Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
Austrian Centre of Industrial Biotechnology, Vienna, Austria.
J Sep Sci. 2018 Aug;41(15):3051-3059. doi: 10.1002/jssc.201800257. Epub 2018 Jul 1.
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.
我们开发了一种用于串联单链抗体白喉毒素（免疫毒素）浓度测定的新型分析方法。该方法基于具有蛋白 L 配体作为结合部分的聚甲基丙烯酸酯整体柱。测试了不同的缓冲液用于洗脱蛋白 L 结合的免疫毒素，4.5 M 盐酸胍效果最佳。我们优化了洗脱条件和方法序列，得到了一种快速、稳健的方法，运行时间<10 分钟。如果任何过程决策都依赖于这些数据，那么快速测定免疫毒素是至关重要的。我们确定了该方法的性能，检测限为 27μg/mL，定量限为 90μg/mL。在 100 至 375μg/mL 的范围内证明了该方法在残留分析、精密度和重复性方面的有效性。高效液相色谱法的短运行时间和易用性特别适用于过程分析工具方法。与免疫毒素相关的生物工艺，如果可以根据免疫毒素水平调整发酵或其他过程参数，将受益于该方法以达到尽可能高的纯度和生产率。
