Monolith affinity chromatography for the rapid quantification of a single-chain variable fragment immunotoxin

Area of Science:

• Analytical Chemistry
• Biotechnology
• Biopharmaceutical Analysis

Background:

• Immunotoxins are critical biopharmaceuticals requiring precise concentration monitoring.
• Existing analytical methods may lack the speed and robustness needed for real-time bioprocess control.

Purpose of the Study:

• To develop a novel, rapid, and robust analytical method for determining immunotoxin concentration.
• To validate the method's performance for application in bioprocess analytical tools.

Main Methods:

• Development of a high-performance liquid chromatography (HPLC) method utilizing polymethacrylate monoliths functionalized with Protein L ligands.
• Optimization of buffer conditions for efficient elution, with 4.5 M guanidinium hydrochloride identified as optimal.
• Streamlining of the method sequence to achieve a total runtime under 10 minutes.

Main Results:

• Achieved a lower limit of detection (LLOD) of 27 μg/mL and a lower limit of quantification (LLOQ) of 90 μg/mL.
• Demonstrated method validity, precision, and repeatability within the 100–375 μg/mL range.
• Established a fast (<10 min) and robust analytical procedure suitable for process monitoring.

Conclusions:

• The developed HPLC method provides a fast and reliable means for immunotoxin concentration determination.
• This method serves as a valuable process analytical tool, enabling real-time adjustments in bioprocesses for enhanced purity and productivity.
• The method's speed and ease of use are particularly beneficial for immunotoxin-related bioprocesses requiring dynamic control.