Alphalyse

Alphalyse offers Host Cell Protein (HCP) analysis service by the unique SWATH LC-MS approach. Most importantly, the analysis provides identification and quantification of each individual HCP. Additionally, the analysis also gives you the total HCP content (ng HCP/mg DS) in each sample.

HCPs are the most important process-related impurity in recombinant biopharmaceuticals. In patients, the HCPs may affect drug safety and efficacy and some HCPs may cause adverse effects. Moreover, the drug product stability can be reduced by HCPs with enzymatic activity, such as protease and lipase activity.

We are experts in HCP analysis by mass spectrometry in the following areas:

Identify Host Cell Protein for bioprocess optimization >

• Identification of HCP in each process sample.
• Moreover, physical properties of each HCP (amino acid sequence, MW, pI, hydrophobicity).
• Finally, a comparison of HCPs between process steps and HCP clearance.

Analysis of HCP levels in pre-clinical and clinical batches >

• Identification of HCPs in GMP batches.
• Documentation of HCP clearance for PAT and process validation.
• Absolute quantification of specific HCPs.

Quantification of HCPs of concern by MRM >

• Identification of HCP of potential concern in drug substance.
• Moreover, feature analysis of identified HCPs.
• Monitoring and quantification of specific HCP of concern based on your risk assessment.

Comparison of process related impurities in multiple lots of biosimilar and originator >

• Quantification of individual HCPs in biosimilar and originator.
• Identification of single HCP in biosimilar and originator.
• Finally, a comparison of HCP in biosimilar and originator.

ELISA coverage analysis >

• Evaluation of your ELISA assay.
• Validation of HCP coverage of generic or process-specific ELISA polyclonal antibodies.

Do you want to monitor residual proteins added during manufacturing?

Mass spectrometry can quantify specific proteins that are utilized in the manufacturing process, with a customized process-related impurities analysis. This is especially important in the development of gene therapy products.

Common process residuals include added growth factors, Benzonase® Nuclease, Protein A, enzymes for site-specific PEGylation, aminopeptidase, etc. Downstream purification aims to reduce these process additives in the drug substance, but standard analyses cannot detect trace amounts.

Therefore, Alphalyse offers analysis development for your residual protein of interest, in both final drug substance and process samples with low-to-sub ppm detection limit.

Alphalyse probably offers the most detailed impurity analysis available on the market. Where most other suppliers offer analysis based on ELISA kits or ELISA antibodies, we use advanced SWATH mass spectrometry.

How does ELISA compare to Mass Spectrometry (LC-MS)?

As with an ELISA, LC-MS provides you with the total impurity/HCP content (ng HCP/mg DS) in each sample. But with LC-MS you also get the ID and quantity of each individual HCP. And it is fast – just 8-12 weeks – and without the need for a null-cell line.

Additionally, if you already use ELISA kits or antibodies, we can help you measure their HCP-antibody coverage.

If no commercial ELISA HCP assay exists for your host cell line, mass spectrometry is the solution for detecting and measuring host cell proteins, as well as other process-related impurities.

Applicable to all types of samples and expression systems

The approach can be applied to all types of process samples, from harvest to final drug product, and all types of expression systems, including E. coli, CHO, yeast, HEK, adenovirus, etc. The only requirement is that the host cell proteome must be sequenced and available – in public databases or in your possession.

Send us a request to start a dialogue on how the method can be applied to your samples and expression system.

Identify your proteins by mass spectrometry analysis

Protein identification using mass spectrometry is a powerful tool. You can use it identify or verify a single protein, and also for identification of multiple proteins in complex samples. The analysis works for both in-gel samples or in-solution samples. The service includes a digest of protein material using a protease followed by MS and MS/MS sequencing.

Protein identification is made by matching the obtained data to the public protein database from UniProt. This is the best database, as it contains more than 100 million known protein sequences from thousands of organisms.

Multiple protein ID >
First, we perform an in-gel or in-solution trypsin digest, followed by a peptide analysis by nanoLC-MS/MS. Afterwards, we search your data against a protein sequence database, with more than 100 million protein sequences.

Single protein ID >
We start with an in-gel trypsin digest and peptide analysis by MALDI MS/MS. Then we search your data against a sequence database, with more than 100 million protein amino acid sequences.

