Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

1. Purification of antibody

NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M acetic acid. The buffers and resin (Protein A) are dependent on the specific antibody purified. Column volume is equivalent to the bed height of the resin. The amount of mobile phase used is determined in terms of column volume.

1. Initializing the purification system
   1. Open the software attached to the purification system. Using manual instructions, equilibrate the column with the equilibration buffer at a flow rate of 2 mL/min for 40 min. Stop the manual run after equilibration.
   2. In the fraction collector, place 15 mL conical tubes to collect purified antibody eluate and 50 mL conical tubes to collect flow-through during high salt wash. Ensure that the fraction collector is reset to the start position by opening and closing the fraction collector before the beginning the run. The fraction collector is maintained at 7 °C.
      NOTE: The fraction collector can be reset manually under the Fraction Collector tab in Settings, for both 15 mL and 50 mL tubes.
2. Sample injection
   NOTE: The harvested cell culture fluid used in the following procedures has been obtained from chinese hamster ovary cells cultured in automated micro-bioreactors⁹.
   1. Add the 0.22 µm filtered harvest cell culture fluid to an empty 12 mL syringe whose nozzle end is capped.
   2. Holding the syringe with the nozzle facing down, insert the syringe plunger till a small portion of the plunger is in. Making sure that the fluid is not leaking, turn the syringe with the nozzle facing up and remove the cap.
   3. Still holding the syringe with the nozzle facing up, push the cylinder to dispel any air until the cell culture fluid is at the tip of the nozzle. Insert the syringe nozzle into the manual injection port on the purification system and twist to tighten.
   4. Push down on the plunger until all the sample is injected and is visible in the attached 10 mL large volume sample loop.
   5. Open the saved method file. Save the result file in the required location and specify file name when prompted. Hit run after the sample has been injected into the large volume sample loop.
3. Running the purification method
   1. Select the saved method and click run when prompted by instrument software (step 1.2.5).
      NOTE: The system is set up to run the following steps. The user needs not do anything while the instrument is running.
   2. Equilibrate the column with three column volumes (CVs) of equilibration buffer at a flow rate of 2 mL/min. Once the column is equilibrated, the system, using the large volume sample loop, will inject the sample onto the column at a flow rate of 1 mL/min.
   3. A drop in UV signal at 280 nm indicates that the sample is finished loading. Wash the column with equilibration buffer at a flow rate of 2 mL/min until the UV signal drops below 25 mAU.
   4. Use four CVs of 25 mM Tris with 1 M NaCl at pH 7.5 to perform a secondary high salt wash at a flow rate of 2 mL/min. The system fraction collector will collect any protein/DNA that comes off the column during the salt wash in 50 mL tubes.
   5. Apply five CVs of elution buffer at a flow rate of 1 mL/min to elute the antibody off the column. Collect the eluate in 15 mL tubes based on UV signal; when the UV 280 signal is above 35 mAU, collection starts; collection ends when the signal drops below 50 mAU; this is called peak cutting.
      NOTE: Peak cutting ensures normalization of elution profiles and to avoid elution peak tailing which may contain protein aggregates²⁴.
   6. Wash the column using three CVs of equilibration buffer. The run ends after the wash step.
   7. After elution immediately neutralize the purified protein using 1 M Tris base to a pH of ~5.5. Measure the protein concentration using a microvolume UV-Vis spectrophotometer at 280 nm and 260 nm and store at 4 °C.
   8. Concentrate the purified antibody using centrifugal units (step 2). Then subject the purified antibody to glycan analysis using LC-MS and after aggregation profile analyze using SEC-MALS (steps 3 & 4).
      NOTE: The purified antibody should not be frozen without further buffer exchange as frequent freeze-thaw cycles can cause aggregation and precipitation.

2. Concentration of purified antibody

NOTE: The Tris-acetate buffer is 0.1 M acetic acid neutralized with 1 M Tris Base to a pH of ~5.5.

