Optimize monoclonal antibody development with icIEF-UV/MS

Monitoring product quality attributes (PQAs) throughout monoclonal antibody (mAb) development is vital to ensuring drug safety and efficacy. During the manufacturing process, heterogeneity can occur due to enzymatic cleavage and chemical posttranslational modifications (PTMs). In addition, process changes can happen during manufacturing which can result in PTMs that impact product quality. By integrating new technology early in the cell line development pipeline, it is possible to improve product quality and manufacturing efficiency.

Dr. Kristen Nields, Senior Scientist at Johnson & Johnson Innovative Medicine, works on preparing stable cell lines for clinical manufacturing. By incorporating the Intabio ZT system from SCIEX into its cell line selection processes, the Cell Line Engineering and Analytical Sciences team at J&J Innovative Medicine was able to identify PTMs earlier in clone selection allowing them to integrate a mitigation strategy to move that clone forward, saving time and resources downstream.

Assessing charge heterogeneity throughout mAb development is essential to understanding the native state of the mAb and the impact of any PTMs including deamidation, C-terminal lysine truncation, glycation, and sialylation.

“C-terminal lysine clipping is a common phenomenon occurring during the bioproduction of mAbs and leads to variable amounts of final process-related charge variants,” explains Nields. “C-terminal lysine will appear as a basic proteoform, which we tend to cleave off during method validation. The only way to do that would be enzymatically, and that can become costly during method validation. Different carboxypeptidase can also provide variability once the method is validated and can create supply chain issues when you have an out-of-spec product.”

Capillary isoelectric focusing (cIEF) offers a high-resolution separation of protein charge variants and is commonly used to monitor product quality attributes. However, how to identify and characterize different PTMs from these charge variant peaks in a single assay has been challenging in the past.

“The Intabio ZT system is having a positive impact on the number of investigations required and the turnaround time for identifying and characterizing PTMs,” says Nields. “The Intabio ZT system directly integrates imaged capillary isoelectric focusing (icIEF) separation with 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.”

Navigate clone selection with ease

Discussing a recent application of the Intabio ZT System, Nields explains how her team was able to identify a genetic variant earlier in the cell line development process. “Recently, in one of our cell line selections for the top 8 clones we tested, there were some basic proteoforms present that typically we would assign as a C-terminal lysine based on the literature. The basic peak ended up being a genetic sequence variant. Because of these findings, we were able to take that clone and find a mitigation strategy to move that clone forward since it was already selected for the manufacturing process.”

Nields continues, “We were able to work with regulatory ahead of filing to say how we would drive the program forward, even though there was a genetic mutation within that clone. This genetic mutation typically would have been caught later in the process and then there would have been a certain amount of work that would need to be done to confirm that it is a genetic mutation with additional studies to support regulatory filing. With the Intabio ZT, we were able to catch it right after cell line selection.”

Dr. Kristen Nields
Johnson & Johnson Innovative Medicine

Process intensification with increasingly diverse therapeutic formats

The color of a protein therapeutic under process development, and any changes to that color require close monitoring and is a potential critical quality attribute. Using a multi-attribute methodology (MAM) to track the cause of color change increases efficiency in this monitoring by enabling rapid hot spot analysis for the continued development of the drug.

“We are increasingly seeing custom colors in new therapeutics being brought to market,” states Nields. “Using the Intabio ZT system with an electron activated dissociation (EAD)-based 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 the strive towards process intensification and trying to get the most protein out of our bioreactors to reduce overhead costs.”

Orthogonal approaches to monoclonal antibody development

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 require a multitude of key characteristics to be tested.

“Using icIEF-UV/MS we can look for the presence of unmodified protein as well as characterize any PTMs that occur in acidic and basic proteoforms,” says Nields. “This analysis is performed alongside peptide mapping, intact mass reduced mass analysis to look at oscillation patterns, and size exclusion chromatography to look at heterodimer purity to help with downstream purification strategies.”

As an initial screen intact protein analysis will 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,” says Nields. “Following an initial screen, we evaluate clonal productivity based on viable cell density, leading to the characterization of the top 24 clones.”

“Early material two comprises a pool of the top 8 clones,” continues Nields. “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."

Enhanced charge heterogeneity monitoring

Looking to the future, the team at Johnson & Johnson Innovative Medicine hopes to incorporate icIEF-UV/MS analysis into other aspects of the cell line development workflow. “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,” explains Nields. “This will help decrease full time equivalent (FTE) work required, which will also help reduce overall costs and speed up the process.”

Nields concludes, “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.”