Background:
While α-gal is present as major carbohydrate in non-primate mammals, anti-α-gal is the most abundant natural immunoglobulin in humans. α-gal glycosylation of therapeutic antibodies may thus have an impact on their clinical properties, especially regarding their potential for allergic reactions. Murine production cell lines such as SP2/0 are known to lead to α-gal glycosylation, whereas CHO cell lines usually lack this property. In this regard, we tested anti-GD2 antibodies ch14.18 derived from murine SP2/0 and CHO cell lines.
Methods:
A detailled comparative head-to-head glycan profiling of ch14.18/CHO and ch14.18/SP2/0 (including dinutuximab) was performed. The glycans were cleaved from the antibody backbone enzymatically using PNGase F and were labelled with a 2AB fluorescent tag for determination. Labelled glycans were seperated using Hydrophilic Interaction Chromatography (HILIC), and the identity of the eluted glycans was determined based on the calculated Glucose Unit (GU) values as well as by ESI-QTOF mass spectromety.
Results:
The presence of α-gal structures in ch14.18 manufactured from murine SP2/0 cell lines was clearly demonstrated. None of these structures were identified in any of the CHO cell derived ch14.18 batches. Whereas levels of up to 8.17% of α-gal structures were determined for ch14.18/SP2/0 (a product comparable to that used within COG study ANBL0032 (Yu, 2010)), a level of 1.12% was determined for dinutuximab.
Discussion:
The glycosylation analysis of ch14.18 antibodies derived from SP2/0 or CHO cells confirmed literature data regarding α-gal glycosylation. SP2/0 derived ch14.18 batches contain α-gal although percentages may differ considerably. No α-gal whatsoever was found in ch14.18/CHO. Clinical consequences of α-gal glycosylation of ch14.18 may relate to their allergic potential. As published in the EPAR for Unituxin (dinutuximab) out of 798 neuroblastoma patients 81.1% reported an allergic reaction-related event, 29% were reported severe (Grade 3-4) and 18% had anaphylactic reactions. Out of 514 neuroblastoma patients treated with ch14.18/CHO, 20% reported an allergic reaction-related event (including 0.8% with anaphylactic reactions), 6% were reported to have severe (Grade 3-4) allergic reaction-related events.