1. Insert 100 kDa filters into centrifuge tubes.
2. Wash the filters with 500 µL of double distilled water. Centrifuge for 10 min at 14,000 x g at room temperature (RT). Repeat this step twice. Discard filtrate.
3. Transfer the rinsed filters to fresh centrifuge tubes and add 500 µL of sample to each filter. Centrifuge for 10 min at 14,000 x g.
4. Invert the filter into a fresh spin tube. Centrifuge for 2 min at 1,000 x g to collect the concentrated sample.
5. Determine the sample concentrations using a UV-Vis spectrophotometer. Blank the spectrophotometer using a solution of Tris-acetate buffer. Use a protein extinction coefficient of 1.37 mL•(mg•cm)^-1 at 280 nm for a 1% (% m/v) IgG solution.
6. Use the concentrated sample to prepare 12.5 µL of 2 mg/mL working solution for glycan analysis and 30 µL of 3.5 mg/mL working solution for SEC-MALS.
   NOTE: The protocol can be paused here. Samples should be refrigerated at 4 °C. At 2 mg/mL concentration, these samples should be stable for at least three months at 4 °C while higher concentrations might precipitate.

3. Analysis of N-glycans using mass spectroscopy

1. N-Glycan labeling and isolation
   1. Start with antibody concentrations of 2 mg/mL in an appropriate buffer such as neutral sodium phosphate, citrate or HEPES buffer. Prepare an intact mAb standard (such as NIST mAb) at 2 mg/mL to process alongside the experimental samples to serve as a positive control.
      NOTE: The antibodies should be in a final buffer containing no SDS and less than 0.1 mM nucleophiles (such as Tris, DTT, glycine or histidine). SDS in the sample buffer must be removed. If nucleophiles are in the buffer, then dilute them down or perform a buffer exchange since they will interfere with the kit. The general protocol is provided with the glycan kit.
   2. Dilute 7.5 µL of the antibodies with 15.3 µL of LC-MS-grade water in 1 mL tubes provided with the kit and then denature using 6 µL of 5% solution of an enzyme-friendly and MS-friendly surfactant at 90 °C for 3 min.
   3. Cool the samples for 3 min to room temperature (RT). Then, add 1.2 µL of PNGase F and incubate for 5 min at 50 °C.
   4. After cooling 3 min to RT, label the cleaved N-glycans by adding 12 µL of fluorescent tagging reagent dissolved in anhydrous dimethylformamide (DMF) and wait for 5 min. Dilute the labeled N-glycan mixture with 358 µL of acetonitrile (ACN).
   5. Place a hydrophilic interaction chromatography (HILIC) plate in a vacuum manifold with shims and waste tray. Use a multichannel pipette for large numbers of samples.
   6. Condition the wells with 200 µL of water, where the vacuum is adjusted so the liquid will take 15-30 s to pass through the HILIC resin. Equilibrate with 200 µL of 85% ACN prior to loading the ACN-diluted labeled glycan mixture (400 µL), applying the vacuum after each new liquid is added to the wells. Wash the resin with 600 µL of 1% formic acid (FA)/90% ACN twice.
   7. Replace the waste tray with 600 µL collection tubes. Elute the labeled N-glycans with SPE elution buffer (3 elutions of 30 µL each) into the collection tubes. Dilute the pooled elutions with 310 µL of DMF/ACN sample diluent. Pipette the samples into auto sampler vials to be ready for fluorescence (FLR)-MS analysis.
      NOTE: These samples are stable when stored at -80 °C for at least 1 month. Store the HILIC plate in its original packaging, taped shut and inside a desiccator for future use.
2. LC-MS analysis of labeled N-glycans
   1. Analyze the labeled N-glycan elution samples on an Ultra Performance Liquid Chromatography (UPLC) system coupled to a fluorescence detector and quadrupole time-of-flight (Q-ToF) mass spectrometer. Use a column approved for chromatographic separation of the labeled glycans and heat to 60 °C during separations.
      NOTE: The column must be flushed with 60% acetonitrile and 40% H₂O before use: 50 CVs before the first use or 20 CVs if the column has been used before.
      1. Use 50 mM ammonium formate (AmF) (made with mobile phase concentrate) and 100% LC-MS-grade ACN for the mobile phases. The AmF is sensitive to pH changes and is usable for 1 month after mixing. Set the initial flow rate to 0.4 mL/min, with the LC gradient providing increasing AmF during the elution phase.
      2. Set the FLR detector to measure at EX 265/EM 425 nm with a sampling rate of 2 Hz. Set the Q-ToF to MS1 positive ion sensitivity mode, with a mass range of 100-2,000 daltons (Da), a scan time of 0.25 seconds and continuum data acquisition. Use leucine enkephalin (2 ng/µL in 50% ACN/0.1% FA) for the internal mass reference, in the "Do NOT apply correction" mode.
         NOTE: The internal mass reference correction will be applied later during data processing.
      3. Re-suspend the dextran ladder sequentially in 22.5 µL of H₂O, 25 µL of DMF and 52.5 µL of ACN. Prepare 10 µL aliquots for storage at -80 °C, as the ladder is not stable for more than 24 h at higher temperatures (room temperature, 4 °C). The dextran ladder degrades after more than one freeze-thaw cycle.
      4. Place the samples in the auto sampler set to 10 °C. Load a vial of dextran ladder along with samples, as the retention time information of the ladder will be used for assignments while the mass information used to validate identifications. Use 10 µL injections for the samples and 7.5 µL injections for the ladder. Inject samples in triplicate. Run the loaded method.
3. N-Glycan identification for LC-MS data
   1. Perform data processing with a program optimized for hydrophilic interaction chromatography fluorescence mass spectrometry (HILIC-FLR-MS) data.
   2. Apply internal mass reference corrections within the program. Designate the dextran ladder injections as "Standard" in Sample Information. In the Analysis Method, set the Separation Compound retention times to those of the ladder compounds that were detected during the run.
   3. To ensure that Area % will be returned for identified glycans, modify the Analysis Method: Under the Processing tab, click Quantitation Settings -  Calibrate and set "Calibration curve fit type" to "Relative response (%)".

