Cell and Developability Sciences Group at Johnson & Johnson Innovative Medicine
For this case study the Cell and Developability Sciences group at J&J encountered an unusual split-peak profile in their established icIEF screening method. To further investigate what the split peak was, the team utilized the integrated icIEF-UV/MS workflow on the Intabio ZT system.
Learn how simultaneous pI separation and mass ID of an unknown charge variant enabled the team to focus peptide mapping resources to confirm a high abundance of trisulfide linkage and free thiol quickly.
| Problem | During lead candidate selection for development prioritization, the established icIEF screening method showed an unusual, split main peak in the profile from an unknown cause. |
| Hypothesis | The unidentified split peak could be attributed to trisulfide bonds or free thiol groups. |
| Goal | Determine the source of peak splitting and evaluate the suitability of clone candidates for production. Confirm the source of peak splitting by peptide mapping. |
| Outcome | Established unknowns as trisulfide linkage and free thiols. |
| Impact | An unstable product was halted prior to cell line development entry, saving considerable resources and prioritizing support for a more suitable candidate. |
The Cell and Developability Sciences group at J&J Innovative Medicine has the primary goal of producing high-quality cell lines that will go forward onto manufacturing for clinical drug substances. The group works to perform characterization with new and established technologies to define quality attributes so they can inform process development.
This workflow enables direct correlation of pI shift to intact mass, allowing for mass spec identification of unknown charge heterogeneity profiles in minutes.
In this case, intact mass analysis on the Intabio ZT system indicated that the split peak observed during icIEF could be attributed to trisulfide bonds (via the +33 Da present in the Main Peak A shoulder) and/or free thiol groups or disulfide shuffling (via the +2 Da shift of Main peak B).
To support and confirm the icIEF-UV/MS identification, peptide mapping via LC-MS/MS was performed utilizing collision-induced dissociation (CID) and electron activated dissociation (EAD) on the ZenoTOF 7600 system.
CID fragmentation exhibited an unexpected presence of trisulfide linkage (~20%). However, with EAD, a two pair of dominant peptide fragments [(P1 and P1+32 Da) and (P2 and P2+ 32 Da)] provided confident trisulfide peptide confirmation. The EAD fragmentation patterns demonstrated distinctive linkage patterns which reduced false positives and improved data confidence.
This anomalous linkage could cause product stability issues, reducing its developability. Further analysis by non-reduced peptide mapping confirmed the thiol isoforms.
Gaining more information at earlier stages in the development pipeline is critical to optimizing process and increasing efficiency. The Intabio ZT system enables more information at the intact level, providing detail as to why an unexpected peak was observed during screening. If conventional techniques, like IEX/MS or sample fractionation followed by mass spec, had been utilized, this process could have taken up to 3 months. The cell and developability sciences group was able to complete the work in about 2 weeks instead.
Peptide mapping is a necessary confirmatory step in fully characterizing a CQA. However, it can be a resource and time-intensive workflow, and so typically is not widely employed in the early phases of development. Acquiring mass spec ID on a charge variant in minutes allowed for the team to focus peptide mapping on a more defined target region of the protein and confirm the presence of trisulfide linkage and di-sulfide shuffling.
The comprehensive confirmation that the molecule exhibited complex and unexpected linkage patterns was used to create an early mitigation strategy before the candidate went further into the development pipeline, where mitigation would have been more challenging and resource-intensive.
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Learn how Kristen Nields drives process improvement by implementing an icIEF-UV/MS workflow on the Intabio ZT system into the cell line development toolbox.
These bonds are responsible for the folding, stability and function of proteins, so it is important to understand the correct formation to ensure the biotherapeutic maintains its intended structure and efficacy1.
Mapping disulfide bonds helps identify and correct improper disulfide linkages, which can lead to protein aggregation or misfolding. Regulatory agencies like the FDA and EMA require detailed characterization of disulfide bonds in biotherapeutics. Proper mapping ensures compliance with these regulations1. Mis-paired disulfide bonds can have a significant impact on the development pipeline, including reduced efficacy, increased immunogenicity, stability issues, and regulatory setbacks, with the most critical impact being patient safety.
This helps to optimize production processes and improve overall efficiency2. Typically, liquid chromatography-mass spectrometry (LC-MS) workflows are employed to confirm disulfide scrambling in biotherapeutics. However, traditional LC-MS methods can sometimes be limited in their ability to provide high-confidence mapping of disulfide linkages.
To address these limitations, alternate fragmentation methods, like EAD on the ZenoTOF 7600 system, are often preferred. EAD offers high-confidence mapping by selectively cleaving disulfide bonds, which reduces ambiguities and false positives in identifying disulfide linkages3. This advanced technique provides more accurate and reliable data, which is critical for the detailed characterization required in drug development.
LC-MS workflows can be time-consuming and are typically deployed later in the development process. While this strategy provides critical information needed to develop molecular knowledge that can inform future projects, it would be ideal to perform these analyses earlier.
Utilizing orthogonal techniques like stand-alone cIEF-UV on the BioPhase 880 system for high-throughput screening of clones and then integrated icIEF-UV/MS on the Intabio ZT system for quick identification of anomalies provides information to guide the next analytical steps.
With more targeted information, it makes it easier, and more plausible, to utilize advanced analytical techniques like mass spectrometry for characterization in these early stages of drug development.
References
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