Toinon Audrey, Fontaine Christelle, Thion Laurent, Gajewska Beata, Carpick Bruce, Nougarede Nolwenn, Uhlrich Sylvie
Sanofi Pasteur, 1541 Avenue Marcel Merieux, 69280 Marcy L'Etoile, France.
Sanofi Pasteur, 1541 Avenue Marcel Merieux, 69280 Marcy L'Etoile, France.
Biologicals. 2018 Jul;54:1-7. doi: 10.1016/j.biologicals.2018.05.006. Epub 2018 May 31.
Recombinant proteins expressed in host cell systems may contain host cell proteins (HCP) as impurities. While there is no clear evidence of clinical adverse events attributable to HCP, HCP levels and profiles must be documented to meet regulatory requirements and to understand the consistency of the biological product and manufacturing process. We present a general strategy for HCP characterization applied to a recombinant protein antigen, Hepatitis B surface antigen (HBsAg) used in a multivalent vaccine.
Polyclonal antisera raised against HCPs in process fractions from a mock preparation of the HBsAg yeast expression host, Hansenula polymorpha, were used to develop a quantitative sandwich ELISA to measure HCP content in batches of purified recombinant HBsAg. Batches were also subjected to SDS-PAGE and LC-MS/MS to identify detectable proteins. Batch consistency was further assessed by SDS-PAGE/densitometry purity analysis and by the ratio of specific HBsAg content (by ELISA) to total protein.
Using the quantitative HCP ELISA, the HCP content showed no discernable trend in multiple HBsAg batches manufactured over a 5-year period. All batches were ≥95% pure by SDS-PAGE/densitometry, with consistent HBsAg/total protein ratios. In addition to the expected HBsAg antigen protein, LC-MS/MS analysis of three HBsAg batches identified several yeast proteins, none of which are known to cause adverse reactions in humans.
Analysis of multiple HBsAg batches showed consistent HCP content and identification profiles, as well as product purity and specific antigen content, demonstrating consistent manufacturing process. Recombinant vaccines, unlike therapeutic products, are administered infrequently with only small amounts of protein injected at a time. With limited potential for adverse reactions to small quantities of HCPs in purified recombinant vaccine antigens, and considering the relevant regulatory guidelines, we conclude that once consistent manufacturing process has been demonstrated, routine HCP testing in recombinant vaccine antigens is no longer required.
在宿主细胞系统中表达的重组蛋白可能含有宿主细胞蛋白（HCP）作为杂质。虽然尚无明确证据表明HCP会导致临床不良事件，但必须记录HCP的水平和概况，以满足监管要求，并了解生物制品和生产工艺的一致性。我们提出了一种用于HCP表征的通用策略，该策略应用于一种重组蛋白抗原，即用于多价疫苗的乙型肝炎表面抗原（HBsAg）。
用针对来自HBsAg酵母表达宿主多形汉逊酵母模拟制备物的工艺馏分中的HCP产生的多克隆抗血清，开发一种定量夹心ELISA，以测量多批纯化重组HBsAg中的HCP含量。各批次还进行SDS-PAGE和LC-MS/MS分析，以鉴定可检测到的蛋白质。通过SDS-PAGE/密度测定法纯度分析以及特异性HBsAg含量（通过ELISA）与总蛋白的比率，进一步评估批次一致性。
使用定量HCP ELISA，在5年期间生产的多批HBsAg中，HCP含量没有明显趋势。通过SDS-PAGE/密度测定法，所有批次的纯度均≥95%，HBsAg/总蛋白比率一致。除了预期的HBsAg抗原蛋白外，对三批HBsAg的LC-MS/MS分析鉴定出几种酵母蛋白，其中没有一种已知会在人类中引起不良反应。
对多批HBsAg的分析显示，HCP含量和鉴定概况、产品纯度和特异性抗原含量一致，表明生产工艺一致。重组疫苗与治疗性产品不同，使用频率低，每次仅注射少量蛋白质。鉴于纯化重组疫苗抗原中少量HCP引起不良反应的可能性有限，并考虑到相关监管指南，我们得出结论，一旦证明生产工艺一致，重组疫苗抗原中就不再需要进行常规HCP检测。
