A 蛋白质序列的变异分析SVA (Sequence Variant Analysis )

一般应用于生产抗体蛋白药之前，对表达细胞株的筛选。可检测低至0.1%的突变。

B 蛋白质突变点检测 PMI (Protein Mutation Identification)

一般应用于生物工程中的酶等未知蛋白的序列分析或者表达蛋白中的突变氨基酸检测，可测出100%准确的蛋白氨基酸序列。

During 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) before making the decision to proceed one clone into production.

It becomes more and more clear that mutation can occur during storage and sequence variable is not rare in many clone collections.  Since the mutated clones may have some growth advantage, even minor amounts of SV (e.g. >0.1%) in a production clone may lead to altered bioactivity and higher immunogenicity.

Detection and identification of low level SVs are still very challenging. To have a high-confident SV screening, we have developed dedicated process and proprietary software tools for screening SV in clone selection process. With our SV technology we are able to identify sequence variants of with ratio as low as 0.01% and our screening cover the entire mAb/protein sequence.

SVA service based on our SV-technology contains the following features:

1.    100% sequence coverage based on triple digestions.

2.    A systematic approach based on highly reproducible peptide mapping method and ultra-sensitive NanoLC-MS/MS platforms.

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

4.    Capable to detect and identify SV at 0.01% of the protein level. 

Process:

1.    Digestion

In insure 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.

2.    Peptide mapping with HPLC-UV/MS/MS

Since the detection of SV in this step depends on a high 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 difference.

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 with protein level > 3% of total.

3.    NanoLC-ESI-MS/MS and Automated SV Identification.

Each digested sample is also subjected to NanoLC-ESI-MS/MS analysis, and  MS/MS data is then searched by our proprietary SVfinder software to identify all  potential sequence variants. All potencial SVs are analysed and validated manually before reporting to our cliens . In this way, a sequence vriant 0.01% of total protein level can be identified.

4.    Quantitation of SV

After we have identified a SV, if that SV is visible in HPLC-UV chromatograph as a distinct peak, we’ll calculate the peak area of SV peptide and that of the original peptide and to determine the ratio. If a SV peptide does not have a distinct chromatographic peak, we’ll use extracted ion chromatogram (EIC) from SV peptide and the original peptide to calculate the ratio of SV.

Service Terms:

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

2.    Turn-around Time: 4 weeks.

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

网址：www.prottech.com.cn  

电话：0512-62956171