Fig 1.
Overview of labelling techniques used in this study.
Amino acid residues schematically represent the majority of the amino acids on which labelling or modification takes place. Reactive group describes the part of the label that conjugates with the corresponding amino acid side chain. The reaction product shows the formed chemical structural formula after the covalent linkage of the label to the protein.
Fig 2.
Chemical structures of labels used in this study. Label Code describes the abbreviations of each label mentioned in this study. Column “Amino acid” shows the corresponding amino acid involved in the labelling process. Alphanumeric links to the modified mAbs are given in the two right-hand columns: mAbA1-4: wildtype mAb; mAbB1-7: research concept mAb.
Table 1.
Chromatographic method used for FcRn affinity chromatography.
Table 2.
Chromatographic method used for heparin affinity chromatography.
Fig 3.
Representative example of a chromatographic separation for subsequent mass spectrometric analysis.
LC: light chain; HC: heavy chain; PNGase F: peptide N-glycosidase F.
Fig 4.
Labelling distribution of mAbA1-4 and mAbB1-7.
(A): light chain; (B): heavy chain; (C) intact protein, according to Poisson distribution; (D): calculated degree of labelling (DoL). DoL1: based on labelling distribution determined by mass spectrometry analysis; DoL2: based on spectroscopic calculation for fluorescence labelled mAbs (Supplementary Information: S2 Formula). Label Code: NSP: N-succinimidyl propionate; AFM: Alexa Fluor 488-maleimide; SCN: Tm-p-SCN-Bz-DOTA; DI: direct iodination; AFN: Alexa Fluor 488-NHS; BHN: Bolton-Hunter-NHS; DN: DOTA-NHS.
Table 3.
Retention times in size-exclusion chromatography.
Fig 5.
SEC of mAbB2 (Alexa Fluor 488-maleimide), denatured with 0.1% formic acid.
Blue line: unlabelled mAbB showed a single peak. Red line: absorbance of modified mAb6 at 280 nm. Green line (494 nm) showed several peaks that indicate (labelled) protein fragments.
Table 4.
Retention times in FcRn and heparin affinity chromatography.
Fig 6.
Negative control of mAbB3 (Tm-p-SCN-Bz-DOTA) using sepharose column without FcRn (blue). The red line shows mAbB3 measured with FcRn affinity chromatography.
Fig 7.
Correlation of FcRn and heparin column retention of labelled antibodies.
Green triangle: mAbA series, blue dot: mAbB series. Left: full graphic, right represents the framed area. Dotted lines indicate a relative retention of 1.00. Label Code: NSP: N-succinimidyl propionate; AFM: Alexa Fluor 488-maleimide; SCN: Tm-p-SCN-Bz-DOTA; DI: direct iodination; AFN: Alexa Fluor 488-NHS; BHN: Bolton-Hunter-NHS; DN: DOTA-NHS.
Table 5.
Calculated relative retention times.