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Pharmaceutical development of biosimilars is primarily focused on meeting the regulatory requirements for analytical comparability of the product's critical quality attributes (CQAs), concerning safety and efficacy, to those of the originator drug of interest. To this end, the early adoption of a systematic science-based approach, as guided by quality-by-design (QbD) principles, is crucial due to the blind starting point where the same insights of an originator developer into the challenges of a given biopharmaceutical and its manufacturing process are lacking. In this study, we devised a pharmaceutical QbD-guided approach to undertake the biosimilar development of Pembrolizumab (Keytruda®), the ace of therapeutic monoclonal antibodies (mAbs) in terms of approved indications and market sales, and its manufacturing process development. Quality target product profile (QTPP) for Pembrolizumab biosimilar product was assembled using publicly available information on Keytruda®. Upon preliminary analyses of four different lots of Keytruda®, the product CQAs and their acceptable ranges of specification were determined via risk assessment based on the relevant pharmaceutical development quality guidelines, particularly those of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). The development and clone selection of Chinese Hamster Ovary (CHO) DG44 cell line was performed using DHFR expression vectors and Methotrexate (MTX) selective pressure. The CHO clone stably expressing relatively higher mAb titer (∼1200 mg/l) in small-scale shake-flask cultures, with the highest similarity of the CQAs charge variants contents (CVCs), N-glycan profile, and biological potency to those of Keytruda® reference standard was selected as the lead clone and the produced Pembrolizumab candidate was named PSG-024. The upstream process (USP) and downstream process (DSP) developments for production were started with the process evaluation screening experiments for the identification of critical process parameters (CPPs) founded upon the prior knowledge on different process stages, input process parameters (iPPs), output process parameters (oPPs), and their impacts on product CQAs. Thereby, screening experiments of USP fed-batch cell culture in 5-liter bioreactor resulted in improvement of PSG-024 expression titer to 2060 ± 70 mg/l and selection of the iPPs feed amount (A), glucose setpoint (B), culture temperature (C), and agitation rate (D) for the optimization design of experiments (DoEs) mainly focused on the CQA acidic CVC and the oPPs mAb expression yield. The USP optimization DoEs using response surface methodology (RSM) yielded valid prediction models and optimal conditions of A = 35%, B = 4.5 g/l, C = 37 °C, and D = 160–220 rpm, which resulted in the final PSG-024 expression titer of 3170 ± 40 mg/l without an excessive rise in acidic CVC. The DSP screening experiments led to achieving the mAb recovery rates of 94% ± 3% and 71.5% ± 3.5% for affinity (capture) and cation-exchange (polishing) chromatography stages, respectively. The capture eluate buffer and viral inactivation conditions were optimized to prevent mAb eluate turbidity and protein aggregation. Moreover, the polishing stage optimization DoEs via one-factor-at-a-time method focused on wash and elution steps for control of the acidic CVC CQA and achieving >80% mAb recovery rate. By shifting to Step elution from the primary salt gradient method and considering an additional intermediate wash step, the maximum mAb recovery of 87% ± 1.5% was achievable while maintaining the CQA acidic CVC within the acceptable range. The consistency of final analytical comparability of PSG-024 demonstrated the effectiveness of the adopted pharmaceutical QbD approach for Pembrolizumab biosimilar development, paving the way for the technology transfer to the client to proceed further development. [Display omitted] [ABSTRACT FROM AUTHOR]