Progress 07/01/12 to 06/30/17
Outputs
Target Audience:The target audiences of this study are serum producers, pharmaceutical companies, vaccine companies, research institutions, and universities where serum is used. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has proven to be a great training opportunity for undergraduate students. A senior student, LaDonya Jackson, was the primary undergraduate student working on this project and was the first author for a oral presentation and a poster at two national conferences respectively. How have the results been disseminated to communities of interest?In addition to the presentation of our research work at two national conferences (one is at 27th National Conference on Undergraduate Research (NCUR), April 11-13, 2013, University of Wisconsin-La Crosse, Wisconsin USA, and another at the ISBioTech 3rd Annual Meeting, March 11-15, 2013, Rosslyn, VA USA), we are planning to publish the research in a peer-reviewed journal. We will also present the discoveries in other conferences. What do you plan to do during the next reporting period to accomplish the goals?To carry out research on the effect of Ab-free serum on hamster embryo culture. The PI is actively pursuing external or internal funding for such projects.
Impacts
What was accomplished under these goals?
The main scientific objectives of this project were to investigate whether antibody-free calf bovine serum (AF-CBS) derived from bovine immunoglobulin knockout cattle is superior to traditional bovine serum, both calf bovine serum (CBS) and fetal bovine serum (FBS), in providing better cell viability and growth, as well as improving production of recombinant proteins by cell cultures. We also aimed to provide research training opportunities for undergraduate students in mammalian cell culture, recombinant production and related techniques. Currently, about 60-70% of recombinant biopharmaceuticals are produced from mammalian cell culture. Bovine serum, either CBS or FBS, is routinely used in cell culture media for promoting cell survival and growth. Furthermore, CBS or FBS have also been used extensively as key reagents in academic research. However, bovine serum produced from wild type cattle contains bovine antibodies and such antibodies may interfere cell growth when used in cell culture and interfere bioassay accuracy when used in biological assays. Therefore, antibody-free bovine serum is needed both for industrial applications and scientific research. To address these needs, BioDak, LLC has produced genetically engineered cattle in which the genes required for antibody (immunoglobulin) production have been genetically inactivated (knocked out). We proposed to study the serum from such antibody-free cattle for its superiority in support cell growth and recombinant protein production in cell cultures. It is possible that the antibody-free bovine serum will become the "next generation" bovine serum to replace the current serum products. To achieve these goals, we utilized two mammalian cell lines that are most commonly used for recombinant protein production: 1) the recombinant CHO (rCHO-ks) cell line that secretes the protein alkaline phosphatase, and 2) the Vero cell line which is the most often used cell line for vaccine production. We cultured each of these two cell lines in standard cell culture medium (Dulbecco's Modified Eagle's medium; DMEM) supplemented either with AF-CBS, FBS, or CBS, and investigated cell yield (growth), cell viability, glucose consumption and lactate formation, and alkaline phosphatase production (in CHO-ks only). We have successfully accomplished all of the planned experiments and addressed all of the questions we had at the initiation of this project. Our results have led to the conclusion that AF-CBS is superior to CBS and comparable to traditional FBS in terms of promoting cell growth, cell viability, and recombinant protein production by both rCHO-KS & Vero cell lines. Specifically, we found that AF-CBS provides about 2-fold more cell yield (a measure of cell growth) and viability than CBS does and is comparable to FBS. In terms of alkaline phosphatase production by rCHO-KS cells, AF-CBS behaves very similar to FBS and results in over 2-fold alkaline phosphatase production per rCHO-KS cell. Because energy metabolism is a critical parameter in measuring cell health and proliferation, we also measured glucose consumption and lactate production. We found that both rCHO-KS and Vero cells consume significantly higher levels (p<0.05) of glucose when the culture media were supplied with either AF-CBS or FBS than supplied with CBS; similarly, lactate production by these two cell lines grown in AF-CBS or FBS supplied media is significantly higher (p<0.05). It is worth noting that in AF-CBS performs also better than FBS. In summary, this project demonstrated that AF-CBS is superior to CBS and is comparable or better than FBS in promoting cell growth, viability and recombinant protein production. In economic terms AF-CBS would be more desirable than FBS for industrial applications and scientific research, because AF-CBS can be produced from calves or adult cattle while FBS is produced from fetuses. Furthermore, because large volumes of AF-CBS can be produced each time, it would also reduce the commonly found batch to batch variations in FBS. Taken together, our investigation has paved the way for more in-depth testing of this novel AF-CBS product to establish it as the "next generation" serum to replace CBS and FBS. Our study has significant implications both for the pharmaceutical industry and for academic research where serum is routinely used.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Target Audience The target audiences of this study are serum producers, pharmaceutical companies, vaccine companies, research institutions, and universities where serum is used. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Opportunities This project has been proven to be a great training opportunity for undergraduate students. A senior student, LaDonya Jackson, was the primary undergraduate student working on this project and was the first author for an oral presentation and a poster at two national conferences respectively. How have the results been disseminated to communities of interest?Dissemination In addition to the presentation of our research work at two national conferences (one is at 27th National Conference on Undergraduate Research (NCUR), April 11-13, 2013, University of Wisconsin-La Crosse, Wisconsin USA, and another at the ISBioTech 3rd Annual Meeting, March 11-15, 2013, Rosslyn, VA USA), we are planning to publish the research in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work To carry out research on the effect of Ab-free serum on hamster embryo culture. This will depend on if we are successful in obtaining external or internal funding based on the data collected.
Impacts
What was accomplished under these goals?
