Host Cell Protein Analysis

Soluble SMART Digest (Part no. 60113-101)

(alternative) SMART Digest Mag Beads (Part no. 60109-101-MB)

• Host cell proteins (HCPs) coexpressed during the production of biotherapeutics can affect the safety, efficacy, and stability of the final product.

• Monitoring HCP populations and amounts throughout the production and purification process is an essential part of the overall quality control framework.

• Mass spectrometry (MS) is used as an orthogonal method to enzyme linked immunosorbent assays (ELISA) for the simultaneous identification and quantification of HCPs, particularly for the analysis of downstream processes.

• Improvements in both speed and identification performance that can be implemented for routine analysis to support upstream process development.

“Automated sample preparation using commercially available magnetic beads enables high throughput by reducing the processing time while allowing for high reproducibility and robustness.” - Journal of Pharmaceutical Analysis 11 (2021) 726e731

 Example protocols

Protocol 1

Acetone Precipitation

• 4 times the volume of ice-cold acetone (-20 °C) was added to the sample

• The sample was then centrifuged, the supernatant was discarded, and the protein pellet was washed once with 200 μL of ice-cold acetone.

• After removal of the acetone, the pellet was air-dried on ice for 30 min.

SMART Digest

• The pellet was mixed with ultrapure water to a volume of 50 μL.

• Digestion was carried out as specified in the ThermoFisher SMART digest
  trypsin kit, Soluble (Part no. 60113-101), which included a heat-resistant trypsin and a digest buffer.

• 150 μL of SMART digest buffer and 5 μL of soluble trypsin were added to the 50 μL sample.

• The samples were digested for 40 min at 70 °C on an Eppendorf thermomixer without agitation.

• Subsequently, 0.1 M dithiothreitol was added for a final concentration of about 5 mM and incubated for 30 min at 70 °C while shaking at 300 rpm. After the sample was cooled to room temperature, 0.1 M 2 chloroacetamide (CAA) was added to reach a final concentration of about 10 mM.

• The sample was incubated at room temperature for 10 min while being shaken at 1000 rpm. Finally, the peptides were acidified using formic acid (FA) to approximately pH 2 to quench the digest.

• After digestion, the sample was cleaned up and concentrated by reversed phase SPE

 Protocol 2

Protein A affinity chromatography

• Cells were harvested on day 10 and the supernatants clarified via centrifugation and sequential filtration through 0.45 mm and 0.20 mm filters.

• Purification of the expressed mAb performed using an €AKTA avant 150 chromatography system with a 1 mL HiTrap Protein A HP column.

• Protein concentration is evaluated using an IgG1 standard

SMART Digest

• Samples diluted to 2 mg/mL in water

• Add 15 uL of SMART Digest Mag Beads (Part no. 60109-101-MB) incubate for 45 minutes at 70 °C then cool (on KingFisher)

• Disulfide bond reduction is performed with 10 mM DTT for 30 minutes at 57 °C then alkylation with 20 mM IA in darkness for 30 minutes

• The reaction is quenched with 15.45μL of 100 mM DTT followed by 15.64μL 10% TFA (final concentration 11 mM DTT and 1% TFA)

• Samples are then injected immediately into the LC-MS

 Select publications

• Seidel, J. D., et al. (2024). Development of an optimized LC-MS workflow for host cell protein characterization to support upstream process development. Journal of Proteome Research.

• Strasser, L., et al. (2021). Detection and quantitation of host cell proteins in monoclonal antibody drug products using automated sample preparation and data-independent acquisition LC-MS/MS. Journal of Pharmaceutical Analysis, 11(6), 726–731.