Immunogenicity and safety evaluation of a newly manufactured recombinant Baculovirus-Expressed quadrivalent influenza vaccine in adults 18 years old and Above: An Open-Label, phase III extension study.

In the face of global health threats, there is a growing demand for vaccines that can be manufactured on a large scale within compressed timeline. This study responds to this imperative by delving into the evaluation of FluGuard, a novel recombinant influenza vaccine developed by Nivad Pharmed Salamat Company in Iran. Positioned as a phase 3 extension, the research aimed to evaluate the safety and immunogenicity of FluGuard in volunteers aged 18 and above. The study was conducted as a single-center, open-label clinical trial. All eligible volunteers received FluGuard (2021-2022 Formula) on day 0. Safety assessments occurred at days 1, 4, 7, 14, 28 and 42 post-vaccination. Immunogenicity was measured through seroconversion, seroprotection, and geometric mean titer fold increase in subgroups of 250 volunteers. Among the 4,260 volunteers were screened and assessed for eligibility, 1000 were enrolled. At day 28 post-vaccination, seroconversion rates for A/H1N1, A/H3N2, B/Yamagata, B/Victoria were 53.4 % [95 %CI: 46.7-60], 57.7 % [95 %CI: 51.1-64.3], 54.3 % [95 %CI: 47.7-60.9], and 36.2 % [95 %CI: 29.8-42.6], respectively in volunteers 18 years and above. The most common solicited adverse events were pain at the injection site, malaise, and headache. No suspected unexpected adverse events and adverse events of special interest occurred during the study period. Our findings suggested that FluGuard® exhibits a desirable safety profile and provides sufficient immunogenicity against influenza virus types A and B. However, extended studies are warranted to assess the long-term protective efficacy. Trial Registration: The study protocol was accepted by Iranian registry of clinical trial; https://www.irct.ir; IRCT20201104049265N2.

QbD-guided pharmaceutical development of Pembrolizumab biosimilar candidate PSG-024 propelled to industry meeting primary requirements of comparability to Keytruda®.

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.