Application of Ion Chromatography with Electrochemical Detection in Optimization and Control of Fermentation and Cell Culture - An overview of applications in the analysis of nutrients and

ADVERTISEMENT

Application of Ion Chromatography with Electrochemical Detection in Optimization and Control of Fermentation and Cell Culture
An overview of applications in the analysis of nutrients and metabolites.


BioPharm International Supplements
Volume 24, Issue 5, pp. s12-s20

Amino Acids


Figure 5: Amino acid analysis of (1) a standard amino acid mixture containing 277.5 µM glucose;(2) GMEM medium; (3) GMEM medium + 1% (v/v) peptone; (4) GMEM medium+10% (v/v) FCS + 1% (v/v) peptone. Norleucine was added as the internal standard. Column: AminoPac PA10; elution: as per the procedure described in Ref. 16. (Reprinted from Ref. 13 with permission from Elsevier)
The amino acid compositions of cell culture and fermentation broths can be determined by HPAEC-PAD without sample derivatization (13–15). However, the presence of carbohydrates and alditols in the media may interfere. By optimizing the elution method, Genzel et al. (13) reported that the amino acids were well resolved and also resolved from the predominant carbohydrate, glucose, in GMEM media used in the production of inactivated influenza vaccines (see Figure 5). The authors reported good precision, accuracy, linear range, and low quantitation limit for each amino acid. Similar results were also reported for the supernatants of YPD broth, LB broth, Minimum Essential Eagle's Medium and a serum-free, protein-free hybridoma medium (14, 15).

Antibiotics


Figure 6: HPAEC-PAD chromatograms of a neomycin standard and two selective culture media containing neomycin. Column: CarboPac PA1; flow rate: 0.5 mL/min. The effluent from the column was mixed with 100 mM NaOH before entering into the detector for baseline stabilization.
Selective media are used for the growth of select microorganisms. For example, if a microorganism is resistant to a certain antibiotic, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. Selective growth media for eukaryotic cells commonly contain neomycin to select cells that have been successfully transfected with a plasmid carrying the neomycin resistance gene as a marker. Generally, neomycin is assayed following the procedure described in USPNF General Chapter <81>, Antibiotics—Microbial Assay (17). However, the procedure is tedious and labor intensive, has poor precision, and requires days to complete. Neomycin is an aminoglycoside containing a carbohydrate moiety and, like other carbohydrates, can be assayed using HPAEC–PAD. Figure 6 shows the HPAEC–PAD chromatograms of two selective media containing neomycin. The media also contain glucose, sucrose, and amino acids at concentrations 100-1000 fold higher than that of neomycin. These components also show peaks in HPAEC–PAD. Thus, the resolution of neomycin was a challenge, which was accomplished using a CarboPac PA1 column and a linear elution gradient from 1.0 mM to 3.0 mM NaOH over 25 min.

Conclusion

Monitoring nutrients and metabolites in cell culture media and fermentation broth provides critical pieces of information on their roles in fermentation, and in optimizing, characterizing, and controlling the process to produce safe and efficacious therapeutic products in a reproducible manner. The results presented in this review show that the unique selectivity and sensitivity of IC with ED permit determination of fermentation broth components. This is particularly important for those components which do not have suitable chromophores for detection by absorption measurement. The sample preparation involves simple filtration and dilution of fermentation broth with water. No pre- or postcolumn derivatization is necessary for detection. In addition, the technique provides the ability to analyze the desired components selectively in the presence of others, e.g., amino acids in the presence of large excess of carbohydrates or inorganic cations and anions in the presence of amino acids. These features make IC with ED an effective technique in analysis of fermentation broths in a simple and cost effective manner.

References

1. 21 CFR Parts 210 and 211, Parts 600–610 (Government Printing Office, Washington, DC).

2. Application Note 157, "Comparison of Suppressed to Nonsuppressed Conductivity Detection for the Determination of Common Inorganic Cations" (Dionex Corp., Sunnyvale, CA).

3. W.R. LaCourse, Pulsed Electrochemical Detection in High Performance Liquid Chromatography (Wiley, New York, NY, 1997).

4. J. Weiss, Ed., Handbook of Ion Chromatography, 3rd ed. (VCH Verlag, Weinheim, Germany, 2004).

5. S.J. Fritz and D.T. Gjerde, Ion Chromatography, 4th ed., (Wiley-VCH, Weinheim, Germany, 2009).

6. L. Bhattacharyya and J.S. Rohrer, Eds., Applications of Ion Chromatography for Pharmaceutical and Biological Products (Wiley, New York, NY), in press.

7. L. Joergensen, A. Weimann, and H.F. Botte, J. Chromatogr. 602, 179–188 (1992).).

8. R.S.R. Robinett and W.K. Herber, J. Chromatogr. A 671, 315–322 (1994).

9. R.S.R. Robinett, H.A. George, and W.K. Herber, J. Chromatogr. A 718, 319–327 (1995).

10. Application Note 123, "The Determination of Inorganic Anions and Organic Acids in Fermentation Broths" (Dionex Corp., Sunnyvale, CA 2006).

11. W.K. Herber and R.S.R. Robinett, J. Chromatogr. A 676, 287–295 (1994).

12. Application Note 122, "The Determination of Carbohydrates, Alcohols, and Glycols in Fermentation Broths" (Dionex Corp., Sunnyvale, CA).

13. Y. Genzel, S. Königa, and U. Reichl, Anal Biochem. 335, 119–125 (2004).

14. V.P. Hanko and J.S. Rohrer, Anal. Biochem. 324, 29–38 (2004).

15. V.P. Hanko, A. Heckenberg, and J.S. Rohrer, J. Biomol. Tech. 15, 317–324 (2004).

16. P. Jandik et al., J. Chromatogr. B 732, 193–201 (1999).

17. USP–NF General Chapter <81>, "Antibiotics—Microbial Assay (USP, Rockville, MD).

*The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

Mallinckrodt to Acquire Questcor Pharmaceuticals
April 16, 2014
EMA Warns of Falsified Herceptin Vials
April 16, 2014
PhRMA Report Reveals Growth Trajectories and Policy Factors Affecting Biopharmaceutical Growth
April 11, 2014
American CryoStem and Rutgers University File Joint Patent on Stem Cell Platform
April 11, 2014
Center for Biologics Evaluation and Research Relocates
April 11, 2014
Author Guidelines
Source: BioPharm International Supplements,
Click here