Sequence variant analysis (SVA) is critical in therapeutic protein development as it ensures the absence of genetic mutations during cell culture. Sequence variant analysis aims to identify and quantify all the variant peptides down to very small percentages of their “wild type”. The functionality and efficacy of antibodies and other therapeutic proteins are significantly influenced by their structure.  

Samsung Biologics, located in South Korea’s global biocluster, is an integrated CDMO service provider offering development and manufacturing services from cell line development to final aseptic fill/finish, as well as lab testing services at every stage for biopharmaceuticals. The company’s expansive facilities specialize in the development and manufacture of biopharmaceuticals including ADCs.

Kyoungmin Lee, Senior Scientist of Structure Analytics, Samsung Biologics, discusses the role of sequence variant analysis in drug development, challenges and risks, and the capabilities and tools needed to accelerate protein sequence variant analysis.

Contract Pharma: What role does sequence variant analysis play in biologics drug development?

Kyoungmin Lee: Sequence variants (SVs) in proteins can affect the quality, safety, and efficacy of biologic drugs, and also challenge standard purification processes. It is crucial to identify SVs within proteins as they can lead to misfolding, aggregation, and fragmentation. These variants typically arise from amino acid substitutions due to genetic mutations during cell line development (CLD) or amino acid misincorporations during upstream processing (USP).

Detecting and quantifying these SVs are complex due to limitations in existing analytical techniques. To address this, Samsung Biologics has developed a high-resolution method that can detect both genetic and non-genetic variants at very low levels within a significantly reduced timeline. This innovative approach has been successfully applied during cell line and upstream process development, lowering the risks of unexpected developmental issues and ensuring higher efficiency with better quality in biologic drug production.

Contract Pharma: What are the challenges associated with the in-process sample analysis of biologics?

Kyoungmin: In-process sample analysis is important because the data allow us to monitor and ensure the process is being operated in a desirable manufacturing process. Since some of the quality test results of in-process samples can affect the decisions made on the subsequent process, it is necessary to complete the in-process sample analysis within a short time and obtain reliable results. 

Sequence variant analysis (SVA) is used as one of the in-process sample analysis methods during CLD or USP. Samsung Biologics can perform SVA rapidly through high-resolution liquid chromatography-mass spectrometry (LC-MS) equipment and MS processing software that applies optimized validator algorithms. The resulting data can support the effective progress of manufacturing processes.

Contract Pharma: What are the critical phases of drug development for sequence variant analysis?

Kyoungmin: SVA can be applied in different stages of drug development including CLD and USP development. At the CLD stage, SVA can help select clones without sequence variants derived from genetic mutation, capturing any risk of unsolved issues during development. At the USP stage, SVA can help find amino acid misincorporations arising from cell culture conditions such as lack of certain supplements and provide valuable insight as to how culture conditions should be optimized, leading to more efficient process development.

Contract Pharma: What capabilities and tools are needed to accelerate protein sequence variant analysis?

Kyoungmin: Conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques often yield low-resolution results, generating a vast array of potential sequence variants – as many as 1,000 in one analysis. This requires extensive and meticulous data analysis, typically performed by skilled analysts, to distinguish genuine variants from false positives caused by misidentification.

To address the limitations of traditional analysis in detecting and quantifying SVs, Samsung Biologics developed a cutting-edge LC-MS/MS methodology. The advanced method incorporates ultra-high-performance liquid chromatography (UHPLC) for rapid separations – coupled with a high-resolution Orbitrap mass spectrometer for detection – and utilizes comprehensive protein characterization software with validator algorithms for detailed analysis. Potential false positives can be intuitively identified through this processing software, allowing experienced analysts to quickly and accurately inspect SVA results.

The synergy of these high-end MS acquisition and data processing technologies allows for more precise and efficient detection of SVs, even at very low levels.

By optimizing the workflow, the timeline for SVA has been reduced from the typical 6-8 weeks to just 1-2 weeks for analyzing two to six samples, significantly accelerating the process and reducing the risk of misidentification. This enhanced technique has been applied effectively during both CLD and USP development phases, including the analysis of clonal pools to identify superior clones and the assessment of media and nutrient feed strategies on the formation of SVs.

Contract Pharma: What are the risks posed by sequence variants? How can the risks be addressed to support product development?

Kyoungmin: DNA transcription into messenger RNA and subsequent protein translation are critical phases where mutations can impact both genetic and protein levels. During CLD, genetic mutations may arise during transfection and DNA replication. These mutations can persist through clone selection, affecting subsequent development activities. 

Furthermore, during USP, multiple mechanisms contribute to the creation of SVs at the protein level. The dense cell culture environment can exacerbate cellular stress, increasing the likelihood of tRNA mispairing or ribosomal inaccuracies and leading to the incorporation of incorrect amino acids during protein synthesis. Similarly, a deficiency of certain key nutrients in the media can prompt the translation machinery to substitute an available amino acid for one that is limited or absent, thereby altering the protein’s amino acid sequence.

Although SVs generally constitute less than 0.1% of biotherapeutic molecules, achieving increased titers and productivity can intensify stress on cell lines, potentially creating production errors. Complex proteins, such as bispecific antibodies, fusion proteins, and other advanced modalities, are particularly susceptible to a higher incidence of SVs. This increased risk of SVs may largely be due to the inclusion of repetitive sequences within flexible linker regions, which are susceptible to duplications or deletions via homologous recombination at the repeated sequences.

Samsung Biologics employs SVA during CLD to ensure the selection of more stable cell lines that produce minimal undesired SVs. In addition, SVA of samples cultured for 14 days in the CLD stage can improve the USP process by identifying potential misincorporations caused by nutrient depletion during cell culture. SVA in the USP process pinpoints exact stages within the manufacturing process where errors might occur, enabling targeted interventions.

Contract Pharma: What are the limitations of traditional analyses in detecting and quantifying sequence variants? How can the limitations be addressed?

Kyoungmin: Proteins with incorrect sequences pose significant challenges in removal through standard purification processes, impacting the quality, safety, and efficacy of biologic drugs. Next-generation sequencing is traditionally used to detect and quantify SVs in cell lines, but it is restricted to identifying mutations at the DNA level and does not extend to critical non-genetic variants.

LC-MS/MS is a powerful tool for detecting and quantifying genetic and non-genetic protein SVs during CLD and USP development. This method identifies SVs by comparing observed amino acid sequences against their theoretical expectations. The acquired LC-MS/MS data is meticulously processed to delineate between wild-type and variant peptides, by comparing retention times and the MS/MS spectra and measuring parts per million errors.

The LC-MS/MS technique developed by Samsung Biologics for SVA offers significant advantages in mitigating risks associated with product development using high-resolution equipment and optimized software. In addition, a distinctive aspect of this method compared with common peptide mapping is the application of multiple proteases to facilitate orthogonal digestions, which enhances the reliability of detecting SVs. While trypsin is typically used to cleave proteins at lysine and arginine residues – yielding positively charged peptides conducive to mass spectrometry detection – its use can also create long peptides with ionization challenges. Introducing additional proteases mitigates this issue by producing multiple data sets, which helps confirm the presence of both real and potential false positive variants.

This streamlined method effectively identifies both single-base mutations during CLD and amino acid misincorporations during both CLD and USP development. By integrating SVA at strategic points within the CLD and process development workflows, Samsung Biologics not only reduces development time and costs but also enhances the quality of the final product.

Learn more at www.samsungbiologics.com