The N-glycans were prepared using the GlycoWorks RapiFluor-MS N-glycan kit (Waters) on an
Andrew Alliance pipetting robot (Geneva, Switzerland) as described previously.19 (link) Briefly, 10 µL of diluted infliximab sample was loaded to a 96-well sample plate.
Reagents including RapiGest surfactant, Rapid PNGase F enzyme, and RFMS
labeling reagent were used for protein denaturation, N-glycan release, and labeling,
respectively, by following a modified protocol in the GlycoWorks RapiFluor-MS
N-Glycan Kit Care and Use Manual (p/n 715004793).19 (link) A total of 48 released N-glycan samples were prepared, including 12 samples from
batch 1 of innovator infliximab, 6 samples from each of the other three batches of
innovator infliximab, 6 samples for each of two batches of biosimilar infliximab, and 6
samples from a blank control solution. After the glycan labeling protocol was completed,
0.25 pmol of a high mannose glycan standard (Waters) was spiked into each of six released
glycan samples prepared from innovator batch 1. This group of samples was used to evaluate
the capability of the workflow to capture the relative abundance changes in glycans across
samples.
Andrew Alliance pipetting robot (Geneva, Switzerland) as described previously.19 (link) Briefly, 10 µL of diluted infliximab sample was loaded to a 96-well sample plate.
Reagents including RapiGest surfactant, Rapid PNGase F enzyme, and RFMS
labeling reagent were used for protein denaturation, N-glycan release, and labeling,
respectively, by following a modified protocol in the GlycoWorks RapiFluor-MS
N-Glycan Kit Care and Use Manual (p/n 715004793).19 (link) A total of 48 released N-glycan samples were prepared, including 12 samples from
batch 1 of innovator infliximab, 6 samples from each of the other three batches of
innovator infliximab, 6 samples for each of two batches of biosimilar infliximab, and 6
samples from a blank control solution. After the glycan labeling protocol was completed,
0.25 pmol of a high mannose glycan standard (Waters) was spiked into each of six released
glycan samples prepared from innovator batch 1. This group of samples was used to evaluate
the capability of the workflow to capture the relative abundance changes in glycans across
samples.
