Cell and Developability Sciences at Johnson & Johnson Innovative Medicine
Gaining more information upfront about a therapeutic can reduce risk and late-stage failures by providing critical insights on post-translational modifications (PTMs), or the effects of growth conditions on your cell line.
For this case study, the team encountered multiple CMC programs where a dark brown color at the viral inactivation stage was observed and which became darker at higher concentrations. Learn how the Intabio ZT system was utilized to rapidly uncover the underlying cause.
| Problem | Multiple CMC programs started to observe a dark brown color at viral inactivation stages, which became darker at higher concentrations. |
| Hypothesis | The reaction occurs in the bioreactor during the upstream process and is present throughout the downstream purification process. |
| Goal | Characterize the components that contribute color and understand if the process can be optimized to reduce those components. |
| Outcome | Advanced glycation events were responsible for the color change. |
| Impact | This information was used to formulate a mitigation strategy to minimize business impact and was shared with colleagues in both upstream and downstream teams to optimize processes for future products. |
The mission of this group at Johnson and Johnson Innovative Medicine is to produce high-quality cell lines that will go forward into manufacturing for clinical drug substances. They start to define the quality attributes so that they can inform process development. A key piece of information is the charge heterogeneity of the molecule and this data is provided to process development teams to track molecular integrity and stability.
Understanding how process changes impact product quality is crucial. By gathering detailed information early on, we can reduce risks and late-stage failures, particularly by examining post-translational modifications (PTMs) and the effects of growth conditions on cell lines. This helps in obtaining targeted answers or guiding the next steps in analytical strategies. A key piece of information is the charge heterogeneity of molecules, which helps track molecular integrity and stability.
In this case study, the team observed a dark brown color at viral inactivation stages, which intensified at higher concentrations. They hypothesized that this reaction occurred in the bioreactor during the upstream process and persisted through downstream purification.
To test this hypothesis, cultures were harvested at specific time points (days 5, 7, 11, 13, and 16). The icIEF-UV profile from the Intabio ZT system showed a shift in acidic and basic peaks after day 7.
The icIEF-UV/MS workflow, integrated with the ZenoTOF 7600 system, provided mass identification of PTMs associated with these shifts.1
Analysis of the acidic isoforms revealed that the acidic shift doubled after day 7. This shift was attributed to increased glycation, advanced glycation events, and other PTMs. For basic isoforms, proline amidation was the primary PTM observed in later harvest days. Traditional charge variant analysis methods could take months to identify the cause of such shifts. However, direct integration with mass spectrometry on the Intabio ZT system allowed the team to quickly determine that glycation and advanced glycation events increased after day 7. Later harvest days showed levels of AGEs that could contribute to the observed color change.
While peptide mapping is necessary for full confirmation of PQAs, it is time-consuming. In this case, the team was able to use the information gained in just minutes from the Intabio ZT system to guide where to focus peptide mapping efforts. Peptide mapping on the ZenoTOF 7600 system confirmed that AGEs were responsible for the discoloration and that harvest day impacts acidic isoforms, glycation, and AGEs.2 This enabled the team to develop a mitigation strategy and collaborate with upstream teams, regulatory, and clinical groups for future filings.
Hear how Hirsh Nanda achieved antibody sequencing using the Intabio ZT system.
Discover how Andrew Mahan defined PQAs linked to color change in biotherapeutics during process development.
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.
Accelerating cell line development timelines often involves assuming more risk. To do this effectively and avoid costly consequences, it's crucial to gather detailed information early in the drug development pipeline. This requires in-depth and rapid characterization of protein therapeutics across multiple modalities.
During early development, we focus on clone characterization and verifying critical quality attributes (CQAs). These steps are essential for increasing molecular knowledge of the therapeutic being manufactured. A Biophorum study surveyed 27 scientists to understand cell development timelines and identify areas for acceleration. Clone characterization showed the most variability, ranging from 8 to over 20 weeks.3
Several assays are critical at this stage:
Cell line purity assessment via CE-SDS
Charge variant monitoring via capillary isoelectric focusing
Charge variant identification via integrated icIEF-UV/MS workflows
Implementing and streamlining these workflows earlier in the pipeline provides faster insights into CQAs, enabling better decision-making and pushing the best clone forward.
Identification of PTMs at the intact protein level can increase biopharma efficiency. For this purpose, charge variant analysis is a key assay. Charge variants arise from protein modifications during development, growth, or storage. PTMs like glycosylation, oxidation, deamidation, glycation, and C-terminal lysine residues impact the molecule's efficacy and safety, making them critical CQAs. Capillary isoelectric focusing (cIEF) is the preferred method for charge variant analysis, monitoring acidic or basic shifts in the molecule's pI. However, it is challenging to confirm proteoform identity associated with these shifts. Traditional workflows using ion-exchange chromatography (IEX)- MS or fractionation technologies are time-consuming, taking weeks or months.
The Intabio ZT system integrates icIEF separation, UV detection and mass spec identification on a single platform, reducing charge variant identification time by 95% compared to IEX-MS. This platform provides comprehensive charge variant identification in minutes, not weeks, and allows for the correlation of pI and mass shift to peak identification, confirming common and challenging PTMs like deamidation, sialylated glycans, succinimide, and glycation.
References
SCIEX offers analytical solutions that support the drug development pipeline, from cell line development to commercialization.
Set the pace with confidence. Cell line monitoring and analysis solutions verify the identity, purity, and stability of lead candidates with high throughput strategies that are ready for automation.
Achieve new heights in PTM analysis with confidence and speed. Define CQAs and streamline processes from early to late-stage development with in-depth peptide mapping solutions for next-generation protein therapeutics and standard mAbs.
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