Monitoring specific process-related impurities

During development and manufacturing, residual proteins derived from production, such as Host Cell Proteins (HCPs), growth factors, serums, and processing reagents, must be monitored through each downstream purification step to ensure efficient clearance and purity of the final drug substance. Residuals may cause unwanted biological activity, immunogenicity, or degradation of the therapeutic protein or polysorbate surfactants.

Alphalyse offers development of targeted liquid chromatography mass spectrometry methods (LC-MS). Multiple Reaction Monitoring (MRM) analysis is a highly specific and sensitive LC-MS technique that can target your residual protein(s) of interest in both the final drug product and individual process samples. MRM  enables a low-to-sub ppm detection limit and is advantageous for identifying and quantifying key residuals, even in complex process samples.

Why use LC-MS for process-related impurity analysis?

Document and monitor process-derived residuals

Quantify target proteins throughout the purification process and in the final drug substance

Full validation of assay under GMP

Fully validate your assay according to ICH guidelines for use as a release assay under GMP

Assay targeting polysorbate-degrading HCPs in mAbs

Our standard assay targets the most common lipases and esterases found in monoclonal antibody products

Regulatory authorities demand documentation for the efficiency of downstream purification of specific enzymes and proteins added during process development to ensure patient safety, drug stability, and efficacy.

Mass spectrometry analysis can detect all types of process-related protein residuals, e.g., affinity capture chromatography proteins, growth factors, Benzonase® nuclease, Protein A, enzymes for site-specific PEGylation, aminopeptidase, serum albumin, cytokines, protein-based media components, carrier proteins, and more.

Using LC-MS analysis with Multiple Reaction Monitoring (MRM), unique signature peptides for specific proteins can be identified and quantified even in small amounts. The assay is highly reproducible and, therefore, ideal for batch-to-batch comparison.

The LC-MS assay can be fully validated according to ICH guidelines and performed under GMP conditions as a release assay if needed.

ELISA (enzyme-linked immunosorbent assay) has been the gold standard in the industry for HCP analysis for many years. However, it is restrained by a lack of specificity and a limited possibility to monitor multiple specific proteins in highly complex samples. Developing a process-specific ELISA is time-consuming and typically takes 1 to 1.5 years.

A customized mass spectrometry (LC-MS) assay for MRM analysis can be developed and validated within six months. It may quantify even low amounts of specific proteins utilized during manufacturing.

We can measure up to 20 individual proteins in the same LC-MS run using multiplex analysis. The technique is highly reproducible and provides both the identity and absolute amounts of specific analytes. Furthermore, the method can be validated and qualified according to your specifications.

Polysorbate surfactants are crucial to the shelf-life and efficacy of monoclonal antibody (mAb) products, and their degradants may form particles that are harmful to patients and considered a critical quality attribute outlined by the USP General Chapters <787> and <788>. Consequently, degradation of polysorbates is a major problem in mAb development, resulting in project delays, often in very late stages of project development.

While some host cell proteins (HCPs) – such as proteases that cleave the therapeutic protein – are usually found early on, polysorbate-degrading lipases and esterases can remain undetected in the final drug product until months or years into long-term stability studies – or even after the product has been licensed!

At this stage, modifying the manufacturing process to eliminate these elusive yet dangerous HCPs is costly and time-consuming. Identifying polysorbate-degrading HCPs in a mAb product before they become a problem is the most effective way to reduce their impact on the project timeline.

Video case

Do you experience polysorbate degradation – but don’t know the cause?   

One of our mAb clients struggled with polysorbate degradation, affecting the efficacy and safety of their drug product. The client suspected the problem was a host cell protein – a lipase – which co-purified with the mAb through Protein A purification. But without knowing the identity or characteristics of the culprit, they were unable to remove it from the final drug product.

Case-24: Polysorbate degradation caused by LPL (lipoprotein lipase)

Typical project process

You typically work with
these experts:

Rie Bak Jäpelt, MSc Pharmacy, PhD Analytical Chemistry

Rie Bak Jäpelt

MSc Pharmacy, PhD Analytical Chemistry
Janne Skaarup Crawford, BSc in Cell Biology & Biochemistry

Janne Skaarup Crawford

BSc in Cell Biology & Biochemistry
  • Facilitate meetings

    Project scope

    We like to start with an online meeting to learn more about your project. Based on your needs, you will receive a draft proposal outlining the suggested analyses and expected timeframe. We will develop the MRM LC-MS analysis specifically for your protein and project.

