The absolute quantitation of HCP assays is exceedingly difficult for several reasons. First of all, there is no recognized reference preparation of HCP against which we can calibrate our assays. While ELISA is inherently a quantitative method when applied to a single analyte, ELISAs that attempt to measure simultaneously all of the hundreds of potential HCP impurities in the same well using a single reporter/detector system are at best semi-quantitative assays. Many arbitrary choices and assumptions are used when preparing anti-HCP antibodies and later the choice of material to use when preparing “standards”.

How to obtain the HCP material for immunogen is the first choice that must be made.

• Do we use a null cell line or a mock transfected cell line or one with the actual product plasmid and what differences in HCP might we see from each? 
• At what point in the purification process do we obtain the HCPs for the immunogen or standards?

In almost every case the array and relative concentrations of HCPs in final product are different from the HCP in the ELISA standards.

• How does one assign a total HCP concentration to an indeterminant mixture of many proteins with different molecular weights?
• What effect will the different affinities of antibodies to various HCPs as well as the different concentrations of those antibodies have on the ability of the assay to be quantitative?
• How do we know that we have antibody to all the HCPs present in a given sample type? 

All of these questions and arbitrary choices mean HCP assays will at best be semi-quantitative methods capable of making only relative estimates of HCP concentrations from one sample to the next. We estimate the potential error in reporting an HCP level in a sample that has a different array of HCPs from the standards to be as high as 4-fold even if you have a very good broadly reactive antibody. The commonly expressed theoretical concern is that HCP assays might under-estimate results due to incomplete coverage of all HCPs. In reality, the quantitative errors in HCP assays due to the arbitrary choices and fundamental limitations of the method as discussed above can result in either over-estimation or under-estimation and tend to do so to about the same degree. In our experience, a well generated and affinity purified antibody will react to more than 70% of individual HCPs as demonstrated by traditional 2D Western blot correlated to silver stain. What is more important than the number of individual proteins with antibody reactivity, is the total mass of those proteins that are detected. In our experience and as you might expect from theoretical considerations, the proteins in highest concentration have the best chance of generating an antibody. As such, an antibody with about 70% reactivity to an individual HCP will react with those proteins that account for more than 95% of the total mass of HCP. Given this level of uncertainty in the absolute HCP concentration, the most important criteria by which we can judge any HCP assay is on other objective parameters by which any analytical method should be qualified or validated. Those criteria include specificity and accuracy as demonstrated by sample dilution linearity and spike recovery experiments, precision, robustness, and sensitivity. Provided the assay has sensitivity to detect at least a relative portion of the HCP in your final drug substance and the assay also meets the other objective analytical parameter specifications, such an assay is a valuable analytical method capable of demonstrating process control and reporting relative levels of HCP contamination from lot to lot as a release test.

Due to the impracticality of obtaining real final product HCPs that co-purify with product down to the final step, most of our HCP assays will utilize a source of HCPs from very upstream in the purification process. Those HCPs typically do not come from the actual product cell line but rather a null cell or mock transfected cell line. Once we have decided on the source material for the kit standards we first perform some partial purifications to remove non-HCP materials such as growth media additives, buffer salts and extraction reagents. Our initial approach to calibrate this processed material is to simply perform a BCA total protein assay using bovine serum albumin (BSA) as an arbitrary standard. When standards made with the HCP material calibrated by BCA give reasonable “stoichiometric agreement” with the amounts of antibody used in the ELISA then we feel the calibration by BCA is good enough. What is meant by “stoichiometric agreement” is that we know how much antibody is used in the ELISA. It is the quantity of antibody that actually dictates the analytical range and dose response curve of the ELISA. If we assume an average molecular weight for the total HCPs, then we can reasonably estimate HCP concentrations across the valid analytical range of the assay. When the BCA assay concentrations do not reasonably approximate the ELISA stoichiometry, we process the HCP material further. Such processing involves various purification steps to remove components registering in the BCA assay but that are in fact not HCP or at least not immunoreactive HCPs. This processing might also involve affinity purification against the anti-HCP antibody. The purification stops as soon as the BCA concentration gives a realistic stoichiometric agreement in the ELISA.

