Materials and Methods
Separations were performed on an Acquity UHPLC system (Waters Corporation) equipped with tunable ultraviolet (TUV; for peptide
mapping) or fluorescence (FLR, for released glycan profiling) detectors. All MS measurements were made on a Synapt HDMS system
(Waters). The systems were controlled by MassLynx 4.1 software (Waters); data processing was performed with BiopharmaLynx
v. 1.2 (Waters). Simglycan (v. 2.75) (Premier Biosoft International) was used for glycan identification from a MALDI MS–MS
experiment. The conditions of intact protein analysis and peptide mapping were the same as described in recently published
papers.8,9 The conditions for hydrophilic interaction chromatography-fluorescence (HILIC–FLR) analysis were described elsewhere.10
Results and Discussion
Intact Protein LC–MS Analysis
The intact MS analysis of large proteins such as MAbs requires appropriate preparation but can be performed routinely. The
samples must be thoroughly desalted before MS analysis to obtain a sufficient signal. In our experience, the best results
are obtained with on-line desalting.11
The reversed-phase desalting column was used in an on-line setup to obtain UHPLC–MS data shown in Figure 1. Deconvoluted data
for the innovator and biosimilar MAbs are presented as a mirror plots; the glycoform annotation is based on the deconvoluted
MS signals. One can clearly observe protein mass heterogeneity resulting from the presence of several main protein glycoforms.
Several dominant peaks can be distinguished for each MAb sample; the difference in mass of ~162 Da corresponds most likely
to loss of galactose, G, and ~146 to fucose, F, from the N-linked glycan core structure. The molecular weight of the innovator
MAb is consistent with its expected primary sequence, i.e., its mass corresponds to the sum of the mass of two G0F, G0F/G1F,
two G1F, G1F/G2F, or two G2F glycans (Figure 1, upper trace). Interestingly, the biosimilar candidate shows different glycoform
heterogeneity and all main glycoforms show a mass shift of ~64 Da compared with the innovator drug. This shift is putatively
caused by the presence of unknown PTMs or by differences in the primary amino acid sequence.
Figure 1. Intact MAb MS analysis. Multiply charged spectra were deconvoluted into protein molecular mass and compared using
BiopharmaLynx software. Heterogeneity is caused by the MAb's glycoforms. Notice the mass shift of biosimilar drug candidate
compared to the reference innovator sample.
Further investigation of intact protein mass data suggests the presence of minor glycoforms such as G0/G0F in each MAb (a
mass difference of 146 Da, due to incomplete occupancy of a fucosylation site on one of the two G0F core glycan structures).
In addition, a low-level Man5/Man5 form was detected in both MAbs.
Although the intact protein analysis is fast and useful, it is difficult to quantitatively measure the relative glycoform
ratios, especially for minor glycoform species (Figure 1). Therefore, we performed a partial reduction of MAb samples, followed
by on-line desalting and MS analysis. The deconvoluted masses of light and heavy chains are shown in Figures 2A and 2B, respectively.
Whereas the light chains are identical, the heavy chain data for the innovator and biosimilar MAbs show a distinct mass shift
for all glycoforms. Analyzing the 50-kDa heavy chain alone allows for better assessment of glycosylation heterogeneity (whereas
the information about heterogeneity on the intact MAb molecular level is now lost). Man5, G0, G0F, G1, G1F, G2, and G2F glycoforms
were clearly distinguishable in deconvoluted MS spectra (Figure 2B).
Figure 2. UHPLC–MS analysis of reduced MAb samples. Data were processed by BiopharmaLynx software. A) Light chain of innovator
and biosimilar MAb are identical. B) Heavy chain of biosimilar candidate MAb shows consistent mass shift of ~32 Da for all
protein glycoforms compared to the reference sample. Glycosylation pattern differences were detected.
When comparing the innovator to the biosimilar MAb, the consistent mass shift of ~32 Da is observed for all glycoforms, suggesting
a possible modification of the biosimilar MAb candidate heavy chain. This is consistent with the previously detected ~64 Da
difference in the intact protein mass (i.e., the combined contribution of two heavy chains). In the next section, we describe
a peptide mapping experiment used to pinpoint the origins of the mass shift.