Fig 1.
Mispairing characterization in distinct tsAb producer clones.
Schematic representation of the experimental workflow used to evaluate mispairing profiles in CHO clones producing trispecific antibodies. Clones were cultured in Ambr15 micro bioreactors under fed-batch conditions. The conditioned medium collected on day 12 was clarified and filtered, followed by Protein A purification. The resulting eluates were subjected to bioanalytical characterization using biophysical assays (nDSF and SPR) and further analyzed by SEC-MS and HIC-HPLC.
Fig 2.
Total Ion Chromatogram (TIC) and respective reconstructed mass of the different peaks.
(A) Protein A purified samples a) complete mAb species and (B) clarified samples a) complete mAb species, b) Light Chain 1 (L1) and c) Light chain 2 (L2).
Fig 3.
Correct and mispaired tsAb antibody species produced by CHO producers.
H1L1/H2L2 – correct tsAb form (highlighted in green); H1L1/H2L1, H1L2/H2L2, H2L2/H2L2 – mispaired complete species; H2L2, H2L1, H1L1, H1L2 – half mispaired species. H = Heavy/L = Light chains. *Species not detected in SEC-MS analysis.
Fig 4.
SEC-MS peak areas of Protein A purified and Clarified samples.
Peak areas of (A, B, C) protein A purified samples and (D, E, F) clarified samples of all clones. Panels A and D depict the areas of free LCs detected (LC1 and LC2), panels B and E the half mAbs (H1L1 and H2L2) and panels C and F the complete mAbs (H1L1/H2L2 - correct form, H2L2/H2L2, H1L2/H2L2 and H1L1/H2L1). *No data available for clarified sample of clone H, as no MS signal was obtained.
Fig 5.
Type and distribution of tsAb complete species from distinct samples (A, B, C, D, F, G, H, I and J) analyzed by SEC-MS.
Percentage of complete mAb species calculated for (A) Protein A purified and (B) clarified samples. H1L1/H2L2 corresponds to correct form (blue bars) and remaining mispaired species are depicted in other colors. Threshold line at 80% of complete mAb species. *No data available for clarified sample of clone H, as no MS signal was obtained.
Fig 6.
HIC-HPLC chromatograms of Clarified and Protein A purified samples for both mispairing profiles.
Chromatogram overlay of (A, B) protein A purified samples and (C, D) clarified samples of clones for all clones. Chromatogram overlay of clarified and protein A purified samples for Clone A (E) and clone F (F), as examples.
Fig 7.
Characterization of tsAb thermal stability.
nDSF profiles were obtained from four different clones within two different mispairing groups. (A) clone A and (B) clone D corresponding to lower mispairing and clone F (C) and clone H (D) to higher mispairing clone groups. (E) depicts the thermal stability profile of IgG1 negative control. The melting curves of each sample correspond to 350/330 nm first derivative and the curve shading reflects the standard deviation of the 3 replicates performed. (F) Average Tm values detected and number of melting peak transitions associated for all the clones evaluated, including human IgG1, used as negative control. ND – not determined.
Fig 8.
SPR kinetic characterization of the binary interaction between tsAb and each of the different antigens (CD28, CD3ε/CD3δ, CD38).
Two different SPR sensorgram profiles were obtained for tsAb samples for all the clones from the different mispairing level groups. The figure depicts samples tested against: (A), (B) and (C) CD28, (D), (E) and (F) CD3ɛ/CD3δ, and (G), (H) and (I) CD38, each with ten different dilutions: up to 1 nM for CD28, 10 nM for CD3ɛ/CD3δ, and 100 nM for CD38. Two antibody clones are shown as examples: clone A, representing a lower mispairing clone and clone F representing a higher mispairing clone. (C), (F) and (I) panels display the binding profile of igG1 negative control. (J) Binding kinetic parameters from CD28, CD3ε/CD3δ and CD38 binary interaction analysis. For CD28 antigen the dissociation rate was too slow to calculate the interaction affinity KD.
Fig 9.
Summary of bioanalytical tools used for mispairing screening and characterization.
ProtA – protein A purified samples; Clarif – Clarified samples. Color gradient corresponds to peak areas obtained in SEC-MS analysis: red gradient – LC’s, half-mabs and full mAbs mispaired species; green gradient – full mAb correct form. * No data available for clarified sample of clone H, as no MS signal was obtained.