4. Analysis of antibody aggregation using SEC-MALS

1. Sample preparation
   1. Transfer the 3.5 mg/mL (step 2) diluted protein to a vial with a 150 µL glass insert. Use a gel loading tip to pipet into the bottom bell of the insert to avoid the introduction of bubbles.
   2. Cap the vial with a septa cap and analyze immediately. Store at 4 °C if analyzing later.
2. SEC-MALS configuration and equilibration
   NOTE: Analyze aggregation on SEC-MALS configured with an Ultra High-Pressure Liquid Chromatography (UHPLC) with a MALS Detector and Refractive Index Detector controlled by the MALS software.
   1. Configure a method file in the UHPLC software for control of the UHPLC system, setting the flow rate to 0.4 mL/min with a mobile phase of 1x Phosphate Buffered Saline (PBS) (diluted from 10x), the injection volume to 5 µL, the column temperature to 25 °C, and the Diode Array Detector (DAD) to monitor 280 nm. Set run time to 20 min. Equilibrate the system for at least 4 h prior to any sample analysis.
   2. The interface between UHPLC and Multi Angle Light Scattering – Refractive Index (MALS-RI) detectors requires the use of the analog output on the DAD. Set the DAD attenuation to 1,000 mAU in the DAD method file and AU/UV setting to 1 (UV instrument>Channels>Channel 1).
   3. Turn on the DAD lamp 30 min prior to beginning analysis and set the wavelength to 280 nm. At the same time, purge the Refractive Index (RI) reference cell for 15 min or until the baseline is stable and then close the reference cell.
   4. Configure SEC-MALS software sequence, setting the collection time to 12 min, the injection volume to 5 µL, the dn/dc to 0.185 mL/g, A280 extinction coefficient if previously experimentally determined or to 1.37 mL*(mg*cm)^-1, and the concentration of the sample. Click Run and wait for the "waiting to inject" dialog to appear on the screen.
      NOTE: The A280 extinction coefficient is specific to the protein of interest and should be determined experimentally.
   5. Configure a sample list in the UHPLC software in the same order as in MALS-RI software and submit.
      NOTE: It is important to run a system suitability check before and after a run. Bovine serum albumin typically is used to check for peak broadening, a sign that the SEC column may need cleaning or replacement. The same BSA standard injection can be used to specify signal alignment, peak broadening, and detector normalization.
3. Aggregate analysis with MALS software
   1. Click on the tab marked Procedures. Specify the minimum level of despiking required; none is usually sufficient.
   2. Verify that baselines have been drawn correctly and adjust if necessary, for LS1, LS2, LS3, RI, and UV channels. Set the peak area of interest.
   3. Review the molecular mass distribution to confirm that the called peaks contain particles of similar size.

