Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

We have developed a highly sensitive method to detect those low-abundance host cell proteins from the drug product using the protein enrichment beads. This approach help us to find the root cause and the risk associated with the drug product. There are multiple ways to improve the detection limit of HCP analysis.

And targeted methods including, but not limited to Protein A depletion, the limited digestion, the molecular weight cutoff, the immunoprecipitation, the targeted way to detect the host cell protein, including liquid chromatography, multiple reaction monitoring, and the nano liquid chromatography parallel reaction monitoring. The major challenge associated with HCP analysis is the significant dynamic range between antibody drug and host cell proteins. So most the methods aim to reduce amount of antibody and the enriched HCP before LC-MS analysis to decrease the dynamic range between antibody peptides and HCP peptides.

So ProteoMiner technology and limited digestion protocol significantly improved the detection limit of HCP analysis compared to other methods. It's non-biased compared to immunoprecipitation, and can be applied to different drug module compared to Protein A depletion method. This protocol is also simple and straightforward.

The ProteoMiner technology and limited digestion protocol help us identify several HCPs that degrade polysorbate and the fragment antibody drug. In combination with the nano LCMPRM method, the enrichment method can also provide quantitative results to find the biological relevant concentration of problematic host cell proteins. Begin By appropriately diluting the monoclonal antibody drug products for host cell protein enrichment.

To dilute drug products with concentrations above 50 milligrams per milliliter, add deionized water to 15 milligrams of the monoclonal antibody in a two milliliter microcentrifuge tube. For drug products with concentrations below 50 milligrams per milliliter, transfer 20 milligrams of it to a 10 kilodalton centrifugal filter device. Centrifuge the contents at 14, 000G for 25 minutes at room temperature to leave approximately 100 microliters of solution remaining in the tube.

Then, add 350 microliters of 10 millimolar histidine buffer having a pH of six into the filter. Following which, vortex the tube before centrifuging it at 14, 000G for 25 minutes at room temperature. Invert the filter and place it into the sample collection tube.

Centrifuge the sample at 1, 000G for five minutes at room temperature, retrieving the entire solution in the collection tube. Add 200 microliters of 10 millimolar histidine buffer to the filter and pipette up and down to recover the remaining protein sample. Transfer the entire solution into the same collection tube, vortex, and spin down as demonstrated previously.

After measuring the sample concentration by NanoDrop, adjust it to 50 milligrams per milliliter by adding the histidine buffer. Lastly, transfer 300 microliters of the sample into a two milliliter microcentrifuge tube. To begin, remove the top and bottom caps from the spin column of the commercial protein enrichment kit, and retain those for future use.

Place the uncapped spin column in a two milliliter microcentrifuge tube. Centrifuge the setup at 1, 000G and room temperature for 30 to 60 seconds to remove and discard the storage solution. Add 200 microliters of wash buffer to the enrichment beads in the spin column, and mix by pipetting up and down several times.

After centrifuging the spin column as demonstrated previously, discard the collected buffer. Replace the bottom cap, and add 200 microliters of water to the spin column before replacing the top cap. Mix the prepared aqueous bead slurry by pipetting it up and down before transferring 40 microliters of it to the pre-prepared monoclonal antibody drug product sample.

Incubate the tube by rotating it at room temperature for two hours. Next, make holes in a suitable sized frit using a 16 gauge needle, and insert it into the tip end of a 200 microliter tip. Transfer the sample with beads to the prepared tip and place it in a two milliliter microcentrifuge tube with a hole in the top cap.

Centrifuge the tip in the tube to remove the solution. Wash the beads by adding 200 microliters of wash buffer to the tip. Remove the wash buffer by centrifuging the tip in a two milliliter microcentrifuge tube.

Next, wash the beads in the tip with 200 microliters of water and centrifuge as shown previously to remove the water. Now, add 10 microliters of elution buffer to the tip and thoroughly soak the beads in it by pipetting the slurry up and down 10 times. Transfer the tip into a new 0.5 milliliter microcentrifuge tube with a hole in the top cap

Add 1.5 microliters of trypsin solution into the eluent and let it digest overnight at 28 degrees. Then, add 1.5 microliters of 25 milligrams per milliliter of dithiothreitol to the sample, and incubate it at 90 degrees for 20 minutes. Next, incubate the sample with 1.5 microliters of 0.25 molar iodoacetamide at room temperature in the dark for 20 minutes.

After that, add 3.5 microliters of 10%trifluoroacetic acid or TFA and vortex the sample for two minutes. Centrifuge the sample at 14, 400G for 10 minutes at room temperature to precipitate SDC and SLS. Collect the acidified supernatant for desalting.

Add 50 microliters of Buffer B to the GC desalting tip. Then, place the tip in a holder and centrifuge at 1, 000G for three minutes at room temperature. Add 50 microliters of Buffer A to the tip and spin the tip as demonstrated previously.

Now, add the acidified sample to the tip. Centrifuge the tip in the holder at 500G for six minutes at room temperature. Following this, wash the tip by adding 50 microliters of Buffer A to it and spinning it at 500G for three minutes at room temperature.

Elute the peptide from the GC desalting tip by adding 50 microliters of Buffer B and centrifuging the tip in a new tube at 500G for three minutes at room temperature. After that, dry the collected eluent using a vacuum concentrator. Resuspend the dried eluent in 30 microliters of 0.1%formic acid solution.

Measure the ultraviolet absorbance of the peptide mixtures at 214 nanometers using a spectrophotometer. Finally, transfer 10 microliters of the digested sample into a liquid chromatography sample vial, and analyze one microgram of the sample using nano LC-MS/MS. The total ion chromatogram profiles of host cell proteins revealed that compared to direct digestion, most of the major monoclonal antibody peptides were either reduced or eliminated after the demonstrated protein enrichment coupled with limited digestion protocol.