GE Healthcare Bio-Sciences AB, Downstream Bioprocessing, Björkgatan 30, SE-75184 Uppsala, Sweden.
GE Healthcare Bio-Sciences AB, Downstream Bioprocessing, Björkgatan 30, SE-75184 Uppsala, Sweden.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jun 15;1118-1119:194-202. doi: 10.1016/j.jchromb.2019.04.018. Epub 2019 Apr 18.
A rapid and sensitive liquid chromatography-mass spectrometry assay was developed and used to quantify emetic cereulide peptide exotoxin, which can be related to possible Bacillus cereus contamination in monoclonal antibody (mAb) bioprocess feeds. The assay limit of detection was 0.05 ng/mL (1 fmol injected) and limit of quantification 0.16 ng/mL (3 fmol injected) over a standard curve with >3 orders of magnitude linear dynamic range. The assay allowed quantification of toxin removal in an established two-step mAb purification process consisting of Protein A affinity chromatography followed by multi-modal anion exchange chromatography. Toxin content was ascertained in process stream sample fractions as well as on the Protein A affinity column. An optimized analytical method allowed separation of cereulide toxin from other mAb cell culture components within 6 min. Spiking experiments showed that samples should be collected in high (80% v/v) content acetonitrile to reduce nonspecific losses of the cereulide. The majority of mAb purification process-associated cereulide was detected in the Protein A flow through fraction, whereas only residual amounts were found in wash, strip, and elution fractions. Column cleaning-in-place (CIP) procedures were evaluated to prevent carryover between affinity capture cycles. No carryover was detected between cycles, however trace amounts of cereulide were extracted from the Protein A resin. Increasing the CIP NaOH concentration from 0.1 M to 0.5 M, and contact time from 15 min to 1 h, improved removal of residual cereulide from the resin. Applicability of CIP clearance of cereulide during Protein A chromatography was confirmed with three different mAb feeds. Post Protein A polishing, via target flow through on a multi-modal anion exchange chromatography column, resulted in a product pool with no detectable cereulide. Approximately 5 logs of reduction in cereulide concentration was obtained over the two-step chromatography process. Cereulide contamination is well known and of concern in food processing, however this research may be the first LC-MS quantification of cereulide contamination, and its clearance, in biopharmaceutical mAb processing. The analytical method may also be used to rapidly screen for cereulide contamination in upstream cell culture process streams, prior to downstream product purification. This will allow appropriate measures to be taken to reduce toxin exposure to downstream bioprocess raw materials, consumables and equipment.
建立并应用了一种快速灵敏的液相色谱-质谱法来定量分析呕吐性的环十肽呕吐毒素，这种毒素可能与单克隆抗体（mAb）生物工艺进料中存在的可能的蜡样芽胞杆菌污染有关。该检测方法的检测限为 0.05ng/mL（1fmol 进样），定量限为 0.16ng/mL（3fmol 进样），标准曲线的线性动态范围超过 3 个数量级。该检测方法可定量评估两步 mAb 纯化过程中呕吐毒素的去除情况，该过程由 Protein A 亲和层析和多模式阴离子交换层析组成。毒素含量在工艺流样品级分以及 Protein A 亲和柱上进行了测定。优化的分析方法可在 6 分钟内将呕吐毒素与其他 mAb 细胞培养成分分离。加标实验表明，为减少呕吐毒素的非特异性损失，样品应在高浓度（80%v/v）乙腈中收集。在 Protein A 流穿级分中检测到大多数与 mAb 纯化过程相关的呕吐毒素，而在洗涤、洗脱和洗脱级分中仅检测到残留量。评估了在位清洗（CIP）程序以防止亲和捕获循环之间的交叉污染。在循环之间未检测到交叉污染，但从 Protein A 树脂中提取了痕量的呕吐毒素。将 CIP 氢氧化钠浓度从 0.1M 增加到 0.5M，接触时间从 15 分钟增加到 1 小时，可改善树脂中残留呕吐毒素的去除效果。通过在多模式阴离子交换层析柱上进行目标流穿，对 Protein A 层析过程中 CIP 清除呕吐毒素的适用性进行了验证，得到了无呕吐毒素可检测的产物池。经过两步层析过程，呕吐毒素的浓度降低了约 5 个对数级。呕吐毒素污染在食品加工中是众所周知且令人担忧的，然而，这项研究可能是首次对生物制药 mAb 加工过程中的呕吐毒素污染及其清除情况进行 LC-MS 定量分析。该分析方法还可用于在下游产品纯化之前，快速筛选上游细胞培养工艺流中的呕吐毒素污染情况。这将有助于采取适当措施，降低下游生物工艺原料、耗材和设备中暴露于毒素的风险。
