Mass Spectrometry is quickly becoming a very valuable tool in host cell protein analysis. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) detects, identifies, and quantifies individual Host Cell Proteins (HCPs). Mass spectrometry (MS) is an orthogonal method to HCP ELISA that provides in depth data about specific HCPs and improves HCP analysis. For process purification, ELISA can tell you the total host cell protein to drug substance ratio, and MS supplements HCP analysis this data with the identity and quantity of individual problematic host cell proteins. HCP identity is needed to determine their isoelectric points to guide optimization of chromatography conditions to eliminate targeted host cell proteins in drug substances.
Due to advances in mass spectrometer resolution and scanning speed, biopharmaceutical companies can monitor which HCPs are enriched or persist through downstream purification processes, facilitating purification process development and process validation. MS also provides a second level of coverage analysis in addition to 2D PAGE or 2D-DIGE.
The major factor limiting the application of MS in the detection of downstream HCPs is sensitivity and specificity. In most well purified drug substances, HCP content will be composed of numerous individual HCPs that in total are less than 100 ppm. MS discrimination of the drug substances at mg/mL levels from individual HCP at low ppm concentrations is problematic. Cygnus Technologies combines MS analysis with Antibody Affinity Extraction (AAE™) as a sample preparation step to deplete drug substance and concentrate HCPs, making mass spectrometry a powerful method for detection and identification of downstream HCPs.
Cygnus MS Workflow for HCP Analytics
In MS based HCP analysis, each sample type is a unique consideration. In general, samples are enriched for HCPs by AAE, denatured, alkylated, reduced, tryptic digested, and concentrated.
Custom LC-MS methods are developed to the specific host cell line, drug substance, sample matrix and purification process. Dependent on sample type, Data Independent Acquisition (DIA) or Data Dependent Acquisition (DDA) methodology may be used.
DIA methods are well suited for upstream discovery applications to determine the overall HCP profile, whereas DDA methods can be used during later bioprocess purification steps to track problematic HCPs. After method development, samples are run by liquid chromatography mass spectrometry upon a high-resolution accurate mass spectrometer.
MS provides PPM (parts per million), IEP (isoelectric points), and MW (molecular weight) data relative to a drug product, thus, informing bioprocess scientists which HCPs are co-purifying at certain steps and the absolute purity of the drug substance.
By using a broadly reactive polyclonal antibody AAE method as an enrichment step for MS, we can identify more HCPs than by prospective or targeted MS methods alone. Along with Ph.D.-level HCP experts, Cygnus Technologies is home to an advanced and powerful Mass Spectrometer offering comprehensive assay qualification and sample testing services.