1. Protein purity and integrity after protein A affinity and SEC
Protein purity and integrity after protein A affinity and SEC. A, SDS gel electrophoreses under nonreducing (left) and reducing (right) conditions with single pMHCI-monovalent IgG and single pMHCI-bivalent IgG after protein A affinity chromatography and SEC...
Protein purity and integrity after protein A affinity and SEC. A, SDS gel electrophoreses under nonreducing (left) and reducing (right) conditions with single pMHCI-monovalent IgG and single pMHCI-bivalent IgG after protein A affinity chromatography and SEC. Nonreducing gel shows the expected size of the 145 kDa single pMHCI-monovalent IgG and 193 kDa of the single pMHCI-bivalent IgG. Reducing gel shows the pMHCI-IgG HC fusion (95 kDa) and the antibody light chain (25 kDa) for both constructs. In addition, the single pMHCI-monovalent IgG consists of a truncated antibody heavy chain (26 kDa) lacking the variable domain and the single pMHCI-bivalent IgG consists of the antibody heavy chain (50 kDa). B, analytic SEC of the single pMHCI-monovalent (left) and bivalent IgG (right) after protein A purification. Percentages of aggregates and monomeric product are indicated in the schemes. C, mass spectroscopy (ESI-MS) of single pMHCI-monovalent IgG (left) and single pMHCI-bivalent IgG (right). Main peak under deglycosylated, nonreducing conditions is the expected product consisting of the antibody light chain, the truncated antibody IgG knob and the pMHCI-fused antibody IgG hole heavy chain (left) and two antibody light chains, and the antibody IgG knob and the pMHCI-fused antibody IgG hole heavy chain (right). Minor peaks are PNGaseF, which was used for deglycosylation and adducts of phophate/sulfate, or one or two hexoses. D, mass spectroscopy (ESI-MS) of single pMHCI-monovalent IgG (left) and single pMHCI-bivalent IgG (right) under deglycosylated and reducing conditions. All peptide chains have the expected mass. Antibody heavy chains are lacking the C-terminal lysine. Other modifications are indicated.
Martina Schmittnaegel et al. Mol Cancer Ther 2016;15:
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