Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej Understanding unfolding and refolding of the antibody fragment (Fab). I. In- vitro study Kayanat Gani a,b , Rahul Bhambure a,b, *, Prashant Deulgaonkar a,b , Deepa Mehta a,b , Manoj Kamble a a Chemical Engineering and Process Development Division, CSIR - National Chemical Laboratory, Pune, India b Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India HIGHLIGHTS • Fab refolding follows a three-state folding mechanism. • No signifcant variation in free energy change for Fab unfolding at 15-30 °C. • The intermediate formation is an overall rate-limiting step in Fab refolding. • Inter-domain disulfde bond formation critically depends upon temperature. • Designed a kinetic reaction model for scalable Fab refolding. ARTICLE INFO Keywords: In-vitro refolding Antibody fragment Two-state and Three-state models rHu Ranibizumab Refolding kinetics ABSTRACT In-vitro protein refolding is a major rate-limiting step in the large scale production of antibody fragments ex- pressed using a microbial source like E. coli. This investigation is focused on understanding the in-vitro unfolding and refolding of the multi-domain protein involving inter-domain disulfde linkage, like antibody fragment (Fab). Solubilization behavior of the inclusion bodies and unfolding events of Fab fragment (Biosimilar rHu Ranibizumab) were studied using nano-diferential scanning fuorimetry (nano-DSF). Fab unfolding behavior was studied by ftting experimental data with the two-state and three-state thermodynamic model. Based on the Fab unfolding understanding, a two-stage design of experiment (DoE) strategy was used for the optimization of the in-vitro refolding condition of a Fab fragment. Refolding yield of 56.03 ± 1.15 % was achieved using the optimized oxidative refolding conditions maintained by appropriate dilution factor and redox reagent ratio. Refolding kinetics of the rHu Ranibizumab was analyzed using a three-parameter kinetic model showing rate constant k 1 :7.05e -6 l/mg.min, k 2 :0.57 l/mg.min, and k 3 :310.19 l/mg.min. Based on observed refolding kinetics, it was concluded that the Fab refolding follows a three-state mechanism with the refolding intermediate/(s) formation from light and heavy chain of the Fab fragment as an overall rate-limiting step. The method described here is a useful tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter- domain disulfde bonds. 1. Introduction Monoclonal antibodies (mAbs) and antibody fragments (Fabs and scFv) have gained enormous importance in the biopharmaceutical in- dustry. mAbs and Fabs are the key therapeutic molecules used in the treatment of various immune-oncology diseases. So far, around 450 monoclonal antibodies have entered the clinical study, 54 of which were antibody fragments. Among these, three have been approved in the United States (rHu Abciximab, rHu Ranibizumab, and rHu Certolizumab pegol), and one has been approved in China, whereas, 19 (35%) are currently in the clinical study and 31 (57%) have been dis- continued [1,2]. Antibody fragment (Fab) is the multi-domain protein https://doi.org/10.1016/j.bej.2020.107764 Received 6 May 2020; Received in revised form 27 July 2020; Accepted 17 August 2020 Abbreviations: rHu, Recombinant humanized; IBs, Inclusion bodies; Fab, Antibody fragment; scFv, Single-chain variable fragment; mAbs, Monoclonal antibodies; IgG1, Immunoglobulin G1; GuHCl, Guanidine hydrochloride; EDTA, Ethylenediaminetetraacetic acid; OD, Optical density; IPTG, Isopropyl β-D-1-thiogalactopyr- anoside; SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis; RP-HPLC, Reverse-phase – High-performance Liquid Chromatography; CD, Circular dichroism ⁎ Corresponding author: Chemical Engineering and Process Development Division, CSIR - National Chemical Laboratory, Dr. Homi Bhabha Road, Pashan, Pune, 411008, India. E-mail address: rs.bhambure@ncl.res.in (R. Bhambure). Biochemical Engineering Journal 164 (2020) 107764 Available online 20 August 2020 1369-703X/ © 2020 Elsevier B.V. All rights reserved. T