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During the clone selection process for the production of therapeutic antibodies and proteins, it is often necessary to screen for potential unintended amino acid substitutions, (also called sequence variants - SV), process-related impurities and product-related impurities before making the decision to proceed with one clone into production.

It is becoming more and more apparent that mutations can occur during storage and sequence variants are not rare in many clone collections.  Since the mutated clones may have some growth advantage, even minor amounts of SV (e.g. <0.1%) during production the presence of SV may lead to altered bioactivity and higher immunogenicity.

 

Detection and identification of low level SVs is still very challenging. To facilitate high-confidence SV screening, we have developed dedicated processes and proprietary software tools for screening SV during the  clone selection process. With our SV technology we are able to identify sequence variants at levels as low as 0.01% and our screening covers the entire mAb/protein sequence.

 

SV analysis service based on our SV-technology involves the following features:

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  • 100% sequence coverage based on triple digestions.

  • A systematic approach based on a highly reproducible peptide mapping method and ultra-sensitive Nano LC-MS/MS platforms.

  • A dedicated SVfinder software that can identify SV with any amino acid substitution 

  • Capacity to detect and identify SV at 0.01% of the protein level.

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As part of this service, we will also identify, characterize and quantify process and product-related impurities, including:

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  • Host cell proteins

  • The truncated protein products

  • Protein aggregations

  • Modified forms of protein products

  • Residual impurities from manufacturing processes

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The procedure for impurity analysis depends on the nature of the impurity and  the specific requirements of the client.  We often need to design an optimized process by using various separation and characterization techniques, such as chromatography, electrophoresis and mass spectrometry.

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Process:

1.    Digestion: In order to ensure 100% sequence coverage we use trypsin, chymotrypsin & Lys-C as three default enzymes for the digestion of IgG mAb. The use of triple digestions also increases the confidence of the analysis.

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2.    Peptide mapping with HPLC-UV/MS/MS: Since the detection of SV in this step depends on a highly reproducible chromatogram, an analytic C18 column (2.1 mm ID) is used for HPLC separation. For each of the 3 digested samples, visual comparisons of the separation profiles from different clones are carried out to check for any differences.

Any detectable differences will be analyzed based on MS and MS/MS data from the corresponding chromatographic peak. Based on our experience, this procedure can identify SV at protein levels of >3% of total.

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3.    Nano LC-ESI-MS/MS and Automated SV Identification.

Each digested sample is also subjected to Nano LC-ESI-MS/MS analysis and  MS/MS data is then searched using our proprietary SVfinder software to identify all  potential sequence variants. All potential SVs are analysed and validated manually before reporting to our clients . In this way, a sequence variant at levels as low as  0.01% of the total protein amount in the sample can be identified.

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4.    Quantification of SV

After we have identified a SV, if that SV is detectable in HPLC-UV chromatography as a distinct peak, we will calculate the peak area of SV peptide and that of the original peptide to determine the ratio. If a SV peptide does not have a distinct chromatographic peak, we use extracted ion chromatograms (EIC) from SV peptides and the original peptide to calculate the ratio of SV.

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Service Terms:

  • Sample Requirements: Due to a higher amount of sample usage in HPLC-UV based peptide mapping and 3 digestions, we need > 200uµg of each mAb clone or protein sample . The sample should be of >98% purity, and should not contain any antibody contamination.

  • Turn-around Time: 15 -20 days.

  • Shipping: To minimizing modifications of protein in the sample, a low-temperature shipping is required.

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