This protocol addresses the analysis of limited samples obtained from microbioreactors to extract vital information about the critical quality attributes of the products. Another advantage of these techniques is that they use minimal times of analysis to determine the key attributes that dictate the quality parameters of the manufactured product. Demonstrating procedure will be David Powers, Talia Faison and Phillip Angart, research fellows from my laboratory.
Begin by opening the software attached to the purification system and placing 15 milliliter conical tubes to collect the purified antibody eluate and 50 milliliter conical tubes to collect the flow-through during the high salt wash into the fraction collector. Next, add 0.22 micrometer pore filtered harvested cell culture fluid to an empty 12-milliliter syringe with a capped nozzle. After removing the cap, insert the syringe nozzle into the manual injection port of the purification system.
Twist the syringe to tighten it in place and depress the plunger until the entire volume of the sample has been injected and is visible in the attached 10 milliliter large volume sample loop. Select the saved method and click Run when prompted by the instrument software. When all of the antibody has been eluded, immediately neutralize the purified protein with one molar tris base to a pH of about 5.5.
To concentrate the purified antibody by centrifugation, place 100 kilodalton filters into filtrate collection tubes. Wash the filters with 500 microliters of double distilled water, then centrifuge. Repeat filter wash two times.
After the second wash, discard the filtrate and transfer the rinsed filters to new centrifuge tubes. Next, add 500 microliters of sample to each filter for centrifugation. At the end of the spin, invert the filter into a new collection tube to obtain the concentrated sample with a final centrifugation.
For N-glycan labeling and isolation, dilute 7.5 microliters of each concentrated antibody sample. With 15.3 microliters of liquid chromatography mass spectrometry or LCMS-grade water in one-milliliter tubes from the kit, and denature the antibodies with six microliters of a 5%solution of an enzyme-friendly and mass spectrometry-friendly surfactant at 90 degrees Celsius for three minutes. At the end of the denaturation, allow the samples to cool to room temperature for three minutes before adding 1.2 microliters of peptide N-glycosidase F for a five-minute incubation at 50 degrees Celsius.
After cooling the samples for three minutes to room temperature, label the samples with 12 microliters of fluorescent tagging reagent dissolved in anhydrous dimethylformamide for five minutes at room temperature. At the end of the incubation, dilute the labeled N-glycan mixture with 358 microliters of acetonitrile and place a hydrophilic interaction chromatography plate in a vacuum manifold with shims and waste tray. Condition the wells with 200 microliters of water with the vacuum adjusted to 10 to 15 kilopascals to ensure that the liquid will take 15 to 30 seconds to pass through the hydrophilic interaction chromatography resin.
Equilibrate the wells with 200 microliters of 85%acetonitrile for 15 to 30 seconds before loading 400 microliters of each labeled glycan mixture to each well, applying the vacuum after each new liquid is added. When all of the samples have been added, wash the resin two times with 600 microliters of 1%formic acid and 90%acetonitrile per wash and replace the waste tray with 600-microliter collection tubes. Then elude the labeled N-glycans with three 30-microliter volumes of spectrometry elution buffer and dilute the pool dilutions with 310 microliters of dimethylformamide in acetonitrile sampled eluent.
To analyze the labeled N-glycan elution samples on an ultraperformance liquid chromatography system coupled to a fluorescence detector and quadruple time of flight mass spectrometer, use 50 millimolar ammonium formate and 100%LCMS grade acetonitrile for the mobile phases. Set the initial flow rate to 0.4 milliliters per minute, with the LC gradient providing increasing ammonium formate during the elution phase. Set the fluorescence detector to measure an excitation of 265 nanometers and an emission of 425 nanometers with a sampling rate of two hertz.
Set the quadruple time of flight to MS1-positive ion sensitivity mode with a mass range of 100 to 2, 000 Daltons, a scan time of 0.25 seconds, and a continuum data acquisition. Then load the samples into the auto sampler set to 10 degrees Celsius and run the loaded method. To desalt a sample in preparation for charge variant analysis, snap off the bottom stopper of a 0.5 milliliter desalting column, loosen the top stopper, and place the desalting column in a 1.7 milliliter centrifuge tube.
Transfer the new column into a new microcentrifuge tube and add 80 microliters of a 3.5 milligram per milliliter antibody solution to the top of the column. Align the column to the original orientation and centrifuge the column. Then discard the desalting column and thoroughly mix the concentrated sample.
Dilute the sample to a two milligram per milliliter concentration in 25 microliters of ultrapure water in a single well of a 96-well plate and add five microliters of labeling buffer and five microliters of labeling reagent to the well. After a 10-minute incubation at room temperature protected from light, mix 60 microliters of reagent-grade water with the sample and cover the plate with a plate seal for centrifugation. To prepare the charge variant chip, remove the storage solution and wash wells one, three, four, seven, eight and 10 with water.
Replace the water with pH 7.2 running buffer and add 750 microliters of running buffer to the buffer tube. Press Unload Plate on the instrument user interface and remove the seal from the 96-well plate. Insert the plate and buffer tube into the indicated spots on the GX2 sample tray and press Load Plate.
Place the chip into the chip chamber, close the lid to the chip chamber, select the HT protein charge variant assay when prompted, and click Run. Purification of the harvested cell culture sample from the automated microscale bioreactor by fast protein liquid chromatography allows the characterization of the critical quality attributes of the purified proteins by various downstream analytical methods, as demonstrated. N-glycan data from Chinese hamster ovary-produced monoclonal antibodies processed by mass spectrometry should appear similar to these representative chromatograms.
Size exclusion chromatography multiple angle light scattering can be used to assess the aggregation profile and molecular weight of the antibody. The small quantity of sample and the importance of the aggregation are critical quality attributes for making this technique a highly valuable complementary analytical tool to the automated microbioreactor system. The result of micro capillary zone electrophoresis is an electropherogram and can be used to show the charge variant profile for a monoclonal antibody, a unique signature for the protein being investigated that is highly sensitive to the operating pH.
Amino acid consumption can also be monitored to determine whether the depletion causes changes in the critical quality attributes of the antibody. It is important to ensure a proper and efficient purification of the manufactured product, as the analytical steps can only be carried out with a properly purified protein. The product can also be subjected to peptide mapping and stability testing.
But once the protein's been used for one characterization method, it typically cannot be used for another. These techniques allow the analysis of products produced from small volume, high throughput bioprocessing screening platforms to understand the influence of bioprocessing parameters on product quality. Some of these techniques use concentrated perchloric acid, formic acid, N-dimethylformamide, all of which are hazardous and should be handled carefully while wearing the proper protective equipment.
