A Highly Sensitive LC-MS/MS Method for Targeted Quantitation of Lipase Host Cell Proteins in Biotherapeutics

Abstract

Identification and accurate quantitation of host cell proteins (HCPs) in biotherapeutic drugs has become increasingly important due to the negative impact of certain HCPs on the safety, stability, and other product quality of biotherapeutics. Recently, several lipase HCPs have been identified to potentially cause the enzymatic degradation of polysorbate, a widely used excipient in the formulation of biotherapeutics, which can severely impact the stability and product quality of drug products. In this study, we identified three lipase HCPs that were frequently detected in Chinese hamster ovary (CHO) cell cultures using shotgun proteomics, including phospholipase B-like 2 (PLBL2), lipoprotein lipase (LPL), and lysosomal acid lipase (LIPA). A targeted quantitation method for these three lipase HCPs was developed utilizing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with high-resolution multiple-reaction-monitoring (MRMhr) quantitation. The method demonstrated good sensitivity with low limit of quantitation (LLOQ) around 1 ng/mL, and linear dynamic range of three orders of magnitude for the three lipase HCPs. It has been applied for the characterization of process intermediates from various in-house monoclonal antibody (mAb) production. In addition, the method has also been used to evaluate the robustness of clearance for one of the lipase HCPs, PLBL2, under different column purification process conditions.

Ultrasensitive Quantification Method for Understanding Biologically Relevant Concentrations of Host Cell Proteins in Therapeutics

“…Detection of low-abundance HCPs in drug products is a major challenge owing to the extremely large dynamic range barrier between HCPs and drug products. This challenge has been overcome with the recent development of HCP analysis technologies in the past few years. ,,,− ,− The lowest detection limit has been improved to 0.002 ppm with the PDLM method developed by Zhang et al Nevertheless, absolute quantification of HCPs that may potentially have negative effects on drug product quality when present at subppm or low-ppm levels still remains a challenge in the pharmaceutical industry. An LC-MRM-based method developed to overcome this challenge has enabled the detection of three high-risk lipases at low-ppm levels in mAb samples. , However, accurate quantification of HCPs at subppm levels remains difficult to achieve but is desirable for evaluating the potential effects of certain HCPs.…”

“…An LC-MRM-based method developed to overcome this challenge has enabled the detection of three high-risk lipases at low-ppm levels in mAb samples. 19,23 However, accurate quantification of HCPs at subppm levels remains difficult to achieve but is desirable for evaluating the potential effects of certain HCPs. We have shown that subppm levels of lipases or esterase cause PS degradation in drug products during long-term storage.…”

“…Recently, orthogonal mass spectrometry-based approaches have been reported to quantify HCPs in mAbs. , Because mass spectrometry-based methods can enable both the identification and quantification of individual HCP species, they provide comprehensive quantitative information for downstream processing of HCPs. Various combinations of liquid chromatography and mass spectrometry, including two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS), liquid chromatography-parallel reaction monitoring (LC-PRM), , liquid chromatography-multiple reaction monitoring (LC-MRM) targeted quantification, , and ion mobility mass spectrometry coupled with multidimensional liquid chromatography, have been used in the quantification of HCPs. Although the quantification limit has been greatly improved in recent years, quantifying individual HCPs at extremely low levels (<1 ppm) remains a challenge.…”

“…19,20 Because mass spectrometry-based methods can enable both the identification and quantification of individual HCP species, they provide comprehensive quantitative information for downstream processing of HCPs. Various combinations of liquid chromatography and mass spectrometry, including two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS), 21 liquid chromatography-parallel reaction mon-itoring (LC-PRM), 9,22 liquid chromatography-multiple reaction monitoring (LC-MRM) targeted quantification, 19,23 and ion mobility mass spectrometry coupled with multidimensional liquid chromatography, 24 have been used in the quantification of HCPs. Although the quantification limit has been greatly improved in recent years, quantifying individual HCPs at extremely low levels (<1 ppm) remains a challenge.…”

The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation

“…utilized a targeted LC–MS/MS with high-resolution multiple-reaction-monitoring quantitation method for three lipases PLBL2, LPL, and LIPA for the characterization of process intermediates [ 52 ]. This method demonstrated good sensitivity with lower limit of quantitation (LLOQ) around 1 ng/mL (translated to 0.8 to 2.2 ppm for the three lipases in a product matrix), and linear dynamic range of 3 orders of magnitude for the three lipase HCPs [ 52 ]. The LLOQ of those current targeted HCP quantification approaches is still relatively high compared with the low concentration for these active PSDE species to remain active.…”