5. Charge variant analysis

1. Sample preparation and labeling
   1. Start with 80 µL of a 3.5 mg/mL antibody solution.Desalt the sample using a 0.5 mL Desalting Column (7k MWCO). Prepare the column by first snapping off the bottom stopper, then loosening the top stopper, and placing it in a 1.7 mL micro-centrifuge tube. Centrifuge the desalting column for 1 min at 1,500 x g.
      NOTE: Mark the exterior of the column with a dot so that it can be placed in the original orientation for the next steps.
   2. Transfer the column to a new micro-centrifuge tube. Add the 80 µL of diluted protein to the top of the column. Align the column to the original orientation. Centrifuge for 2 min at 1,500 x g. Remove the sample from the centrifuge, discard the desalting column and mix the sample well.
      NOTE: Desalting is only required if the sample matrix contains primary amines, excipients that will perturb the sample electrophoresis, or other incompatible substances.
   3. Dilute the sample to a final concentration of 2 mg/mL in a volume of 25 µL and add 5 µL of the labeling buffer (see Table of Materials: Charge Variant Reagent Kit) in the 96-well plate. Prepare the labeling reagent by diluting the necessary amount of labeling reagent (see Table of Materials: Charge Variant Reagent Kit) 1:30 in dimethylformamide. Incubate the sample for 10 min at room temperature away from light.
      NOTE: It is important to thaw and then immediately use this reagent and use it within 10 min of mixing with DMF.
   4. After incubating, add 60 µL of reagent grade water and mix well by pipetting. Cover the plate with a plate seal and centrifuge the plate at 1,000 x g for 1 min.
2. Preparing the charge variant chip
   1. Prepare the Charge Variant chip by removing storage solution and washing wells 1, 3, 4, 7, 8, and 10 with water. Then replace the water with pH 7.2 running buffer (see Table of Materials: Charge Variant Reagent Kit).
   2. Add 750 µL of pH 7.2 running buffer to the buffer tube and place the buffer tube in the indicated spot on the upper left-hand corner of the sample tray. Now remove the plate seal from the 96-well plate, press Unload Plate on the instrument user interface, and insert the plate into the GXII sample tray.
      NOTE: pH 7.2 buffers were used for this analysis. pH 5.6-7.2 buffers may be used depending on the protein pI. When using lower pH buffers, longer sample run times may be required.
   3. Press the Unload Chip button on the user interface. Ensure that the electrodes are free of any particles, and if not, clean with a lint free swab. When inserting the chip make sure that the window in the center of the chip is free of particles or smudges. If necessary, clean with a lint-free soft cloth.
      NOTE: When working with capillary electrophoresis chips, remove buffer by vacuum aspiration, followed by the immediate addition of the next solution to prevent wells from drying out. To minimize the introduction of bubbles, practice reverse pipetting technique. When handling the chip, be mindful of the fragile capillary extending from the bottom of the chip, making sure that it does not dry out and does not break through rough handling.
   4. Close the lid to the chip chamber and select the HT Protein Charge Variant assay. Click the Run button. Follow the prompts to select the sample wells, plate type, assay time (68, 90, or 100 s), and file name. Click Start at the end of the prompts.
   5. Chip cleanup requires the washing of each well 2x with water, followed by the addition of Storage Buffer (see Table of Materials: Charge Variant Reagent Kit). Once in storage buffer, replace the chip in the instrument and, when prompted, select the HT Protein Charge assay. On the main screen select Wash on the user interface. Once completed, remove the chip, wipe down the electrodes with water and a lint-free swab, and store the chip at 4 °C.
3. Charge variant analysis
   1. Open the instrument analysis software. Import the run by going to File>Import Data File… and clicking on the desired *.gxd file. Only the name will be carried over to software, so renaming the wells is advantageous (Tools > Sample Name Editor). Select the files to be exported by holding down shift while selecting the files. Click File>Export… and select the Raw Data box and then the AIA Format box.
   2. Open the Browse Projects tab within the analysis software. Click on Database>Import Data… and select the exported *.CDF files.
   3. Once imported, navigate to the Injections tab, select the files to be analyzed, right click and go to Process… In the window that pops up, select the check box next to Process and select the "Use specified processing method" radio box and the desired processing method from the drop-down box. In the drop-down box immediately below labeled "How:" select Calibrate and Quantitate. Once processed, navigate to the Results tab and check the integration of the chromatograms.
      NOTE: The processing method needs to be verified for each method. As a starting point, the parameters used for the current processing method are included in Supplementary file.
      NOTE: Exporting of the data can be done in the form of a report or only the peak quantification may be exported. These can be done the same time as processing or from the results window.

