High-throughput quantification of glycoprotein sialylation
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2011.
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | eng |
| Published: |
Massachusetts Institute of Technology
2011
|
| Subjects: | |
| Online Access: | http://hdl.handle.net/1721.1/65762 |
| _version_ | 1826204745406087168 |
|---|---|
| author | Markely, Lam Raga Anggara |
| author2 | Daniel I.C. Wang. |
| author_facet | Daniel I.C. Wang. Markely, Lam Raga Anggara |
| author_sort | Markely, Lam Raga Anggara |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2011. |
| first_indexed | 2024-09-23T13:00:25Z |
| format | Thesis |
| id | mit-1721.1/65762 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T13:00:25Z |
| publishDate | 2011 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/657622019-04-10T07:58:02Z High-throughput quantification of glycoprotein sialylation Markely, Lam Raga Anggara Daniel I.C. Wang. Massachusetts Institute of Technology. Dept. of Chemical Engineering. Massachusetts Institute of Technology. Dept. of Chemical Engineering. Chemical Engineering. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2011. Cataloged from PDF version of thesis. Includes bibliographical references (p. 98-105). Sialic acid can improve qualities of therapeutic glycoproteins, such as circulatory half-life, biological activity, and solubility. In production of therapeutic glycoproteins, a high-throughput method (HTM) is required for process monitoring and optimization to ensure consistent and optimal sialic acid content. The HTM is also required for cell clone screening in cell line development. Current methods for quantifying sialic acid, however, require chromatographic separation that is time consuming and cannot rapidly analyze many samples in parallel. Here we develop a novel HTM for quantifying glycoprotein sialylation. Using chemical reduction, enzymatic release of sialic acid, and chemical derivatization, the HTM can accurately, rapidly (15 min), and specifically analyze many samples in parallel. It requires only 45 piL of sample and has a quantitation limit of 2 ptM sialic acid. We validated the HTM for monitoring sialylation of recombinant interferon-gamma (IFN-y) produced in Chinese Hamster Ovary (CHO) cell culture. The HTM was accurate in monitoring sialylation of IFN-y in batch CHO cell cultures. Furthermore, we used the HTM to study the effects of feeding ManNAc, Cu 2+, and Mn2+ on sialylation of glycoproteins produced in CHO-IFN-y cell cultures. We found that feeding these chemicals increased sialylation from 20 to 36 mg sialic acid/g protein in batch CHO cell cultures. Moreover, a quadratic least square model predicts that the feeding 2 mM ManNAc and 100 pM Cu2+ will increase the sialylation to 41 +/- 4 mg sialic acid/g protein, close to the experimental value of 35 ± 5 mg sialic acid/g protein. We also used the HTM to study intraclonal variability in glycoprotein sialylation. We found that there was significant variability in sialic acid content and productivity. The sialic acid content varied from 1 to 70 mg sialic acid/g protein, and was negatively correlated with the productivity. Overall, we have developed a novel HTM and demonstrated its versatility for various applications in bioprocesses. The HTM can measure sialic acid content of hundreds of samples in 15 minutes, while conventional methods require more than one day per sample. Thus, the HTM is an important analytical tool for producing therapeutic proteins with consistent and optimum sialylation. by Lam Raga Anggara Markely. Ph.D. 2011-09-13T17:49:08Z 2011-09-13T17:49:08Z 2011 2011 Thesis http://hdl.handle.net/1721.1/65762 749124927 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 105 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemical Engineering. Markely, Lam Raga Anggara High-throughput quantification of glycoprotein sialylation |
| title | High-throughput quantification of glycoprotein sialylation |
| title_full | High-throughput quantification of glycoprotein sialylation |
| title_fullStr | High-throughput quantification of glycoprotein sialylation |
| title_full_unstemmed | High-throughput quantification of glycoprotein sialylation |
| title_short | High-throughput quantification of glycoprotein sialylation |
| title_sort | high throughput quantification of glycoprotein sialylation |
| topic | Chemical Engineering. |
| url | http://hdl.handle.net/1721.1/65762 |
| work_keys_str_mv | AT markelylamragaanggara highthroughputquantificationofglycoproteinsialylation |