  • Protein sample


    After signing the final project proposal, we will contact you for details about your samples. We will inform you of an estimated report delivery date as soon as we receive your samples.

  • SWATH LC-MS analysis and protein quantification using amino acid analysis


    A project leader will oversee the project and send you status updates by email at regular intervals.

    A typical analysis includes:

    • Identification of up to 3 unique signature peptides for each protein for quantitation.
    • Standard curves and linearity assessment for a dilution series of the quantification standards and, if possible, the residuals in your specific samples.
    • Absolute quantification of the proteins of concern by MRM LC-MS.
    • Reporting of the determined protein amounts and analysis details.
  • 5000 mass spectrometry reports


    You will receive the report by email, the analysis report includes:

    • Objectives, description of analytical procedure, results, and conclusions.
    • List of identified and quantified impurities in each sample.
    • Selected raw data, e.g., excel sheets. Additional raw data may be provided to you upon request.
  • Follow up on reports

    Follow up

    Upon completion of the project, your team is invited to go through the results at an online meeting.

Curious to know more?

Leading edge technologies

Whatever challenge or question you may have, we are here to help you solve it. One of our protein analysis experts will discuss the best analysis approach or method for your project by email or online meeting – without obligation.

Client stories

13 residual biocatalyst

Video: Residual biocatalysis in small molecule API

If your process uses added enzymes, residuals is a significant concern
Analyzing individual A549 HCPs by mass spectrometry

Analysis of individual human residual proteins

US-based C&GT developer: "We can follow the reduction of specific A549 HCP in our adenovirus-based product"
Getting Gag/Gag-Pol ratio to control viral capsid formation

Quantifying lentivirus and residual proteins in one assay

UK C&GT division: "We can now monitor the consistency of our viral capsid formations in lentivirus-based products"
Comparing viral protein quantities in AAV-based products

Comparing viral protein quantities in AAV batches and DS

Gene therapy CMO: "We could compare and document batch consistency and locate any differences in AAV products"
HCP MS analysis for gene therapy products

Analyzing heterogeneous HCP mix from multiple species

Gene therapy developer: "We went from inconsistent, low-coverage ELISAs to reproducible and detailed HCP analysis"

The advantages of LC-MS MRM analysis of residual proteins

  • Fast development of a protein-specific assay

  • Highly sensitive – sub-ppm level

  • Extensive dynamic range – up to 4-5 orders of magnitude

  • Accurate and reproducible quantification in very complex samples

  • Can be fully validated accordingly to ICH guidelines and used as a release assay under GMP

  • Multiplexing for monitoring up to 20 proteins in one assay

What clients say

Testimonial Alphalyse
"At first, I did not think an HCP-MS assay would be reproducible. I was wrong! And now we have used it to compare batch data during 5 years of process development."

CMC Project Lead
Scandinavian mAb developer
Testimonial Alphalyse
"The analysis showed that the new ELISA standard made the calibration slope shift, resulting in a misinterpretation of the total amount of HCPs in the drug sample"

Director QC
EU-based biotech
Testimonial Alphalyse
“I found the group at Alphalyse knowledgeable, easy to work with, and helpful in
planning the study”

VP CMC and Quality
US Biotech
Testimonial Alphalyse
“We found out why our PG13 ELISA kit had such low coverage of our gene therapy’s HCPs”

Chief Scientific Officer
Top10 Pharma Company
Testimonial Alphalyse
“Extremely professional service, high-level quality of the results, and excellent communication”

Program Manager
European Biotech
Testimonial Alphalyse
"Previously, we had no idea which clearance methods would be most efficient. With Alphalyse results, we could intelligently remove the HCPs"

Senior Manager, Process Development
Clinical-stage biotech company
Testimonial Alphalyse
"The mass spec results were extremely detailed and almost identical between replicates and can probably replace ELISA as the HCP analysis approach for our future C&GT projects"

Chief Scientific Officer
European biopharma company
Testimonial Alphalyse
"With the Gag to Gag-Pol ratio determined, we can monitor the consistency of viral capsid formations in our lentivirus-based product"

Head of CMC, C&GT division
UK pharma company
Testimonial Alphalyse
"We now use a mass spectrometry assay to follow the reduction of specific A549 HCPs in our downstream process"

Sr. Research Associate
US-based C&GTs developer
Testimonial Alphalyse
"Honestly, we were astonished to see the mass spec results: The detailed report enables us to optimize our CMC process and remove specific HCPs if we find it relevant."

