Over 3000 E-Learning topics / 5000 Articles & Applications
 

Glycoprofile

The only common glycosylation site on immunoglobulin G (IgG) isoforms used in mAbs is N-linked in the Fc region, on the asparagine residue at position 297 (Asp-297, highlighted on Figure 1). This is associated with peptide T45 which is produced following trastuzumab digestion with trypsin. It has already been discussed in greater detail that the glycoprofile of a mAb is a key variable and a Critical Quality Attribute (CQA) that needs careful examination and monitoring to ensure batch-to-batch reproducibility. Differing glycoprofiles can dramatically affect efficacy, half-life, and biopharmaceutical safety. In the specific production batches of Herceptin, peptide T45 was glycosylated in over 99% of instances (Figure 2).

trastuzumab with glycosylation site highlighted

Figure 1: Representation of trastuzumab with glycosylation site highlighted (Asp-297).


Glycosylation status of Herceptin peptides

Figure 2: Glycosylation status of Herceptin peptides. 250 x 2.1 mm, 2.7 µm C18, mobile phase A: 0.05% TFA, mobile phase B: 0.05% TFA in acetonitrile, flow rate 300 µL/min, UV 214 nm, gradient 1-45 %B 2-35 minutes, 60 °C


The differing glycoforms, or glycoprofile, are represented as a number of unresolved peaks in the RPLC peptide map, rather than a single peak (outlined in dashed box in Figure 2). Further examination of this peak cluster allows fine details relating to the glycosylation profile to be extracted. In Figure 3 the specific asparagine residue in peptide T45, the glycosylation site, is highlighted in RED.

The importance of assessing the glycoprofile, in terms of ensuring batch-to-batch reproducibility, has already been discussed, especially as this is one of the most difficult parameters to control/match during biosimilar development. However, at the peptide level using RPLC, a non-ideal mode of chromatography for qualifying and quantifying polar glycans, a near full approximation of the glycoprofile can be observed.  

The biantennary branched glycans typical for IgG based mAbs, with the main glycoforms that are coupled to Herceptin, are also shown in Figure 3. It is worth noting that even though is not an ideal technique for glycan separation, the high resolving power of RPLC permits partial resolution of the G1Fa and G1Fb isomers bound onto peptide T45. As expected, an increase in the number of polar glycan units reduces retention in RPLC.

Herceptin peptide T45 glycoprofile

Figure 3: Examination of the Herceptin peptide T45 glycoprofile

 

A certain degree of glycosylation variability is expected, which will be written into the product specification and be regulatory acceptable. In the Herceptin batch example there is a clear increase in fucosylation, detected via the G0F to G0 ratio, across the production batches of Herceptin  (Figure 4). All this detailed and intricate information is available with the glycans still bound onto the peptide backbone.

Comparison of the peptide glycoprofile of different Herceptin production batches

Figure 4: Comparison of the peptide glycoprofile of different Herceptin production batches

 
loading data
loading data
loading data
loading data
loading data
loading data
loading data
 
 
 
 
 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

loading data

loading data

loading data

 

loading data


loading data