Fast Analysis of Intact Therapeutic Monoclonal Antibody Trastuzumab

This article, from Issue 13 of the Analytix Reporter, demonstrates a high-resolution and reproducible analysis of mAb trastuzumab in less than 5 minutes using a BIOshell™ A400 Protein C4 column. More importantly the optimized method was able to monitor degraded product created by heat stress studies.

Abstract
Although the majority of small molecules analysed by reversed phase have a mass below 1500 Da, there is a growing need to improve the performance of HPLC columns for the separation of therapeutic proteins and protein drug conjugates. This application note demonstrates a fast and reproducible reversed phase method with high-resolution for the analysis of intact therapeutic monoclonal antibody, trastuzumab. Separation and quantification were achieved using a BIOshell™ A400 Protein C4 column in less than 5 minutes, and more importantly, the optimized method was able to monitor degradation compounds created by heat stress studies.

Introduction
Over the past few years, monoclonal antibodies (mAbs) have become the best-selling drugs in the pharmaceutical market, and in 2018, eight of the top 10 best-selling drugs worldwide were biologics. The global therapeutic monoclonal antibody market was valued at approximate $115 billion in 2018 growing up to $300 billion by 2025. And although as of December 2019, 79 therapeutic mAbs have been approved by the US FDA for sales worldwide, there is a significant potential for the number to increase. HPLC is a well-established method for the analysis of intact mAbs by Size Exclusion and Ion Exchange chromatography. However, technological advancements in the field of Reversed Phase (RP) have made them promising tools for the analysis on intact proteins.

Here, we have demonstrated the suitability of the BIOshell™ A400 Protein C4 column for a fast and high-resolution separation of intact trastuzumab using RP- HPLC. The retention time and area precision of the method were excellent, demonstrating the suitability of the column. Further, we also showcase quantification and robustness that is highly suitable for biopharmaceutical QC applications.

For experimental conditions, results and discussion, refer to the full article.

Conclusion
Analysis of intact mAbs provides a first level of interrogation of size, post-translational modification and heterogeneity. RP-HPLC analysis of mAbs requires large pore sizes, a hydrophobic stationary phase, and appropriate chromatographic methods. In this application note a simple LC-UV method for the analysis of intact trastuzumab was showcased. Using a BIOshell™ A400 Protein C4 column, a high-resolution and rapid separation of intact trastuzumab was developed. Area and RT precision of the method were excellent and showed the reliability of method. The calibration curves with nine standard concentrations of trastuzumab had excellent coefficient of linearity values displaying that the method was quantitative and accurate. The LOD and LOQ for trastuzumab were found to be 0.125 µg/mL and 0.25 µg/mL, respectively, indicating the method was sensitive. In addition, heat stressed studies demonstrated that the BIOshell^™ A400 Protein C4 column was able to monitor degraded mAbs and the method could be used for stability studies.