Acknowledgements

characteristics of the SEC column changes by 10% or more, it is prudent that cleaning is necessary. The acceptance criteria can be more stringent as needed. Similar cleaning of the

If cleaning is necessary, it is better to try cleaning the column in reversed direction at half the flow rate while the column is detached from the detector to prevent detector damage since the proteins have a tendency to adhere to the quartz material of the detector. Since the dirty materials may remain trapped over the frit outside the column, cleaning in reversed flow direction may be the easiest way to clean the column. Once the column is cleaned using the vendor suggested protocol, the analyst must qualify the column using the QC method and QC

It is always recommended to read the operational conditions and specification sheet (OCS) before the column is used. Occasionally, we may intend to use the column outside the specification for a short period of time, but it is the responsibility of the analyst to monitor if the column is functioning properly. For example, in Section 2.5, it has been shown that an analysis could be repeated to monitor the stability of the antibody over time using a TSKgel SuperSW mAb-HTP column at 0.75 mL/min. The used flow rate (0.75 mL/min) was higher than the recommended maximum flow rate of 0.5 mL/min, as mentioned in the operational conditions and specification (OCS) sheet for this column. Though the column could successfully be used at this higher flow rate without compromising the column health, the effect of the higher flow rate on the column lifetime for prolonged use was not investigated. It is always wise to operate within recommended maximum flow rate to remain in safe side in order to prolong the lifetime of the column. It is always necessary to keep an eye on the back pressure, peak

HPLC system and analytical chromatography columns are costly. Method development is time-consuming and costly too. It is important to employ an HPLC system that is optimized with regard to extra-column band broadening to take full advantage of the high column efficiency that can be obtained on analytical columns. For any troubleshooting situation, the problem is one of the following: [1] the column, [2] the sample, [3] the mobile phases, [4] the instrumentation, and [5] personal errors. The same is true for size exclusion chromatography related troubleshooting issues too. Proper maintenance of the column and instrument is much more needed and important to avoid the troubleshooting in the beginning. Many troubleshooting issues can be avoided by taking proper handling the top three major components of any HPLC analysis. The importance of the use of a protein standard mixture and the standard mAb to monitor the column health from time to time is always a good idea as mentioned in brief in Section 2.5. Maintaining a SEC column is an art and good performance, which will

Size-exclusion chromatography is a great tool for the purification of monoclonal antibodies. The secondary interaction of the stationary phase needs to be taken care of by controlling the

whole system including the flow cell may be necessary.

pass criteria under normal flow direction.

170 Antibody Engineering

parameters, and so on to monitor the column health.

result in a prolonged column lifetime.

10. Conclusions

Yoshimi Hashimoto, Toshiaki Nishi, Kazuaki Muranaka, Kosuke Araki, Yasutami Mitoma, Shigeru Nakatani, Hasegawa, Sachiyuki, Oscar Yamasaki, Toru Satoh – Tosoh Corporation, Separation Centre, Japan for their contributions in a number of references and overall support. Tosoh Bioscience LLC and Tosoh Corporation, Tosoh Bioscience LLC, Tosoh Bioscience Gmbh.
