Bierley S T, Raineri R, Poiley J A, Morgan E M
IEM Section, Pathology Associates International, Durham NC, USA.
Dev Biol Stand. 1996;88:163-5.
The presence of retroviral contamination is of vital concern in the manufacture of cell culture-derived biopharmaceuticals. These cell lines usually have A- or C-type retrovirus-like particles which are visible by transmission electron microscopy (TEM) even when infectivity (IF) or reverse transcriptase activity (RTA) cannot be demonstrated. The supernatant of the post-production cell cultures, therefore, also needs to be evaluated by TEM for viral burden. A major question, however, is how to establish a quantitative viral load estimate for the evaluation of a purification process. The FDA recommends that a purification process for viral contaminants remove or inactivate 3-5 logs over the estimated viral burden. Viral particles are difficult to identify and quantify, however, by conventional negative staining. We present a comparison of infectivity assay, reverse transcriptase assay, negative staining, and thin sectioned TEM. These assays were performed on four samples. Ultracentrifuged sediments of cleared cell-culture media were measured, fixed and processed. Thin sections were evaluated by TEM and the number of viral particles estimated by morphometric derived quantification. Retrovirus particles were easily identified and quantified when examined by TEM as compared to negative staining and correlated with the other viral assays (IF, RTA). These results demonstrate that the TEM thin section method was a superior technique to negative staining for estimating viral particle load in cell-culture supernatant. To validate further the plastic embedding with thin sectioning, we evaluated cell culture supernatants (pellets) for retroviral burden at various dilutions, from two cell lines. Morphometric determinations were made of the number of viral particles present per unit volume and compared to results obtained by infectivity assay. Since the morphometric calculation for viral density assumes even distribution of viral particles, we also evaluated and calculated viral counts on multiple thin sections taken throughout selected pellets.
逆转录病毒污染的存在是细胞培养衍生生物制药生产中至关重要的问题。这些细胞系通常含有A或C型逆转录病毒样颗粒，即使无法证明其感染性（IF）或逆转录酶活性（RTA），通过透射电子显微镜（TEM）也可见。因此，生产后细胞培养物的上清液也需要通过TEM评估病毒载量。然而，一个主要问题是如何建立定量病毒载量估计值以评估纯化过程。美国食品药品监督管理局（FDA）建议，针对病毒污染物的纯化过程应在估计的病毒载量基础上去除或灭活3至5个对数级。然而，通过传统的负染色很难识别和定量病毒颗粒。我们对感染性测定、逆转录酶测定、负染色和超薄切片TEM进行了比较。这些测定在四个样品上进行。对澄清的细胞培养基的超速离心沉淀物进行测量、固定和处理。通过TEM评估超薄切片，并通过形态计量学衍生的定量方法估计病毒颗粒数量。与负染色相比，通过TEM检查时逆转录病毒颗粒很容易被识别和定量，并且与其他病毒测定（IF、RTA）相关。这些结果表明，对于估计细胞培养上清液中的病毒颗粒载量，TEM超薄切片法是优于负染色的技术。为了进一步验证塑料包埋和超薄切片，我们评估了来自两个细胞系的不同稀释度的细胞培养上清液（沉淀物）的逆转录病毒载量。对每单位体积中存在的病毒颗粒数量进行形态计量学测定，并与通过感染性测定获得的结果进行比较。由于病毒密度的形态计量学计算假设病毒颗粒均匀分布，我们还评估并计算了在选定沉淀物中多个超薄切片上的病毒计数。
