Case study - Repeatability

Background

inicialClient requested repeatability determination of our CE-SDS-MW measurements for a representative monoclonal antibody.

 

Challenge

  • Pipetting, small volume viscous liquids
  • Inadequate heating, overheating
  • Avoid method-induced sample fragmentation or aggregation due to pH issues
  • Removal of insoluble species

Solution

Our analysts are dedicated to providing high quality service- strictly adhering to the requirements set forth in good pipetting guidelines. Albeit, the specifications from the pipette vendors and those mentioned in ISO8655-2 notes specify validity only when pipetting water at room temperature, many laboratories suffer analytical variances due to inaccurate volume dosing. Keeping this in mind, our coworkers always optimize and validate the methods at each step of the project.


Incubation of proteins for denaturation and alkylation or reduction is a potential source of method-induced degradation. While protocols suggest the time and desired temperature for incubation, the ramping time, initial temperature and post incubation conditions often lack strict controls. Our team added adequate, well detailed steps to the standard CE-SDS-MW protocol to minimize such variances caused by incomplete denaturation.


At higher pH values, the SDS-protein binding is enhanced. On the other hand, higher pH may cause alterations due to disulfide bridge reshuffling. Our team pays special attention to each protein sample to optimize the pH values for each of these steps.

Results

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Figure 1. Recorded electropherograms of repeated CE-SDS-MW measurements of Humira.

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Table 1. Statistical evaluation of migration time repeatability

Conclusions

Our optimized methods offer excellent relative standard deviation (RSD) values for migration time (0.6%), which is well below the RSD value reported by the vendor of the kit used. These results allowed the client to assess the protein molecular mass more accurately, resulting in valuable advances in the evaluation of size heterogeneity, purity and the manufacturing consistency of their biologics product. While not deemed necessary in this specific client’s case an automated Ferguson plot analysis is also an option to obtain precise molecular mass data.


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