Anaquant HCP analysis I Protein characterisation I Protein analysis
Introduction & strategy
Many therapeutics are nowadays produced in cells, but during the Downstream process (DSP), Host Cell Proteins (HCPs) could be co-purified with the biotherapeutic. Some known HCPs can affect drug safety or efficiency either because of their high immunogenic power, either due to their ability to degrade the final product (Drug or adjuvant). Consequently, it is mandatory to control the presence of such HCPs in drug substance (DS) or after the last DSP steps.
LC-MS became the orthogonal method to ELISA for Host Cell Protein quantification (HCP quantification) in biotherapeutics. Batch to batch comparison or DSP evaluation through Host Cell Protein analysis are more and more obtained using this analytical method. Broad approaches using Data dependent or Data independent MS acquisition modes (DDA, DIA) correspond to classical methods to obtain great information such as HCPs identities. But their use could require being associated with a validation method to deliver expected and robust outcomes. Targeted mass spectrometry analysis, using selected/multiple reaction monitoring (SRM/MRM) offers capability to measure and quantify a finite set of proteins with high sensitivity, specificity, and reproducibility.
Information regarding the presence of critical Host Cell Proteins in Drug Substance but also at different steps of the DSP can support the regulatory files. In this study, we developed a powerful solution using the READYBEADS technology associated with the SCOUT-MRM method to screen 74 critical HCPs in different matrices. A DS made up of a therapeutic antibody, another DS made up of a therapeutic genetically modified protein and a cell culture harvest from another therapeutic antibody production, all produced in CHO cell line, were screened using the developed HCP targeted SCOUT-MRM method.
Method
74 critical HCPs were extracted from an in-house HCP database and other knowledge obtained in the literature, including the immunogenic PLBL2 lipase, the HTRA1 protease and the BIP proteins. 82 peptides were screened by SCOUT SRM approach and the 82 corresponding heavy internal standard peptides were adsorbed on READYBEADS. Since READYBEADS technology offers valuable reproducibility, relative quantification of Host Cell Proteins signals could be compared between different samples.
Figure 1: HCP Targeted method implementation
In MRM, an upper limit of targeted compounds is reached when the duty cycle becomes detrimental to the signal-to-noise ratio and precision of measurements. To ensure peak definition with at least eight acquisition points per peak, mass spectrometer providers have developed the principle of scheduled acquisition. With this acquisition mode, the compounds are monitored within a detection window centred on their expected retention time (RT). Since the retention time depends on the acquisition process and shifts can occur due to sample matrix effects, it is difficult to implement multiplexed MRM assays based on scheduled acquisition mode for routine analysis all along the Downstream Process due to huge differences between samples after each purification step. That’s why SCOUT-MRM acquisition mode was developed. The multiplexed method was built thanks to RT indexation relative to Scout peptides. SCOUT-MRM relies on the monitoring of a set of transitions (a group) successively triggered by detection of Scout peptides. The number and relative retention time of Scout peptides are carefully chosen to ensure that no more than 120–150 transitions are concurrently monitored in a group. On a peptide level, it corresponds to 20–25 peptides, considering that six transitions are commonly tracked per peptide (endogenous and internal standard). Consequently, potential RT shifts don’t impact detection of a set of transition, therefore the peptides, and in the end, the HCPs. (Figure 2). SCOUTBEADS coated with sentinel molecules called Scout were developed allowing fast, easy and reproducible addition of the Scout peptides.
Figure 2: From the initial DS sample to individual HCP results using SCOUT-MRM analysis
Results
As an example, one peptide of interest (from P14851 PPIA HCP) was detected at 16.5 minutes in the therapeutic antibody DS using scheduled MRM method (Figure 3 Top). Since the initial scheduled MRM method was built on the corresponding matrix, an RT shift occurred (more than 1.5 minutes) during the analysis of the same peptide in the matrix of the therapeutic protein DS. Consequently, the peptide of interest was not properly detected (Figure 3 bottom left). Once SCOUT assay easily designed using SCOUT BEADS was implemented, the matrix effect also occurred on SCOUT peptides RTs. However, the monitoring of the peptide of interest was triggered only after SCOUT peptide detection within an adjusted SCOUT MRM window and then detected properly (Figure 3 bottom right).
Figure 3: Comparison between scheduled MRM method and SCOUT-MRM method for PPIA HCP peptide screening in two different DS produced from CHO cell line. Top, PPIA peptide signal observed in therapeutic mAb DS using scheduled MRM. Bottom Left, PPIA peptide signal observed in therapeutic protein DS using scheduled MRM; chromatographic peak was not complete. Bottom Right, PPIA peptide signal observed in therapeutic protein DS using SCOUT-MRM; chromatographic peak was complete.
SCOUT-MRM method was further validated on therapeutic mAb harvest sample. The sample was treated as presented in Figure 2 and analysed either using scheduled MRM method, either using SCOUT-MRM method. The SCOUT-MRM assay condition offers a substantial recovery gain. As shown in Figure 4, when using a classic scheduled MRM method built from a DS sample matrix, only 51 peptides corresponding to 45 HCPs were detected whereas using SCOUT method, all of them were highlighted (82 peptides corresponding to 74 HCPs).
Figure 4: Comparison of data acquisition on Harvest fluid between a scheduled MRM method built on DS sample matrix (Left) and using SCOUT-SRM method (right).
Concluding remark
SCOUT-MRM allows achieving a robust highly multiplexed MRM assay. The entire SCOUT-MRM solution using HCP Targeted BEADS associated with SCOUT READYBEADS® allows to specifically validate the critical HCPs identification and comparison between different samples and matrices. Thanks to SCOUT-MRM, the analytical method does not need to be developed specifically for each matrix but could be easily adapted whatever the therapeutic drug or the purification step to screen.
ANAQUANT offers the possibility to screen critical HCPs in any samples using a simple robust method that could be easily routine implemented.