6. Amino acid analysis

1. Setting up the standard curve for absolute amino acid quantification by LC-MS
   1. Prepare the extended amino acid (EAA) mixture by dissolving 59.45 mg of Asn, 59.00 mg of Hyp, 65.77 mg of Gln, and 91.95 mg of Trp in 25 mL of 0.1 N HCl. The final concentration of each amino acid in the EAA mixture is 18 nmol/µL.
   2. Prepare the internal standard (ISTD) stock solution by dissolving 58.58 mg of Nva and 44.54 mg of Sar in 50 mL of HCl.
   3. Prepare the complete amino acid standards by combining the amino acid stock solution containing Ala, Asp, Arg, Cys, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val at 1 nmol/µL each with the EAA mixture for final amino acid concentrations of 900, 225, 90, 22.5, and 9 pmol/µL. Add the prepared ISTD stock to the amino acid standards for a final concentration of either 90 pmol/µL or 900 pmol/µL to create "low" and "high" internal standards to use as positive controls for the method.
   4. Place the amino acid concentrations in sample vials into the autosampler of the UPLC. Generate a calibration curve (9 to 900 pmol/µL) in the instrument software based on the amino acid standard concentrations using the following instructions.
   5. Use the Q-ToF in electrospray ionization (ESI) positive sensitivity mode coupled to a UPLC for intact amino acid analysis. For chromatographic separation, use a normal phase column made for amino acid separations. Prepare the following buffers with mass spectrometry grade reagents: A = acetonitrile + 0.1% formic acid and B = 100 mM ammonium formate. Set the LC flow rate to 0.6 mL/min and column temperature to 40 °C.
   6. Use the following 15-minute gradient conditions for amino acid separations: 14% B (0-3 min), 14-100% B (3-10 min), 100% B (10-13 min), 100-8% B (13-14 min), 8% B (14-15 min).
   7. Use the "Sample Type" and "Conc A" column listings in the MS acquisition program to create a calibration curve for future analysis of crude bioreactor media in the quantitation program. To make these columns appear in acquisition program, use the Customize Display… command when right-clicking on the top menu bar.
   8. The Sample Type for the amino acid standards will be "Standard" while the media samples will be "Analyte". Fill in the "Conc A" column with the numerical concentrations of the standards in the required units (ex: pmol/µL).
   9. Run the prepared amino acid standard concentrations at least twice. Validate that the UPLC instrument and mass spectrometer are working properly by checking the ISTD peaks.
   10. Use the "Edit Method" option in the quantitation application to create the quantitation method (*.mdb file). Define all the amino acids of interest in the quantitation application, such as the compound name, m/z value and expected retention time. Change the integration parameters for the method here.
   11. Use the created amino acid method on the standard samples to create the calibration curve. This curve can be exported to a *.cdb file for use with the media samples using the Export>Calibration… command.
   12. In the quantitation application, save the desired layout to a *.qlt file to apply to future datasets using "Save Layout As…". Name (Injection name), Area and Conc are the most important output columns.
2. Amino acid analysis of crude bioreactor media by LC-MS
   1. Centrifuge crude bioreactor media at 1,962 x g for 5 minutes and pass through a 0.22 µm filter.
   2. Follow up with a perchloric acid cleanup to remove protein and particulate matter: mix the filtered bioreactor media with 0.4 N HClO₄ at a 1:1 ratio and centrifuge at 14,700 x g for 5 min at RT. Collect the clarified media in autosampler vials.
      NOTE: Adjust the injection volume as needed to make the amino acid concentrations fall within the calibration range. Depending on the instrument, the injection volume can be adjusted between 0.1 µL and 10 µL.
   3. Run media samples in triplicate by LC-MS. Use "Process Samples" under the quantitation program along with the method (*.mdb) and calibration file (*.cdb). The method and calibration curve will be automatically applied to the crude media samples by the quantitation application once all the injections are completed.
   4. To export data for analysis in another program (such as a spreadsheet), use the "Yazdır" command and create an *.xps or *.pdf file.