC 53789 in the production of cryptophycin. The results of these experiments
also indicate that the optimal media for both strains of algae is Normal BG-11 followed by A3M7 and Modified BG-11. When cryptophycin is produced commercially from these strains of algae, the Normal BG-11 media should be used to optimize production. We are also making progress in the second specific aim of improving the process for cryptophycin production. A series of experiments have been designed and executed to study the cryptophycin production as a function of lifecycle in GSV 224. Figure one illustrates cell density as a function of time for the GSV 224 culture between day 10 and day 20. These results reflected typical exponential growth with an estimated growth rate of 0.1608 g/L*h. Cell growth appeared to plateau shortly after day 15. Figure 1: Cellular Growth Rate of GSV 224 Figure two shows the cryptophycin fraction isolated from GSV 224 during this growth period. The production of cryptophycin also appeared to plateau after day 15. Figure 2: Isolated Cryptophycin Density of
GSV 224. These results suggest that GSV 224 should be harvested shortly after day 15 of inoculation, when both of the cell growth and the cryptophycin production will plateau. As a result, this production process will retrieve cryptophycin in half of the usual lifecycle (30 days), increasing speed of production and reducing production costs.
Impacts
Because cryptophycin is a very complex molecule, it has been quite a challenge to synthesize this drug economically. For drugs with complex structures, it is always far more cost-effective to isolate them from natural sources than to synthesize them chemically. Taxol is a good example. Although taxol can be chemically synthesized, it is far less expensive to isolate the natural precursor and convert it into taxol by semi-synthesis. It might be possible for us to adapt a similar approach to address the problem involved in the cryptophycin production. However, extensive studies on the biosynthesis of cryptophycin are much needed to evaluate this biosynthetic approach. Since this project started in October of 2003, we have improved the separation process of the bioactive components from the crude extract of algae so that this procedure can be scaled-up easily in commercial production. By comparing the growth and cryptophycin production yields of different blue-green
algae, we have selected GSV 224, which produced at least twice as much of cryptophycin as other strains did, for optimal biosynthesis of cryptophycin. Studies on the lifecycle of GSV 224 resulted in early harvest, cutting the cultivation period by half. Our research has played an important role in the negotiations between the universities and other private companies to heavily invest in the pre-clinical and potentially clinical developments of cryptophycin. Developing high value products such as cryptophycin will improve the competitiveness of local micro-algae industry.
Publications
- Back, S. and Liang, J. 2005. Production of Cryptophycin from Blue-Green Algae. Journal of Young Investigators. Volume 12.
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