Reversed-Phase Analyses of Monoclonal Antibodies Using High Temperatures

Monoclonal antibodies (mAbs) are immunological active proteins, which bind specifically to certain cells or proteins. This will stimulate the immune system to attack those targets.

mAbs are very important for the treatment of different kinds of cancer and autoimmune diseases. Nowadays, a broad variety of therapeutical antibodies are available on the market and several more are in research and development.

Due to their molecular weight of about 150 kDa, intact antibodies are usually analyzed by IEX, SEC, or HIC. In addition, reversed‑phase methods are an easy tool as well.

However, a lack of sensitivity and resolution has been a hurdle in the past. With modern reversed phases addressing the requirements of these analytes, it is easy to find a suitable method.

Successful analysis in reversed-phase mode for mAbs is enhanced by employing a temperature stable (> 60 °C), widepore stationary phase.

This application note shows how to achieve robust chromatographic results for two commercially available mAbs: Adalimumab (Humira®) and Bevacizumab (Avastin®).

The increase in temperatures leads to higher sensitivity and a sharper peak of Adalimumab at temperatures >60 °C (Figure 1).

Bevacizumab shows robust results starting from 70 °C, whereas at lower temperatures no peak was detected (Figure 2).

With YMC-Triart Bio C4, elevated temperatures can easily be applied, due to stability up to 90 °C. In addition, the surface comprising 30 nm/300 Å is beneficial for resolution. In combination with a wide pH range of 1–10 YMC-Triart Bio C4 is a well-suited tool for any mAb (U)HPLC method.

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