This study was aimed at process characterization and improving quality of purification of erythropoietin α, a biopharmaceutical agent. In biopharmaceutical manufacturing, quality should always be targeted to ensure safety and efficacy. Design‐of‐experiments–based approaches have been explored to rapidly and efficiently achieve an optimized yield and an increased understanding of a product and process variables affecting the product's critical quality attributes in the biopharmaceutical industry; this system is known as the quality‐by‐design approach. Changes in three critical process parameters—buffer pH, flow rate, and loading amount—were evaluated. Process characterization was conducted on a scaled‐down model previously validated by comparison with data from a large‐scale production facility. Seven critical quality attributes—relative aggregate content, host cell protein, host cell deoxynucleotides, endotoxin, Z‐value (N‐glycan score), relative content of charge isomers, and step yield—were analyzed. Multivariate regression analysis was performed to establish statistical prediction models for performance indicators and quality attributes; accordingly, we constructed contour plots and conducted a Monte Carlo simulation to clarify the design space. As a result of the optimization analysis of the purification process, it was confirmed that proven acceptance ranges were optimized as follows: loading amount (mg/mL) 0.4–4.0, buffer pH 7.0–8.0, and flow rate (mL/min) 0.5–1.6.

This study is aimed to identify the process characterization of the cell culture of the biopharmaceutical erythropoietin alpha. In biopharmaceutical manufacturing, ensuring safety and efficacy should always be target qualities. The “quality by design” initiative provides guidance on pharmaceutical development to facilitate design of products and processes that maximizes the product′s efficacy and safety profile while enhancing product manufacturability. Fundamental to this approach is an understanding of the relationship between the quality attributes of the product and their impact on safety and efficacy. The DoE approach focuses on quality‐by‐design (QbD). To implement QbD, we report on a case regarding the production of erythropoietin alpha using cultured Chinese hamster ovary cells. Changes in the four critical process parameters (CPPs) of RPM, pH, dissolved oxygen, and temperature were evaluated. To evaluate the DoE, the three CQAs of Z‐value (N‐glycan score), host cell protein content, and erythropoietin alpha concentration (titer) were monitored and analyzed. Multivariate regression analysis between CPPs and CQAs were used to identify the design space needed to satisfy the targeted CQAs. We used QbD techniques and found optimal conditions for the cell culture process of erythropoietin alpha. Monte Carlo simulation was used under the optimized conditions and the set points were verified. As a result, it was confirmed that the optimal operational range was RPM 191.1–209.0, bioreactor temperature 32–34 °C, pH 7.0–7.2, and dissolved oxygen 24.9–35.1. This scientific understanding facilitates establishment of an expanded design space. In these situations, opportunities exist to develop more flexible regulatory approaches.

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