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.