Determination of physicochemical properties according to ICH Q6B guidelines

Alphalyse offers a range of analytical services for determination of Physicochemical Properties. They have been designed according to appendix 6.1.2 in the ICH Q6B guidelines Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.

You can choose between following services

Molecular weight by UV-LC ESI-MS >
We determine the exact mw of proteins present in your drug sample.

Extinction coefficient >
UV-spectrophotometry (A280) determination of the extinction coefficient of your drug substance protein.

1d and 2d gel electrophoresis >
We determine the electrophoretic pattern of your drug sample by 1D or 2D SDS PAGE separation.

Liquid chromatographic pattern >
We use UV-HPLC separation to determine the liquid chromatographic pattern of your drug sample.

Structural characterization of antibodies, ADC's and biosimilars

Are you looking for a professional antibody characterization service?

Alphalyse offers a whole range of analytical services for structural antibody characterization of monoclonal antibodies. We perform our tests according to the ICH Q6B guidelines – Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.

Our optimized analytical antibody characterization services are suitable for monoclonal antibodies, antibody-drug-conjugates and also for antibody biosimilars.

You can choose and combine your preferred analysis package from the following services:

Molecular weight of mAb >
We determine the exact molecular weight of both intact and reduced monoclonal antibodies.

Disulfide analysis of mAb >
We determine the number and also the position of disulfide bridges in monoclonal antibodies.

Peptide mapping of mAb >
We confirm the theoretical amino acid sequence of your monoclonal antibodies by peptide mapping.

De-novo sequencing of mAb >
We determine the exact amino acid sequence of your monoclonal antibodies by de-novo sequencing.

Edman sequencing of mAb >
We can sequence up to 30 residues from the N-termini of your antibody by N-terminal Edman degradation.

Antibody-drug conjugates >
We determine the exact distribution of drug-to-antibody ratio (DAR). In addition we find the exact attachment site(s) of the drug molecule.

Structural characterization of biopharmaceuticals according to ICH Q6B guidelines

Are you looking for analytical services for structural protein characterization?

All of our structural protein characterization services are conducted according to appendix 6.1.1 in the ICH Q6B guidelines. (Test Procedures and Acceptance Criteria for Biotechnological/Biological Products). Our protein scientists use their expertise to provide you with high quality characterization of proteins. Furthermore, we use the newest technology and advanced equipment.

Our experts analyze your samples and present the data results in a detailed report. You can then easily use the data in your further studies of the protein.

You can choose between following protein characterization services:

Amino acid composition >
We determine the amino acid composition of your drug substance protein by amino acid analysis.

N-terminal Edman degradation >
We determine up to 25 N-terminal amino acids from your drug substance protein by Edman degradation.

N- and C-terminal sequencing >
We determine of up to 80 N- and C-terminal amino acids in your drug substance protein by MALDI-ISD.

Peptide mapping against sequence >
We confirm the theoretical amino acid sequence of your drug substance by LC-MS/MS peptide mapping.

We can also combine our other analysis services to a make a complete characterization package fitting your needs.

We determine the number and position of glycosylations in your drug substance protein.

Determination of post translational modifications according to ICH Q6B guidelines

Alphalyse offers a number of analytical services for determination of post translational protein modifications according to the ICH Q6B guidelines Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.

You can choose between following services:

Disulfide bridge analysis >
We determine the number and position of disulfide bridges in your drug substance protein.

Glycosylation analysis >
We determine the number and position of glycosylations in your drug substance protein.

Accurate protein concentration determination of specific proteins in purified or complex samples

What is the protein concentration in my sample?

This is a key question to answer in protein projects. The appropriate protein concentration analysis depends very much on the sample complexity. It also depends on if the quantification needs to be absolute in picomoles & micrograms or just relative to other proteins & samples.

To accommodate this need, we offer a range of protein concentration determination services, including:

• Absolute quantification of the purified protein batch.
• Relative quantification of multiple proteins in complex samples.
• Absolute quantification of single, individual proteins in complex samples.

Our protein concentration determination analyses include:

Quantitative Amino Acid Assay (AAA) >
Triplicate analysis ensures a very accurate protein or peptide concentration determination.

