Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.

The humanized monoclonal antibody (mAb) against the colon and ovarian cancer cells has been successfully expressed in NS0 cell line. This humanized mAb can be used to detect and treat human colorectal cancer. Historically, medium supplemented with serum were widely used in animal cell culture. Howev...

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Main Author:	Ab Latif, Nurzila
Format:	Thesis
Language:	English English
Published:	2011
Subjects:	Serum-free culture media Monoclonal antibodies
Online Access:	http://psasir.upm.edu.my/id/eprint/26510/2/IB%202011%2030R.pdf
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spelling	my-upm-ir.265102014-10-28T02:07:07Z Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody. 2011-12 Ab Latif, Nurzila The humanized monoclonal antibody (mAb) against the colon and ovarian cancer cells has been successfully expressed in NS0 cell line. This humanized mAb can be used to detect and treat human colorectal cancer. Historically, medium supplemented with serum were widely used in animal cell culture. However, the used of serum may leads to virus and prion contamination and gives problems for the downstream process due to its unknown and undefined component. The aim of this study is to develop a suitable serum free culture medium for the production of the humanized mAb. Two types of basal medium, DMEM and IMDM were tested as suitable basal medium for the growth of NS0 cell. Cells were cultured in both basal medium supplemented with different concentration of fetal bovine serum (FBS) (1, 3, 5, 7, 10 (v/v)), 4mM of L-glutamine and 10 μg/ml of antibiotic. The cell concentration and viability of the NS0 cells were determined using tryphan blue exclusion method and ELISA was used to determine the mAb titers of the cultures. The results showed that the higher growth and mAb productivity were observed in DMEM supplemented with 10 % (v/v) FBS. The concentration reached 4.76 x 106 cells/mL compared to only 1.95 x 106 cells/mL in IMDM supplemented with 10 % (v/v) FBS medium. The highest antibody titer, 12.89 μg/ml, was obtained from culture condition of DMEM supplemented with 7 % (v/v) FBS. The other objective of this study is to optimize a serum free medium for the NS0 cell line. Different ratios of DMEM/Ex-cell medium were studied. Results showed that the most suitable ratio for the cell line to grow and producing antibody is in 5:5 DMEM/Ex-cell medium. In order to replace serum in cell culture medium, three component; insulin, SyntheChol and L-glutamine were tested using design of experiment (DOE). By using the 2 level factorial design screening method, the significant of the parameters are determined. Then further optimization was done using Central Composite Design (CCD) by augmenting the previous design using 8 additional runs. Experiments were done in T-flask with working volume of 5 mL. Results showed that several combinations gave good but not an optimum result for the responses. This is due to the narrow ranges of parameters that have been used during experimenting. A larger scale of culture (50 mL) using the optimize condition suggested by the software has substantially increased the viable cell concentration and antibody production by 75 % and 48 %, respectively compare to the non-optimal condition. Serum-free culture media Monoclonal antibodies 2011-12 Thesis http://psasir.upm.edu.my/id/eprint/26510/ http://psasir.upm.edu.my/id/eprint/26510/2/IB%202011%2030R.pdf application/pdf en public masters Universiti Putra Malaysia Serum-free culture media Monoclonal antibodies Institute of Bioscience English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Serum-free culture media Monoclonal antibodies
spellingShingle	Serum-free culture media Monoclonal antibodies Ab Latif, Nurzila Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.
description	The humanized monoclonal antibody (mAb) against the colon and ovarian cancer cells has been successfully expressed in NS0 cell line. This humanized mAb can be used to detect and treat human colorectal cancer. Historically, medium supplemented with serum were widely used in animal cell culture. However, the used of serum may leads to virus and prion contamination and gives problems for the downstream process due to its unknown and undefined component. The aim of this study is to develop a suitable serum free culture medium for the production of the humanized mAb. Two types of basal medium, DMEM and IMDM were tested as suitable basal medium for the growth of NS0 cell. Cells were cultured in both basal medium supplemented with different concentration of fetal bovine serum (FBS) (1, 3, 5, 7, 10 (v/v)), 4mM of L-glutamine and 10 μg/ml of antibiotic. The cell concentration and viability of the NS0 cells were determined using tryphan blue exclusion method and ELISA was used to determine the mAb titers of the cultures. The results showed that the higher growth and mAb productivity were observed in DMEM supplemented with 10 % (v/v) FBS. The concentration reached 4.76 x 106 cells/mL compared to only 1.95 x 106 cells/mL in IMDM supplemented with 10 % (v/v) FBS medium. The highest antibody titer, 12.89 μg/ml, was obtained from culture condition of DMEM supplemented with 7 % (v/v) FBS. The other objective of this study is to optimize a serum free medium for the NS0 cell line. Different ratios of DMEM/Ex-cell medium were studied. Results showed that the most suitable ratio for the cell line to grow and producing antibody is in 5:5 DMEM/Ex-cell medium. In order to replace serum in cell culture medium, three component; insulin, SyntheChol and L-glutamine were tested using design of experiment (DOE). By using the 2 level factorial design screening method, the significant of the parameters are determined. Then further optimization was done using Central Composite Design (CCD) by augmenting the previous design using 8 additional runs. Experiments were done in T-flask with working volume of 5 mL. Results showed that several combinations gave good but not an optimum result for the responses. This is due to the narrow ranges of parameters that have been used during experimenting. A larger scale of culture (50 mL) using the optimize condition suggested by the software has substantially increased the viable cell concentration and antibody production by 75 % and 48 %, respectively compare to the non-optimal condition.
format	Thesis
qualification_level	Master's degree
author	Ab Latif, Nurzila
author_facet	Ab Latif, Nurzila
author_sort	Ab Latif, Nurzila
title	Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.
title_short	Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.
title_full	Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.
title_fullStr	Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.
title_full_unstemmed	Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.
title_sort	optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.
granting_institution	Universiti Putra Malaysia
granting_department	Institute of Bioscience
publishDate	2011
url	http://psasir.upm.edu.my/id/eprint/26510/2/IB%202011%2030R.pdf
_version_	1747811545463652352

Optimization of serum free medium for the production of humanized anti-cea monoclonal antibody.

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