As mAbs are glycoproteins, it is also necessary to characterize the glycan portion of each product. Typically, mAbs have one
N-linked glycan consensus sequence (asparagine–Xxx–serine or threonine, where Xxx can be any amino acid except proline) within
each heavy chain located in the constant fragment (Fc) region. The light chain component of a mAb is not normally glycosylated.
Since additional N-linked glycan consensus sequences may be present within the heavy chains, the presence or absence of glycosylation should
be confirmed. ICH Q6B requests that "For glycoproteins, the carbohydrate content (neutral sugars, amino sugars and sialic
acids) is determined. In addition, the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile)
and the glycosylation site(s) of the polypeptide chain is analyzed, to the extent possible."
As shown in Figure 1, monosaccharide composition analysis is usually carried out to determine the carbohydrate content of
a mAb. Liquid chromatography and gas chromatrography (often with mass spectrometric detection) are commonly used to define
the carbohydrate content of a mAb. The method(s) used should allow identification and quantitation of the levels of the neutral
sugars (fucose, mannose and galactose), amino sugars (N-acetylglucosamine, N-acetylgalactosamine) and sialic acids (N-acetylneuraminic acid and N-glycolylneuraminic acid) within the product.
Figure 1: Schematic of the normal approaches employed to analyze the glycosylation of a monoclonal antibody (mAb). LC is liquid
chromatography, MS is mass spectroscopy.
Following monosaccharide composition analysis, the next stage of analysis moves into characterization of the oligosaccharides
normally present on each heavy chain. An example of the methodology used for releasing, purifying and analyzing the N-linked glycans from a mAb is shown in Figure 1.
Reduction/alkylation and the specific protease digestion are intended to denature the mAb so that the relatively large enzyme
PNGase F can do an efficient job at removing the N-linked glycans. Once the N-glycans are released, a simple reverse phase cartridge can be used to separate the hydrophilic glycans from the more hydrophobic
peptides. The N-linked glycan fraction can then be analyzed using mass spectrometry and/or liquid chromatography-based techniques.
The matrix-assisted laser desorption ionisation time-of-flight (MALDI–TOF) mass spectrum obtained from analysis of the N-glycans released from a mAb (using the methodology shown in Figure 1) is shown in Figure 2. The major signals observed are
consistent with the commonly encountered mAb glycans G0F, G1F and G2F (see Figure 2).
Figure 2: Raw data obtained from matrix-assisted laser desorption ionisation time-of-flight (Maldi-TOF) mass spectrometric
analysis of the N-glycans released from a mAb. The N-glycans were permethylated prior to analysis.
The high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) trace obtained from analysis of the
N-glycans released from the same mAb product is shown in Figure 3.
Figure 3: Raw data obtained from high-pH anion-exchange chromatography with pulsed amperometric detection analysis (HPAEC–PAD)
of the N-glycans released from a mAb.
Both sets of data can be used to provide a relative quantitation of the N-linked oligosaccharides observed and suggest the structures of the N-linked glycans present. Glycosylation is the most common post-translational modification encountered by the regulatory authorities.
The regulators are aware that glycosylation can be significantly affected by even simple changes in the manufacturing processes,
such as a pH change or a change of growth media. The monosaccharide composition analysis data (in terms of levels of mannosylation,
galactosylation and sialic acid) and N-linked oligosaccharide profiling data obtained from the analysis of various batches of a mAb product are therefore used by
the regulator as part of the assessment of whether a manufacturer has control of the manufacturing process of a mAb. Comparability
of N-glycan profiles from batch to batch suggests that the manufacturing process is under control.