Getting Ahead of Industry Expectations for Biological Therapeutics 测试
By Engaged Expert
Khanh Ngo Courtney, Ph.D.Dr. Courtney’s experience extends from R&D to analytical method development, 验证, implementation, method transfer, and optimization of test methods for the cGMP setting per USP and ICH guidelines. 她是im体育APP Ann Arbor(我们生命科学业务的分析实验室)的总经理. She is a CMC professional in the Biological therapeutic space, and specializes in applying analytical strategies to meet regulatory intent.
Emerging trends in biologics testing may come from increasing Agency expectations, advancement in analytical technologies, or the clients' changing business needs in the current economic landscape. 我们在im体育APP的优势之一是我们的敏捷性和转向能力，并开发满足im体育平台app下载需求的新策略. Throughout this article, 您将深入了解导致im体育平台app下载标准变化的三种不同压力，这些压力最终会影响生物疗法的安全性测试.
机构期望:蛋白质组学用于宿主细胞蛋白分析和免疫原性预测
ELISA(酶联免疫吸附测定法)是测定原料药或制剂中残留宿主细胞蛋白(HCPs)含量的传统方法. In recent years, 对基因和细胞治疗安全性的高度认识和警惕导致了抗体是否足以覆盖所有可用的宿主细胞蛋白的问题. 任何免疫测定的一个主要限制是抗体对其靶蛋白的亲和力的差异. If the affinity is low, 然后目标蛋白必须大量存在才能进行检测. If affinity is high, 然后只需要少量的目标蛋白就能得到阳性结果. An ELISA is essentially the analysis of 10s of thousands of proteins, all with their own independent affinity to different antibodies, 然而，该方法只能给出一个平均信号强度，读取一个96 dash井板. If there isn't enough protein present, or if the affinity is too low, the protein could go undetected, even if it is harmful to the safety of the patient.
与此形成鲜明对比的是, high resolution, 蛋白质组学的高灵敏度自底向上质谱法已成为检测原料药或制剂中宿主细胞蛋白质的更合适的方法, because LC-MS/MS does not have the same affinity biases1 as does ELISA. 最近, FDA接受了一份IND提交，该提交仅提供了显示HCPs的蛋白质组学数据，而没有ELISA方法. 使用蛋白质组学的想法不一定是显示HCPs的绝对清除-因为考虑到LC-MS/MS的敏感性，很难显示HCPs的真正缺失, but to use the data to perform a risk-based assessment of the safety of the drug. 具体来说，蛋白质组学可以用来确定是否存在假定的免疫原性蛋白. 我们距离能够将这项技术用于常规QC批放行测试还有很长的路要走, 而是在工艺开发和工艺性能验证期间用于补充elisa的强大工具.
1NOTE: Bottom-up LC-MS/MS also has its own limitations, 但通常在实验室环境中比抗体靶蛋白的丰度和亲和力更容易控制.
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技术进展:PCR检测支原体和残留宿主细胞DNA
支原体 is a large group of bacteria, consisting of over 120 species, 而且很难检测，因为它们不能被克氏染色，而且非常小. 如果支原体存在于生物原料药或产品中，可能会传播并使患者暴露于多种严重的健康状况. USP <63>, EP Chapter 2.6.7 and 21 CFR 610.30 have been the standards for testing mycoplasma, but all three require up to 28 days of cell culturing, require large batch sizes and volume, and would only be suitable to test for a maximum of 6 species. Since the advancement of PCR, up to 90 species of 支原体 in a sample could be detected quickly using RT-PCR2, 或多达113种绝对定量的液滴数字PCR (ddPCR)在数小时内样品接收3. This quick turnaround is much more suitable for biological drugs, such as cell therapies or personalized medicines, that might have a short shelf life, and need to be transplanted in a patient within days, or even hours, of being manufactured.
ddPCR技术的进步也改变了业界分析宿主细胞残余DNA的方式, 再一次。, a safety component of a biological therapeutic. 定量PCR (qPCR)是定量宿主细胞DNA的传统方法. 然而, qPCR是一种相对定量的方法，它依赖于合成的寡核苷酸标准作为标准曲线，来自样品的DNA将根据标准曲线进行定量. qPCR is also high variability and often required up to 7-10 replicates per run. Quantitation by ddPCR, on the other hand, is an absolute quantitative method based on the droplet generating technology, and takes away the variability of the test. 它不需要标准曲线，因为定量是荧光标记探针阳性液滴的绝对定量. The advancement of ddPCR has significantly reduced supply requirements, human hands-on time, 以及获得证明药物对患者是否安全的结果所需的时间.
2 MycoSEQTM 支原体 Real-Time PCR Detection Kit from Thermo Fisher
3 Vericheck ddPCR 支原体 Detection Kit from BioRad
Business needs and trends: upstream process raw materials routine testing
Raw materials testing in the biologic space is not trivial. 对于上游工艺中使用的大多数常见原材料，如细胞培养基，没有标准的USP专论, media supplements, and more ancillary reagents. 如果公司想要开始与CDMO或测试实验室验证原材料批次，以准备提交BLA, 他们经常面临着高额的前期成本和为上述原材料定制方法开发所需的长时间, followed by method 验证, before they are able to analyze the 1-2 samples per year. As we see an increase in small, 创新, advanced therapeutic and/or personalized medicine companies begin to gain momentum, obtain results and the financial support to fast-track to the BLA, 当他们告诉我们仅仅分析一批原材料就需要多少成本和多长时间时，我们也看到了他们脸上的震惊.
This major bottle neck in getting safe, efficacious drugs to patients is one that needs to be dealt with immediately. One of my favorite parts of my job is talking to our clients about their patients, the mode of action of their therapeutic, and their current business demands. As a service provider, 作为客户的合作伙伴，我们承担起减轻他们负担的责任，这样他们就能专注于安全, efficacious drugs to patients who need them. im体育APP已经接受了挑战，让更多的公司更容易获得非标准原材料测试，这些公司致力于将他们的创新治疗方法提供给患者. Connect with us to learn more about this.
来自监管机构的压力和先进生物制剂疗法批准的增加创造了一个鼓励和促进先进分析技术进步和改进的环境. 此外, 2024年的经济环境给制药公司带来了更大的压力，迫使他们迅速将药物送到床头柜上, fiscally efficiently, all while ensuring safety and efficacy. From a testing lab point of view, 这种压力为探索和实施创新解决方案提供了理由，以满足客户和im体育平台app下载的需求.