Quantification of Host Cell Proteins

Determine HCPs specific to your process
Obtain the exact amount in all purification steps
Results to be used for removal of immunogenic and proteolytic proteins

Technical details, sample info, experts etc.

Do you want to monitor specific HCPs of concern in your drug substance?

Host cell protein quantification of individual HCPs is important. Why? Because some HCP can influence product safety even at very low level.

Such problematic HCPs may thus cause

Immunogenicity: Induce immune response against drug substance or human protein
Enzymatic activity: Degrade the drug substance or constitu...

Do you want to monitor specific HCPs of concern in your drug substance?

Host cell protein quantification of individual HCPs is important. Why? Because some HCP can influence product safety even at very low level.

Such problematic HCPs may thus cause

Immunogenicity: Induce immune response against drug substance or human protein
Enzymatic activity: Degrade the drug substance or constituents in the drug product
Biological activity or homology to the drug substance

Therefore, we offer quantification of specific host cell protein in your sample – by a targeted and sensitive LC-MS/MS analysis based on Multiple Reaction Monitoring (MRM) methods.

The analysis includes identification of unique signature peptides for the specific protein. For host cell protein quantification, we prefer using the intact reference protein if available. Or alternatively, the synthetic isotope-labeled signature peptides.

Next, the reference protein or the labeled peptides are spiked in known amounts into the samples. This then generates standard curves for linearity and calibration.

Our standard host cell protein quantification service includes

Identification of up to 3 unique signature peptides for HCP quantitation.
Standard curves and linearity assessment for a dilution series of the HCP of concern and quantification standards.
Absolute quantification of host cell proteins of concern by MRM LC-MS/MS.
Reporting of the determined protein amounts and analysis details.

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"The most sensitive, yet robust, Host Cell Protein analysis we’ve seen"

Analyses of key protein features, stability and impurity levels

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"We shortened the purification process with at least 1 year"

Detailed host cell protein overview enables targeted elimination of impurities

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Technical description

Introduction

The absolute quantification of specific protein in complex matrices are based on the selection of specific peptides as signature peptides. These are used for assay development based on Multiple-Reaction-Monitoring (MRM). The project are typically divided into three parts:

Part 1: Feasibility study

The purpose of the feasibility study is to investigate if we can develop an assay for quantification of the specific protein in the matrix. The feasibility study includes:

Planning phase. Here we evaluate potential sample preparation protocols and signature peptides, i.e. specific marker peptides, based on the protein sequences.
Digest and LC-MS/MS analysis. This ensures that specific signature peptides are formed and that this/these peptides do not appear as part of miss-cleaved peptides or modified peptides (oxidized, deamidation etc.). The digest is performed with urea as denaturation buffer and using the proteases Lys-C and trypsin.
Short report. An evaluation of the possibility for Alphalyse to develop an assay that fits the purpose of the project. It also includes an estimation of LOQ and LOD.

Calibration curve for MRM quantification

Standard curve for MRM quantification of specific HCP

Part 2: Assay development

Optimizing of sample preparation and digest of the proteins for mass spectrometric identification of released peptides.
Ordering isotope labeled peptide standards (heavy peptides).
Sample preparation and digest of the protein with addition of heavy peptides for quantitative measurements. Selection of optimal product ions for each peptide. Evaluate quantitative results and select quantitative peptides for quantification assay.
Integration of workflow; sample prep. incl., reduction/alkylation, protein digestion, peptide cleanup, LC-MS/MS analysis and MRM measurement. Quantitative measurements using heavy peptides and external calibration curves on the protein reference standard.
Qualification of quantification assay; demonstration of measurement range, linearity, precision and accuracy.

Part 3: Sample analysis.

Finally, we use the assay to measure real samples.

MRM areas of the corresponding non-isotope peptides

Sample preparation

Sample requirements for host cell protein quantification

Protein quantification by mass spectrometry requires a protein in sufficient amounts to obtain good data. It is also important that samples are prepared in a clean laboratory to avoid contamination with human keratin. To develop MRM quantification assays requires the protein in pure form. Additionally, we need a matrix sample without the protein similar to the matrix of the real samples to be analyzed.

Protein samples can be submitted in liquid or lyophilized.

Purify the protein
1. The chromatographic protein/peptide purity should be >90%.
2. Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can be done on samples containing small amounts of salts, urea or detergent. However, the best result are obtained with low buffer strength in volatile solvents without detergents.
3. Minimum amount ~ 1 mg.
Put the protein sample into microcentrifuge tube
1. Use a Eppendorf Safe Lock tube or similar tubes.
2. Lyophilize or speed vac the protein to a solid sample. This ensures protein stability during shipment.
3. Alternatively, freeze or refrigerate to +4 ^oC for cold shipment of the liquid sample.

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