S1), we hypothesize that in homogenate, re-engagement of DsbC with the CH2 domain of mAb1 could be facilitated by recognition of the Ig fold structure and chaperone-substrate like interactions due to lack of Fc glycans. homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction and free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc CH2 region. Our results also imply that lack of glycans in produced antibodies may facilitate DsbC accessibility to the disulfide bond in the Fc CH2 region, resulting in its reduction. Conclusions During antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond Porcn-IN-1 reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction and free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in antibody manufacturing processes. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-022-01892-4. Keywords: Antibody, Disulfide bond reduction, Free thiols, Fc glycosylation, Disulfide bond isomerase C (DsbC), Homogenate hold Background Therapeutic antibodies are predominantly produced in mammalian cells, most commonly in Chinese Hamster Ovary (CHO) cells. Another production host, albeit not as common as Porcn-IN-1 CHO, is (lacks the enzymatic machinery to support post-translational modifications such as glycosylation. Therefore, antibodies produced in lack Fc effector functions, which could be advantageous when the intended therapeutic indication requires only an Porcn-IN-1 antibodys blocking function. Depending on the intended molecules design (e.g., antibody fragments), use of as an expression system may offer some advantages over CHO cells. processes also have faster development timelines and lower production costs. In this work, an host with periplasmic expression was used for manufacturing-scale production of the unique recombinant one-armed monovalent mAb1, which has an ability to block the target receptor without inducing its dimerization and activation. In comparison, the standard format, i.e., a bivalent antibody with the same specificity activates the target receptor leading to the opposite effect than intended. mAb1 is a humanized, monovalent, monoclonal antibody based on the IgG1 framework. It is composed of a full-length heavy chain, a light chain, and a truncated heavy chain that consists of only the hinge region and the CH2 and CH3 domains (Fig.?1). mAb1 is aglycosylated (since its produced in thiol-disulfide oxidoreductases DsbA [3] and DsbC [4], which are periplasmic enzymes responsible for disulfide bond formation and isomerization, respectively. Low levels of free thiols (unpaired cysteine residues) have been observed in naturally occurring IgG antibodies [5, 6] and free thiols are typically present in monoclonal antibodies produced in CHO cells [7, 8]. In antibodies produced in cells producing mAb1. Lack of the Fc glycosylation of mAb1 is an apparent cause of its susceptibility to free thiol formation in the Fc region. Lastly, we suggest Porcn-IN-1 a potential mechanistic explanation for the reduction of disulfide bonds in the Fc CH2 domain by DsbC. Results Variability of the total free thiol content during the development and manufacturing-scale GNAS production of mAb1 During technical development of mAb1 production process, the total free thiol content (exposed and buried free thiols) was routinely determined using an in-house developed reversed-phase high performance liquid chromatography (RP-HPLC) assay, with the Porcn-IN-1 free thiol derivatization under denaturing conditions and UV detection, capable of separating and quantitating free thiol-containing mAb1 variants [9]. Using this assay, a high variability of the total free thiol content.