Accomplishments We have completed all the experiments and are in the process of analyzing the data for a potential scientific publication.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Target Audience The target audiences of this study are serum producers, pharmaceutical companies, vaccine companies, research institutions, and universities where serum is used. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Opportunities This project has been proven to be a great training opportunity for undergraduate students. A senior student, LaDonya Jackson, was the primary undergraduate student working on this project and was the first author for an oral presentation and a poster at two national conferences respectively. How have the results been disseminated to communities of interest?Dissemination In addition to the presentation of our research work at two national conferences (one is at 27th National Conference on Undergraduate Research (NCUR), April 11-13, 2013, University of Wisconsin-La Crosse, Wisconsin USA, and another at the ISBioTech 3rd Annual Meeting, March 11-15, 2013, Rosslyn, VA USA), we are planning to publish the research in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work We are actively applying for funding to further extend the research.
Impacts
What was accomplished under these goals?
Accomplishments We demonstrated that antibody-free newborn calf serum is superior to calf bovine serum supplementation and comparable to traditional fetal bovine serum in terms of growth and protein production in the rCHO-KS& Vero cell line.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target Audience The target audiences of this study are serum producers, pharmaceutical companies, vaccine companies, research institutions, and universities where serum is used. Changes/Problems: Changes/Problems Dr. Kamal Rashid has left USU. Consequently, we are not planning to test the serum on other cell lines. The PI (Zhongde Wang) is in the process of discussing with the current Center for Integrated Biology on finding a new faculty who can replace Dr. Rishad's role as a co-PI for this project. What opportunities for training and professional development has the project provided? Opportunities This project has been proven to be a great training opportunity for undergraduate students. A senior student, LaDonya Jackson, was the primary undergraduate student working on this project and was the first author for an oral presentation and a poster at two national conferences respectively. She is also the first author on the to be published manuscript. How have the results been disseminated to communities of interest? Dissemination In addition to the presentation of our research work at two national conferences (one is at 27th National Conference on Undergraduate Research (NCUR), April 11-13, 2013, University of Wisconsin-La Crosse, Wisconsin USA, and another at the ISBioTech 3rd Annual Meeting, March 11-15, 2013, Rosslyn, VA USA), we are planning to publish the research in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals? Plan of Work To pursue the publication of the work. Potentially work with private sector to commercialize the Ab-free serum that we characterized.
Impacts
What was accomplished under these goals?
Accomplishments We demonstrated that antibody-free newborn calf serum is superior to calf bovine serum supplementation and comparable to traditional fetal bovine serum in terms of growth and protein production in the rCHO-KS& Vero cell line
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audiences of this study are serum producers, pharmaceutical companies, vaccine companies, research institutions, and universities where serum is used. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has provided a great training opportunity for undergraduate students. A senior student, LaDonya Jackson, was the primary undergraduate student working on this project and was the first author for an oral presentation and a poster at two national conferences respectively. How have the results been disseminated to communities of interest? We presented the results from this project at two national conferences, one is at 27th National Conference on Undergraduate Research (NCUR), April 11-13, 2013, University of Wisconsin-La Crosse, Wisconsin USA, and another at the ISBioTech 3rd Annual Meeting, March 11-15, 2013, Rosslyn, VA USA What do you plan to do during the next reporting period to accomplish the goals? We are in the process of summarizing the data and preparing a manuscript for publication in a peer-reviewed journal.
Impacts
What was accomplished under these goals?
1) Major activities completed: we systematically compared Ab-free bovine serum with calf serum and fetal serum on the growth of CHO cells and the production of recombinant protein. 2) Specific objectives met: we accomplished the objectives of testing the effects of Ab-serum on recombinant protein production by CHO cells. 3) Significant results: we found that Ab-free bovine serum is superior to conventional serum in both supporting cell growth and recombinant protein production. 4) Key impacts: this is the first study on the effect of antibody on recombinant protein production in CHO cells. This study is significant because it addresses a question both scientifically and practically important for optimizing cell culture conditions to more efficiently produce recombinant proteins.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Activities: The PI and co-PI 1) have been supervising a senior student and a research associate carrying out the project; 2) submitted an abstract to and accepted by International Society for Bioprocess Technology based on the results. Events: submitted a oral presentation to and accepted by National Conference on Undergraduate Research, 2013 PARTICIPANTS: LaDonya Jackson (undergraduate), Shaun Barnett (research associate), Dr. Zhongde Wang (PI), and Dr. Kamal Rashid (co-PI). TARGET AUDIENCES: Researchers who use bovine serum, especially those who work in bioprocess industry. Entrepreneurs and investors interested in developing new cell culture reagents should also find our data interesting. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Bovine sera are widely used for growing animal cells in culture for the production of recombinant proteins and vaccines. Multiple studies have shown that bovine antibodies against a variety of pathogenic and nonpathogenic antigens are present in both fetal bovine serum (FBS) and calf bovine serum (CBS). The presence of antibodies that are positive to a variety of antigens in bovine sera can affect many aspects of mammalian cell culture investigations. The presence of antibodies against Neospora caninum, for an example, may bind to antibody producing cells during MAB production. The presence of bovine antibodies can block or compete during screening of hybridoma cells. This may lead to false positive results in diagnostic assays and other biological applications. False negatives will also occur when the antibodies in the serum mask contamination by bovine viruses in vaccines. Due to these intrinsic problems with using bovine sera, it was essential to develop safer bovine sera. We demonstrated that antibody-free fetal bovine serum (FBS) performs much better than calf bovine serum (CBS) and similarly to regular FBS in supporting rCHO cell growth, cell viability, glucose uptake, lactate formation and alkaline phosphatase production. This is a very important finding for cell culture as the problems associated with bovine antibodies present in the serum in cell culture can be solved.
Publications
- No publications reported this period
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