Extinction coefficient determination >
Exact quantity of protein determined by amino acid analysis. Calculation of the molar extinction coefficient from dilution series and the protein molecular weight.

Mass spectrometry with quantification by multiple reaction monitoring (MRM) >
Selective quantification of specific proteins in very complex mixtures.

Label-free quantification by LC MS/MS

Triplicate Amino Acid Analysis

for quantification of proteins and peptides (accuracy within +/- 5%)

Did you know that quantitative amino acid analysis can be used to determine the:

• Concentration of a purified protein with known amino acid sequence?
• Total protein amount in a complex sample with unknown proteins?
• Accurate concentration on protein stock solution to calibrate other quantitative analyses, as ELISA assays?

Amino acid analysis is the golden standard analysis for accurate quantitation of proteins and of peptides. As an example, we use the analysis to quantify the total protein amount in a complex sample. Another application is determination of the quantity of purified proteins and peptides.

We can set up an amino acid assay consisting of:

• Triplicate analysis for very accurate protein or peptide concentration determination.
• Comparison of results against your protein or peptide sequence.
• Optional: Combining the amino acid assay with an Extinction Coefficient measurement at 280 nm.

Our specialists analyze your samples with the newest technology and equipment. After completion of the data analysis they collect your results in a report, written in a language easily understood by the non-expert. As a last step, we email the final report to one or more persons of your choice.

for absolute or relative protein quantification

The Alpha-Quant™ assays developed by Alphalyse

• Do you want to quantify specific proteins in a complex mixture?
• Do you not have an ELISA assay or suitable antibody for quantification?

The high sensitivity and specificity of Multiple Reaction Monitoring (MRM) is of great advantage. That is why we use it for selective quantification of specific proteins in very complex mixtures. This technique provides both identification and quantification of the specific analyte. Furthermore, it is complementary to rtPCR, Western blotting and ELISA.

Advantages of Multiple Reaction Monitoring (MRM)

The Alpha-Quant™ assays developed by Alphalyse have several advantages over other protein quantification methods. Advantages compared to methods like HPLC and antibody based ELISA assays include:

• Direct LC MS/MS measurement of analytes. – Not indirect measurement of protein-antibody binding.
• Multiplex analysis. – We can measure multiple peptides/proteins in same LC MS run.
• High reproducibility (5% CV) of relative quantification, when combined with internal standards.

We develop the LC MS/MS analysis specifically to your protein, peptides and project. As we analyze your samples on a fee-for-service basis, please contact us to receive a quote.

Determination of the amino acid sequence of proteins and peptides

Want to confirm the amino acid sequence of a specific protein?

We offer two types of peptide and protein sequencing service: N-terminal protein sequencing service by the Edman procedure and Peptide mapping by mass spectrometry. Use our N-terminal Edman protein sequencing service to determine up to 20 N-terminal amino acid residues from your protein. Our peptide mapping service by mass spectrometry provides detailed information about the full protein sequence. Our results are delivered in 4-8 days in a report that is easy to read and understand.

You can choose from the following Fast Protein sequencing services:

N-terminal Edman degradation >
Get the first 5, 10 or 20 amino acids from the N-terminal.

Peptide mapping against sequence >
Peptide mapping with trypsin and nano LC-MS/MS. Then we search your data against theoretical amino acid sequence.

Need to know more about your protein or peptide?

Maybe you need to know more than 20 amino acids? – We can determine up to 80 amino acids. Have a look at our structural protein characterization services.

Do you want to search against a specific sequence? – Then we offer peptide mapping against sequence.

Protein Characterization Services

Protein Services

Biochemistry & Molecular Biology Services

Biology Services

Tandem Mass Spectrometry Methods Services

Mass Spectrometry Services

Spectroscopy Services

Analytical Chemistry Services

Chemistry and Materials Services

Antibody Services

Structural Biology Services

Host cells used for recombinant protein expression, such as CHO, E. coli and HEK 293, contain thousands of proteins (HCPs and other process-related impurities) that contaminate the purified protein. An HCP assay using ELISA enable fast assessment of the HCP amount in both process samples and the final biopharmaceutical product.

A wide range of commercial impurity/HCP assay kits are available. We offer to analyze your samples, and can also optimize the ELISA protocol to your samples – and provide validation for your process by HCP ELISA coverage analysis.