Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

Abstract

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS(E)), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS(E) peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.

Figure 1

A comparison of deconvoluted masses (processed by BiopharmaLynx 1.2 using MaxEent1) between innovator and biosimilar mAbs. (A) intact mAbs; (B) mirror plot of heavy chain. (C) mirror plot of light chain.

Figure 1

A comparison of deconvoluted masses (processed by BiopharmaLynx 1.2 using MaxEent1) between innovator and biosimilar mAbs. (A) intact mAbs; (B) mirror plot of heavy chain. (C) mirror plot of light chain.

Figure 2

Mirror plots of LC-MS peptide maps from 4-hours tryptic digests of the innovator and biosimilar mAbs. (A) LC-MS (TIC) chromatograms; (B) a zoom view of charge-reduced, isotope-deconvoluted LC-MS chromatograms from 32.0–35.0 min. (C) a zoom view of charge-reduced, isotope-deconvoluted LC-MS chromatograms from 3.5–7.0 min. The zoom chromatograms were processed by BiopharmaLynx 1.2 and the peaks were annotated based on accurate mass MH⁺ of peptides in an automated mode.

Figure 2

Mirror plots of LC-MS peptide maps from 4-hours tryptic digests of the innovator and biosimilar mAbs. (A) LC-MS (TIC) chromatograms; (B) a zoom view of charge-reduced, isotope-deconvoluted LC-MS chromatograms from 32.0–35.0 min. (C) a zoom view of charge-reduced, isotope-deconvoluted LC-MS chromatograms from 3.5–7.0 min. The zoom chromatograms were processed by BiopharmaLynx 1.2 and the peaks were annotated based on accurate mass MH⁺ of peptides in an automated mode.

Figure 3

Charge-reduced, isotope-deconvoluted MS^E spectra (processed by BiopharmaLynx 1.2) of tryptic peptide HT35. (A) HT35 (EEMTK) of the innovator; (B) HT35 (DELTK) of the biosimilar mAb. The colors in the spectra represent: y ions (red); b ions (blue); y or b ions with a water or ammonia lost (green); unassigned ions (grey).

Figure 4

Mirror plots of two zoomed LC-UV chromatograms from 4-hour tryptic digests of the innovator and biosimilar mAbs, (A) from 30.5–36.0 min, (B) from 3.2–7.0 min.

Figure 4

Mirror plots of two zoomed LC-UV chromatograms from 4-hour tryptic digests of the innovator and biosimilar mAbs, (A) from 30.5–36.0 min, (B) from 3.2–7.0 min.

Figure 5

A comparison of LC-fluorescence separation and quantification of 2-AB labeled free glycans and MALDI MS profiling of unlabeled free glycans released from the innovator and biosimilar mAbs. (A) LC-fluorescence chromatograms of 2-AB labeled glycans. (B) Relative contents of 2-AB labeled glycans quantified by integrated peak area of LC-fluorescence chromatograms (C) MALDI MS profiling of unlabeled free glycans.

Figure 5

A comparison of LC-fluorescence separation and quantification of 2-AB labeled free glycans and MALDI MS profiling of unlabeled free glycans released from the innovator and biosimilar mAbs. (A) LC-fluorescence chromatograms of 2-AB labeled glycans. (B) Relative contents of 2-AB labeled glycans quantified by integrated peak area of LC-fluorescence chromatograms (C) MALDI MS profiling of unlabeled free glycans.

Figure 6

Charge-reduced, isotope-deconvoluted MS^E spectra (processed by BiopharmaLynx 1.2) of (A) unmodified and (B) N-deamidated tryptic peptide HT10 (NTAYLQMNSLR). The consistent 1-Da mass increase in y-serious ions from y4 in deamidated HT10 compared to unmodified HT10 clearly indicates the deamidated N site. The meaning of colors in the spectra is the same as described in Figure 3.

Figure 7

A comparison of XIC chromatograms of oxidized (21.6 min) and unmodified (26.0 min) tryptic peptide HT21 (DTLMISR) in the innovator and biosimilar mAbs. The relative quantification of oxidized and unmodified HT21 (based on peak area) is inserted.