Case study

Identifying a polysorbate-degrading lipase in mAbs

Polysorbate-degrading enzymes can affect the stability of mAb products even at trace levels undetectable by standard assays.

Degradation of polysorbates is a significant challenge to the stability and efficacy of monoclonal antibody (mAb) products. Host cell proteins (HCPs) such as lipoprotein lipase (LPL) may co-purify with the therapeutic protein and exhibit significant enzymatic activity even at sub-ppm levels.

One of our mAb clients struggled with Polysorbate 20 degradation in their long-term stability studies. The client suspected the problem was a Host Cell Protein which co-purified with their drug protein at trace levels. They had identified Lipoprotein Lipase (LPL) in early reference samples but could not detect it in the final drug substance.

The client tried everything to identify the cause of polysorbate degradation, including chemically inhibiting potential lipases and developing a process-specific ELISA. However, the ELISA failed to detect LPL in the final substance samples, likely due to LPL binding with the therapeutic protein.

LPL molecule in complex with GPIHBP1.

Lipoprotein lipase

Lipoprotein lipase (LPL) is a Host Cell Protein that we frequently identify in process samples for CHO-produced mAbs. It is an essential enzyme for fat metabolism in mammals, and under the right circumstances, it is also quick to cleave the polysorbate surfactants in mAb products. However, the effect of LPL is not always present, even if the sample contains this HCP, due to the propensity of LPL to unfold in its catalytic domain. In vivo, it forms a complex with a lipoprotein-binding protein (GPIHBP1) to maintain its maximum enzymatic activity. When LPL is not bound to GPIHBP1, its enzymatic activity is low.

The predisposition of LPL to bind with other proteins means that it can turn into a 'hitchhiker' HCP in biologics: Forming a complex with the therapeutic protein, it hitches a ride through the polishing steps to the final product, even bypassing Protein A purification.

Additionally, some mAb proteins bind to LPL so that its catalytic domain is activated. This binding enables LPL to degrade polysorbate (PS) rapidly – and unfortunately, it only requires trace amounts of enzymatically active LPL for catastrophic polysorbate degradation.

Since it is common to use polysorbate to prevent protein aggregation and particle formation in biologics, polysorbate degradation impacts drug efficacy and may cause adverse effects in patients.

For patient safety, developers should reduce the levels of PS-degrading HCPs such as LPL as much as possible. If PS degradation is observed, the cause of degradation must be identified and the purification process optimized using highly sensitive methods, such as LC-MS.

LC-MS MRM Analysis

Based on information from the BioPhorum HCP database and our own database with information on 850,000+ detected HCPs from 2,000+ client samples over 20+ years of protein analysis, we have identified the most common residual polysorbate-degrading enzymes.

To identity the enzymatic HCP causing our client's problem, we performed a quantitative LC-MS MRM analysis targeting 8 specific HCPs in process samples from various steps in the purification process.

Conventional assays such as ELISA can struggle to detect HCPs that form complexes with the drug protein and are also challenged by the abundance of drug proteins masking trace HCPs. Our LC-MS lipase assay, on the other hand, can easily handle samples from the production cell line.

Using a native digest method, the intact drug substance is precipitated while protein impurities are digested into peptides and run through the mass spectrometer at a higher concentration. This method increases peptide identification and lowers the LLOD to 0.1 ppm, enabling detection of harmful HCPs even at trace levels.

A mass spectrometry diagram.
1 quantifier and 1 qualifier peptide for each of the 8 HCPs LLOQ is 0.1 ppm

The analysis identified peptides originating from LPL in all samples.

A list the quantities of 8 polysorbate-degrading enzymes. LPL is seen in all DS batches.
The assay detects Lipoprotein lipase in all DS batches.

The client now uses our MRM lipase assay for process optimization and controlling the levels of specific HCPs of concern, as well as for proactively identifying and addressing polysorbate-degrading HCPs in new projects.

Learn how you can also identify and monitor specific HCPs, e.g., lipases and esterases, in your product.



[1]          Fong et al: “GPIHBP1 and Plasma Triglyceride Metabolism“, Trends in Endocrinology & Metabolism, 2016

[2]          Voss et al: “Mutations in lipoprotein lipase that block binding to the endothelial cell transporter GPIHBP1“, PNAS, 2011

[3]          Arora et al: “Structure of lipoprotein lipase in complex with GPIHBP1“, PNAS, 2019

[4]          Young et al: “GPIHBP1 and Lipoprotein Lipase, Partners in Plasma Triglyceride Metabolism“, Cell Metabolism, 2019


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