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

Peter Satzer, Ralf Sommer, Johanna Paulsson, Agnes Rodler, Romana Zehetner, Klaus Hofstädter, Christoph Klade, Alois Jungbauer

Research output: Contribution to journalArticlepeer-review

3 Citations (SciVal)


We developed a novel analytical method for concentration determination of tandem single-chain antibody diphtheria toxin (immunotoxin). The method is based on polymethacrylate monoliths with Protein L ligands as the binding moiety. Different buffers were tested for elution of the Protein L-bound immunotoxin and 4.5 M guanidinium hydrochloride performed best. We optimized the elution conditions and the method sequence resulting in a fast and robust method with a runtime <10 min. Fast determination of immunotoxin is critical if any process decisions rely on this data. We determined method performance and a lower limit of detection of 27 μg/mL and a lower limit of quantification of 90 μg/mL was achieved. The validity of the method in terms of residual analysis, precision, and repeatability was proven in a range from 100 to 375 μg/mL. The short runtime and ease of use of a high-performance liquid chromatography method is especially useful for a process analytical tool approach. Bioprocesses related to immunotoxin where fermentation or other process parameters can be adjusted in accordance to the immunotoxin levels will be benefited from this method to achieve the highest possible purity and productivity.

Original languageEnglish
Pages (from-to)3051-3059
Number of pages9
JournalJournal of Separation Science
Issue number15
Publication statusPublished - 2018 Aug 1

Subject classification (UKÄ)

  • Analytical Chemistry


  • affinity chromatography
  • antibody-drug conjugates
  • immunotoxins
  • monoliths
  • process analytical tools


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