HCP 检测的校准
由于几个原因，HCP 测定的绝对定量非常困难。首先，没有公认的 HCP 参考制剂可以用来校准我们的检测。虽然 ELISA 在应用于单个分析物时本质上是一种定量方法，但尝试使用单个报告器/检测器系统同时测量同一孔中的所有数百种潜在 HCP 杂质的 ELISA 充其量是半定量分析。在制备抗 HCP 抗体时使用了许多任意选择和假设，然后在制备“标准”时选择使用的材料。

如何获得免疫原的HCP材料是必须要做的第一选择。

我们是使用无效细胞系还是模拟转染细胞系，还是使用带有实际产品质粒的细胞系？我们可以从中看到 HCP 的哪些差异？
我们在纯化过程中的哪个阶段获得免疫原或标准品的 HCP？
在几乎所有情况下，最终产品中 HCP 的阵列和相对浓度都不同于 ELISA 标准中的 HCP。

如何将总 HCP 浓度分配给许多具有不同分子量的蛋白质的不确定混合物？
抗体对各种 HCP 的不同亲和力以及这些抗体的不同浓度对测定的定量能力有什么影响？
我们如何知道我们对给定样本类型中存在的所有 HCP 都有抗体？
所有这些问题和任意选择意味着 HCP 测定充其量只能是半定量方法，只能对从一个样品到下一个样品的 HCP 浓度进行相对估计。我们估计，即使您拥有非常好的广泛反应性抗体，在 HCP 阵列与标准不同的样本中报告 HCP 水平的潜在错误也会高达 4 倍。普遍表达的理论担忧是，由于对所有 HCP 的覆盖不完整，HCP 检测可能会低估结果。实际上，由于上​​述方法的任意选择和基本限制导致的 HCP 测定中的定量误差可能导致高估或低估，并且倾向于以大致相同的程度这样做。根据我们的经验，与银染色相关的传统 2D 蛋白质印迹证明，良好生成和亲和纯化的抗体将与 70% 以上的单个 HCP 发生反应。比具有抗体反应性的单个蛋白质的数量更重要的是检测到的这些蛋白质的总质量。根据我们的经验以及您可能从理论上的考虑，最高浓度的蛋白质最有可能产生抗体。因此，对单个 HCP 具有约 70% 反应性的抗体将与那些占 HCP 总质量 95% 以上的蛋白质发生反应。鉴于 HCP 绝对浓度的这种不确定性水平，我们判断任何 HCP 测定的最重要标准是其他客观参数，任何分析方法都应通过这些客观参数进行鉴定或验证。这些标准包括由样品稀释线性和加标回收实验证明的特异性和准确性、精密度、稳健性和灵敏度。如果该测定对检测最终原料药中至少一部分 HCP 具有敏感性，并且该测定还符合其他客观分析参数规范，则该测定是一种有价值的分析方法，能够证明过程控制并报告 HCP 的相对水平。批次间 HCP 污染作为放行测试。

由于获得与产品共同纯化直至最后一步的真正最终产品 HCP 是不切实际的，我们的大多数 HCP 测定将利用纯化过程中非常上游的 HCP 来源。这些 HCP 通常不是来自实际的产品细胞系，而是来自空细胞或模拟转染细胞系。一旦我们确定了试剂盒标准的来源材料，我们首先会进行一些部分纯化以去除非 HCP 材料，例如生长培养基添加剂、缓冲盐和提取试剂。我们校准这种加工材料的初始方法是使用牛血清白蛋白 (BSA) 作为任意标准简单地执行 BCA 总蛋白测定。当使用 BCA 校准的 HCP 材料制成的标准与 ELISA 中使用的抗体量给出合理的“化学计量一致性”时，我们认为 BCA 的校准已经足够好。 “化学计量一致性”的意思是我们知道在 ELISA 中使用了多少抗体。抗体的数量实际上决定了 ELISA 的分析范围和剂量反应曲线。如果我们假设总 HCP 的平均分子量，那么我们可以合理地估计有效分析范围内的 HCP 浓度的化验。 当 BCA 测定浓度不能合理地接近 ELISA 化学计量时，我们会进一步处理 HCP 材料。 这种处理涉及各种纯化步骤，以去除在 BCA 测定中登记但实际上不是 HCP 或至少不是免疫反应性 HCP 的成分。 该处理还可能涉及针对抗 HCP 抗体的亲和纯化。 一旦 BCA 浓度在 ELISA 中给出真实的化学计量一致，纯化就会停止。

What is the Storage and Stability of ELISA Kits?