Literature
Quantitation of Host Cell Proteins (HCPs) using LC-MS/MS
by Thomas Kofoed, CEO, and Jette Friis Thirup, Head of Business Development, Alphalyse
Challenges in HCP quantitation
Mass spectrometry (MS)-based analysis enables simultaneous identification and quantitation of multiple host cell proteins (HCPs) in a single experiment. However, quantifying HCPs using MS can be challenging because of
- The very complex sample preparation, including Digestion Variability: Proteins are often digested into peptides (e.g., with trypsin) for MS analysis, but incomplete or non-uniform digestion can lead to inconsistent results.
- Losses During Preparation: Proteins and peptides can be lost during sample preparation steps, affecting quantification accuracy.
- MS Peptide-Dependent Ionization: This variability affects their detection and quantification.
- Matrix Effects: Other components in the sample (e.g., salts, lipids, or other proteins) can suppress or enhance the ionization of specific peptides.
- Instrument Limitations, including Dynamic Range: While MS instruments have a wide dynamic range, they may struggle to accurately quantify highly abundant and very low-abundance proteins in the same sample.
- Signal Interference: Isobaric peptides (those with the same or similar mass) and noise from the background can interfere with accurate quantification.
- Fragmentation Variability: The efficiency of peptide fragmentation during tandem MS (MS/MS) can vary, affecting the identification and quantification of peptides.
- Quantification Challenges Lack of Universal Standards: Unlike nucleic acids, no universal standards exist for all proteins. Absolute quantification often requires preparing synthetic peptides or isotopically labeled standards, which can be costly and time-consuming.
- Peptide Selection: Quantification usually relies on specific peptides ("surrogate peptides") as proxies for the whole protein. The choice of peptide impacts the accuracy of the quantification.
- Normalization Issues: Normalizing data to account for technical variations (e.g., injection volume, sample preparation) can introduce errors if not done correctly.
Despite these challenges, there are several effective strategies for quantifying HCPs.
MS-based HCP quantitation methods
The US Pharmacopeia has recently published a new General Chapter <1132.1>, which describes approaches for accurate HCP quantitation using MS.
These include:
1. Analysis of unknown HCPs
Here, HCP quantitation involves calculating the area under the curve (AUC) for precursor peptide ions (MS) or fragment ions (MS/MS) using one of the following methods:
- Hi3 Method: Estimates protein abundance using MS signals from the three most abundant peptides.
- Sum All Method: Aggregates signals from all identified peptides to estimate the HCP abundance. Alphalyse has been using this method since 2019, referring to it as the SumAll™ algorithm.
2. Targeted HCP analysis
The targeted MRM approach offers high sensitivity and selectivity and is ideal for quantifying specific known HCPs.
You will pre-select surrogate peptides for each monitored HCP and use Stable Isotope Labeled (SIL) Peptides to enhance the HCP quantitation accuracy. Alphalyse can perform targeted HCP analysis to GMP standards.
Common HCP quantitation approaches
USP General Chapter 1132.1 suggests using one of the following three methods for calibration to get an accurate HCP quantitation:
Method 1 - Relative to product protein
This method uses the product protein's peptide signals as a relative quantitation standard.
- Advantages: Simple and reagent-free.
- Limitations: It may not be suitable for MS1-level quantitation due to limited dynamic range and ionization efficiency differences. Also, it is not possible to compare results between projects.
Method 2 - Relative to spiked-in proteins
Here, you spike known concentrations of intact proteins into the sample before digestion. Alphalyse has used this method since 2014, achieving very linear and reproducible results.
- Advantages: Standard proteins undergo the same processing as test samples.
- Limitations: Requires well-characterized protein standards that generate unique peptides.
Method 3 - Relative to spiked-in peptides
In this approach, you spike unlabeled or SIL peptides into the sample as calibration standards.
- Advantages: Easier to obtain pure peptides; SIL peptides improve robustness.
- Limitations: Assumes complete digestion and recovery of peptides. It is time-consuming and costly.
Considerations for HCP quantitation accuracy
MS-based HCP quantitation has limitations, including peptide digestion efficiency and ionization variability. Therefore, you should carefully select and validate your method to achieve accurate and reproducible results.
By understanding the strengths and limitations of each approach, it is possible to tailor your strategy to the specific requirements of your HCP analysis.
Alphalyse has been refining the method since 2014; therefore, we see very low variability in the data reported to clients.
FAQ
Accurate HCP quantitation ensures the safety and efficacy of biopharmaceutical products. HCPs, if not adequately removed, can cause immunogenic reactions, affect product stability, and fail regulatory requirements.
Key challenges include digestion variability, ionization variability, matrix effects, and instrument limitations like dynamic range and signal interference. Each of these factors can affect the accuracy and reproducibility of HCP quantitation.
Incomplete or inconsistent digestion of proteins into peptides can lead to inaccurate quantitation. Losses during preparation can also reduce the accuracy of HCP analysis, impacting the identification and quantification of specific proteins.
Mass spectrometry provides a detailed profile of individual HCPs, including those not detected by ELISA. It offers higher specificity, lower detection limits, and the ability to simultaneously quantify multiple proteins in complex samples.
The USP General Chapter <1132.1> suggests three main methods:
- Relative to product protein.
- Relative to spiked-in proteins.
- Relative to spiked-in peptides. Each method has distinct advantages and limitations depending on the project requirements.
Alphalyse’s SumAll™ algorithm aggregates the signals from all identified peptides, providing a comprehensive quantitation of HCPs. This method ensures greater accuracy by accounting for the contributions of all peptides from each protein.
Strategies include optimizing sample preparation to reduce variability, using stable isotope-labeled standards, carefully selecting surrogate peptides, and employing robust normalization techniques to minimize technical variation.
Surrogate peptides act as proxies for their parent proteins. Selecting peptides with high ionization efficiency and specificity ensures accurate quantitation and reduces interference from other sample components.
Alphalyse employs advanced LC-MS/MS techniques, automated sample preparation, and robust workflows validated under GMP conditions. Their extensive experience with diverse biologics ensures reliable and consistent results.
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.