Guide decisions during cell line development with more information at the intact level

Mar 28, 2024 | Biopharma, Blogs, Intabio ZT system, ZenoTOF 7600 system | 0 comments

Read time: 5 minutes

Monitoring product quality attributes (PQAs) throughout monoclonal antibody (mAb) development is vital to ensuring drug safety and efficacy. By adopting orthogonal analytical techniques and integrating new technologies that have the potential to provide more information, it is possible to improve product quality and manufacturing efficiency and make more informed decisions.

Kristen Nields from J&J Innovative Medicines sat down with me for a quick Q&A to discuss strategies to address the unexpected in cell line development.

  • Navigating clone selection with more information up front
  • Probing the cause of color change in a therapeutic
  • Continuous improvements to the cell culture process upstream

We talk about guiding decisions throughout the development pipeline, with information gained from orthogonal methods.  What does that mean for your team?

The cell line development process starts with the transfection of plasmids with the relevant therapeutic targets. Once the cells are transfected, the transient pool material is then investigated to identify an optimal clonal cell line. The complicated biochemistry that occurs in the cell processes requires a multitude of key characteristics to be tested.  Evaluating new technologies can help.

For example, using imaged capillary isoelectric focusing-UV/MS (icIEF-UV/MS) we can look for the presence of unmodified protein and characterize any post-translational modifications (PTMs) that occur in acidic and basic proteoforms.  This analysis is performed alongside peptide mapping, intact mass and reduced mass analysis to look at oscillation patterns and size exclusion chromatography to look at heterodimer purity to help with downstream purification strategies.

How can you better navigate clone selection with the information gained from these orthogonal approaches? 

As an initial screen, intact protein analysis can be performed to ensure that the protein of interest is being produced without any cross-contamination. We know by the sequence the theoretical mass of the protein and in intact protein analysis, we’re looking to ensure that the protein is the one of interest. Following this initial screen, we evaluate clonal productivity based on viable cell density, leading to the characterization of the top 24 clones.

We narrow these down to a pool of eight top clones. From these, we select the clone with optimal bioreactor conditions and product quality, picking a clone with the highest purity via non-reduced and reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS). Peptide mapping aids in detecting contamination, sequence variants and amino acid misincorporation. Genetic, mutation-induced, site-specific misincorporation is diligently screened to ensure the selection of a high-quality clone for further development.

What about surprises?  How can you address the unexpected during early development?

 The Intabio ZT system directly integrates icIEF separation with mass spectrometry on the ZenoTOF 7600 system. This allows users to take an icIEF profile and simultaneously look at all the masses under the peaks so that you can easily identify if a C-terminal lysine is present on the basic peak, or you can look at the acidic proteoform and identify if it is due to glycation. Using this information, you can change your feeding process in the bioreactors, knowing that you might have a higher glycation level in that acidic species.

Recently, in one of our cell line selections for the top eight clones we tested, there were some basic proteoforms present that- based on the literature, we would typically assign as a C-terminal lysine. With the integration of the Intabio ZT system into the workflow, we found that the basic peak ended up being a genetic sequence variant. This information would typically have been identified much later on in the process, but here we were able to catch it right after cell line selection. Because of these findings, we were able to take that clone and find a mitigation strategy to move it forward since it was already selected for the manufacturing process.

You recently presented another use case for the Intabio ZT system related to glycation events. How did this help?

We are increasingly seeing custom colors in new therapeutics being brought to market.  Using the Intabio ZT system with an electron-activated dissociation (EAD)-based multi-attribute methodology (MAM) workflow, we’ve been able to identify advanced glycation end products (AGEs) in the acidic proteoform that have been linked to color changes. We’re now working with our upstream cell culture scientists on ways to improve the cell culture so that we don’t have AGEs in our final product. This ties in with striving toward process intensification and trying to get the most protein out of our bioreactors to reduce overhead costs.

Those are some great examples of how increasing molecular knowledge up front can help guide decisions through the process.  So, what is next for the Intabio ZT system in your group?

 The system becomes very useful for investigations and process development because we can then continue to investigate any changes to charge heterogeneity as we make process improvements.

My hope would be to also use the Intabio ZT system to get site specific so that we can begin to engineer and make knockout cell lines to some PTMs. If we can do a middle-down analysis and look at the site-specific chains and maybe where the PTMs are occurring, then it may be possible to engineer these out.

Looking for more information on this workflow?

You can learn more about the use of the Intabio ZT system and ZenoTOF 7600 system to probe the cause of color change in a biotherapeutic in a series of webinars presented by Kristen Nields and Andy Mahan.

SCIEX released the Intabio ZT system at ASMS 2023.  Visit the Intabio ZT system product page to watch the product video and gain additional insight into how you can better understand the charge heterogeneity profile of your molecule at the intact level.

Additional resources available:
Charge heterogeneity analysis
Intact protein analysis
ADC analysis
Peptide analysis

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Roxana has over 15 years of experience in sales and marketing roles in the MS community. As Senior Global Marketing Manager for protein therapeutics at SCIEX, she specializes in communicating innovations in MS and CE-based workflows to the biopharma community.


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