Director, Regulatory CMC
Oncology-focused biopharmaceutical company
Testimonial Alphalyse
"We didn't want surprises in Phase 2 or 3, so we supplemented the HCP ELISA data with an orthogonal method. We got the results within a few weeks and could quickly implement them in our development process."

VP, Process Development and Manufacturing
Drug discovery company
Testimonial Alphalyse
"Alphalyse developed an LC-MS method for determining product-related impurities in our product. It is the most sensitive yet robust HCP analysis we've seen"

Sr. Director, Pharmaceutical Development
US biotech company
Testimonial Alphalyse
"We hoped our biosimilar product was cleaner, more stable, and safer than the originator. However, the results were so favorable that not only can we use them as documentation for regulatory filing, but also as part of our marketing"

Head of Analytical Development
Developer of a mAb-biosimilar
Testimonial Alphalyse
"Thanks to the ELISA-MS data, we knew which kit to use for the setup and validation of our HCP ELISA assay! Furthermore, we knew exactly which individual HCPs were covered by
the ELISA antibodies"

Director CMC
US Biotech
Testimonial Alphalyse
"Based on the data provided by Alphalyse, we settled on a kit that rovided both excellent coverage of the HCPs overall and the individual HCPs of concern"

Senior CMC specialist

US-based Biotech
Aicuris logo
"Alphalyse provided a very well-designed and executed HCP analysis, fruitful technical discussions, and flexibility in terms of writing the report."

Thore Schmedt, Associate Director
AiCuris Anti-infective Cures AG, Germany
SSI logo
"Alphalyse's HCP analysis saved us the development of an ELISA assay that may not have worked anyway. The HCP team explained test results competently and was very open to discussing the method capabilities."

Max Kristiansen, MSc, Special Consultant Assay Development
Statens Serum Institut (SSI), Denmark
Targovax logo
"They handled the project professionally and rapidly, and the report was very well written, clearly explaining the findings."

Kristiina Hyvärinen, Director QC, viral products
Targovax ASA, Finland
Y-mAbs logo
"We enjoy collaborating with Alphalyse as part of our optimization of manufacturing processes. Not only do we gain access to their hands, but we also get to pick their brains for mass spectrometry knowledge."

Torben Lund-Hansen, PhD, SVP
Head of Technical Operations

Y-mAbs Therapeutics Inc., USA
Savara logo
"Using the Alphalyse LC-MS/MS coverage method in HCP ELISA selection, we estimate a savings of approximately $1M and, likely, one year of development time."

Lars Skriver
Senior Science Officer

SAVARA Aps, Denmark

Knowledge center

How can HCP quantitation using LC-MS be validated?

We can fully validate our MRM method as a quantitative method for the specific impurities and product according to ICH guidelines. Reproducible quantitation of HCPs in the samples is obtained using a robust, automated sample preparation method and adding intact protein standards for normalizing the data. SCIEX services the LC-MS system for installation and operational qualifications (IQ, OQ), and our laboratory procedures are meticulously set up and audited for regulatory compliance with GMP requirements.  

Regulatory compliance can be complex, time-consuming, and costly. Alphalyse offers you the option of saving time and resources by contracting our expert team and top-of-the-line instrumentation for validated analyses.

Which database do you use to search for peptide information? Are there any GMP compliance issues with using protein databases?

We typically pull databases and proteomes from UniProtKB. In addition, we can use your custom host cell strain database if available. Several databases are often combined to ensure the inclusion of all potential proteins. Since we rely on publicly available protein databases, no GMP compliance issues exist.

How do you calculate impurity results in ppm when using LC-MS?

We know the exact amount of each of the seven intact protein standards spiked into the sample of known volume and protein concentration. Therefore, we can use the median response curve of the protein standard to calculate the ppm amount of each host cell protein from its total mass spec signal intensity (sum of all peptides).

Talk to us

Whatever protein-related challenge or question you may have, we would love to help. Our experts can help you decide on the best analytical approach for your project by email or online meeting - providing advice without obligation.

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