This protocol can be utilized to screen the effect of multiple glycosylation enzymes on the glyco-profile of a target protein and contribute towards a better understanding of the enzyme's functionalities. The nature of the protocol allows the screening of glycosylation enzymes in a transient, yet high-throughput manner. Altered glycosylation profiles can lead to improved antibody activities, and therefore increase the efficacy of the final product.
Biopharmaceutical companies closely monitor glycosylation as a critical quality attribute. An aberrant glycosylation is a hallmark of diseases like cancer. Therefore, this method is relevant to pharmacological and biomedical research.
The protocol assumes users to have experience in several experimental techniques, including mammalian cell transfection and Western blots. It's better to start with fewer samples and use stopping points in the first instance. Begin by assessing the density and viability of the Chinese hamster ovary cells.
Then, carefully clean the biosafety cabinet and all equipment with 70%ethanol and an RNase inhibitor solution to avoid contamination. Next, pellet the cells at 100 times G for five minutes and resuspend them in a pre-warmed medium at a cell density of five times 10 to the six cells per milliliter. Transfer eight microliters of the DsiRNA master mix or control to a sterile electroporation cuvette.
Add 800 microliters of the cell suspension to the same cuvette, mix, and deliver the electroporation pulse. Then, transfer the cell suspension from the cuvette to one well of a six-well plate, avoiding foam-like material. Incubate the cells for 10 minutes without shaking.
After incubation, add 800 microliters of pre-warmed media to make a final volume of 1.6 milliliters per well. Grow the transfected cells in the incubator while shaking at 150 RPM. After 48 hours, harvest the supernatant and cells.
After IgG purification, add elution A onto a three-kilodalton molecular weight cutoff centrifugal concentrator, and centrifuge at 13, 300 times G for 40 to 50 minutes at four degrees Celsius. Centrifugation is complete once the residual volume is equal to or less than 50 microliters. Discard the flow-through and add 500 microliters of pre-chilled 1X PBS to dilute the residual supernatant.
Centrifuge the sample again using the same conditions until 50 microliters of residual supernatant remains. To obtain a 100X dilution of the supernatant, add 500 microliters of pre-chilled 1X PBS and repeat the centrifugation process. Concentrate the supernatant into a final concentration of approximately 2.5 grams per liter in 40 microliters to ensure compatibility with the glycan analysis method.
For glycan analysis, transfer 200 microliters of the magnetic bead solution to a 2-milliliter PCR tube. Place it on the magnetic stand to separate the beads from the supernatant and remove the supernatant carefully. Then, remove the tubes from the magnetic stand and add the purified protein sample and vortex.
Next, add the supply denaturation buffer to the sample tube and incubate for eight minutes at 60 degrees Celsius. Keep the sample tubes open for optimal reaction performance. After incubation, add PNGase F and incubate for another 20 minutes at 60 degrees Celsius to cleave glycans from the purified antibodies.
After the release of N-glycans, close the sample tube and vortex. Then, add acetonitrile to the sample tube, vortex, and incubate at room temperature for one minute. Place the sample tubes in the magnetic stand to separate the beads from the solution.
Then, using a pipette, carefully remove the supernatant without touching the beads. Add the fluorophore-containing glycan labeling solution to the sample in a fume hood and mix by vortexing. Following a 20 minute incubation at 60 degrees Celsius with open lids, remove excess dye by washing the sample three times in acetonitrile.
Then, elute the labeled glycans in double-distilled water. Place the sample tube in the magnetic stand and collect the supernatant enriched with purified and labeled glycans. Next, prepare and load all the required standards and samples into the designated tray positions.
Run the glycan analysis protocol and analyze and identify the glycans present in the sample using appropriate software. Western blot analysis showed reduced Fut8 protein expression in cells transfected with a mixture of three Fut8 DsiRNA constructs. Glycan structures from the knockdown cells also showed decreased fucosylation.
This trend was most pronounced in agalactosylated structures and observed to a lesser extent in galactosylated structures. A two-fold reduction in core fucosylation was observed from electroporation using two square-wave pulses compared to a single square-wave pulse without significant differences in cell viability. Overall, increasing short-interfering RNA concentration has a great interference on core fucosylation than increasing the harvest time.
The ratio between water and acetonitrile determines whether glycans are in solution or part of the beads. That's crucial to remember during the washing steps to avoid accidental removal of the labeled glycans from the solution. A follow-up experiment might involve the characterization of purified monoclonal antibodies using cell-based assays to quantify antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.
