Fig. 1

Generation and characterization of PSB205. A The principle of MabPair technology for producing two correctly assembled antibodies from a single mammalian cell line. Top panel, co-expression of two different antibodies in a single production cell line requires the simultaneous introduction of DNAs encoding two heavy chains (HCs) and two light chains (LCs) into the same cell. Under normal conditions, the two HCs can randomly dimerize to form two separate homodimers and one heterodimer species; the two LCs can also pair with either of the two HCs. The random combinations result in a total of 10 possible products generated, but only two of them are the desirable antibody products that contain the cognate HC/HC and LC/HC pairings (yellow-circled ones). Middle panel, using a charge-pair approach (referred as “HC pairing keys”) to correctly control the homodimeric pairing of the HCs of two different antibodies, four undesirable side products containing heterodimeric HCs are eliminated, so 10 combinations are reduced to 6. Bottom panel, applying a combined charge-pair and cysteine-pair approach (referred as “HC/LC pairing keys”) to control the cognate LC/HC pairings, only the two correctly paired and structurally stable products can pass the endogenous quality control system inside cells before they are secreted out. Other byproducts are fully eliminated due to their instability. B Fluorescence-assisted cell sorting plots showing the coexpression of PSB103 and PSB105 in the production cell line after intracellular staining of conjugated anti-hu IgG4- and anti-hu IgG1-specific antibodies, respectively. A panel of orthogonal analytical methods was used to characterize the PSB205 MabPair. The results confirmed the molecular integrity, and no mispaired antibodies were detected in the sequential characterization. C PSB205 size variants were analyzed by size-exclusion high-performance liquid chromatography (HPLC). The chromatogram shows the main peak for the monomers of the two mAbs overlaid, frontal minor peak(s) for high-molecular weight species, and post minor peak(s) for low-molecular weight species (not detected) in PSB205. As a result, the PSB205 purity as defined by the monomers (the main peak) was typically measured as 97–99% for different batches. D Baseline separation of the two mAbs in PSB205 was achieved by the hydrophobic interaction HPLC method. Thus, it served as a tool to determine the concentration ratio of the two mAbs, [anti-PD-1]:[anti-CTLA-4] (w/w). E The intact glycoform mass profile was obtained by liquid chromatography–mass spectrometry (LC–MS) analysis. As a result, the two main peaks at 149,320 Da and 147,610 Da in the deconvoluted mass spectra closely match the G0F/G0F glycoforms of anti-PD-1 and anti-CTLA-4, respectively, with their HC N-terminal Gln converted to pyroglutamic acid and the C-terminal Lys removed