Division of Product Quality Research, OTR/OPQ, CDER/FDA, Silver Spring, MD, USA.
Department of Chemical Engineering, University of Massachusetts Lowell, Lowell, MA, USA.
Appl Microbiol Biotechnol. 2024 Mar 26;108(1):274. doi: 10.1007/s00253-024-13110-9.
The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products' efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs). KEY POINTS: • Molecular integrity may suffer with increasing process intensity. • Galactosylated and sialylated N-glycans may decrease. • Perfusion culture appears to maintain protein charge structure.
制药行业采用了各种策略来提高细胞生产率。这些策略包括工艺强化、培养基改良、克隆选择、培养基补充和细胞遗传工程。然而，提高细胞生产率会对产品质量属性（PQA）产生固有风险。PQA 可能会通过稳定性、生物利用度或体内生物活性影响产品的疗效。制造工艺的变化可能会通过改变 N-糖基化的类型和程度来改变产品的异质性，而 N-糖基化是治疗性蛋白质的 PQA。我们研究了不同细胞密度（代表灌流细胞培养中的工艺强化程度增加）对 CHO-K1 细胞系产生 IgG1-κ 单克隆抗体的影响。该抗体在轻链和重链上均发生糖基化。我们的结果表明，IgG1-κ mAb 的整个蛋白质的糖基化含量增加，G0F 和岩藻糖基化聚糖作为一组增加，而唾液酸化聚糖减少。总的来说，在所有工艺强化水平下，G0F、G1F、G0、G2FS1 和 G2FS2 型聚糖的含量都有显著差异。G2FS2 和 G2 型 N-糖基化主要从轻链而不是重链中定量。可以得出结论，在通过灌流细胞培养进行工艺强化过程中，治疗性蛋白质的产品质量属性存在潜在影响，需要进行评估。由于在灌流细胞培养过程中，产品在整个过程中被收集，因此需要仔细关注批分配，因为 PQA 会受到关键工艺参数（CPP）的影响。要点：
分子完整性可能会随着工艺强度的增加而受到影响。
半乳糖基化和唾液酸化 N-聚糖可能会减少。
灌流培养似乎能保持蛋白质的电荷结构。
