Expressing antigen binding fragments with high titers in a targeted integration <scp>CHO</scp> host by optimizing expression vector gene copy numbers and position: A case study

Tang, Danming; Gunson, Jane; Tran, Eric; Lam, Cynthia; Shen, Amy; Snedecor, Brad; Barnard, Gavin C.; Misaghi, Shahram

doi:10.1002/btpr.3290

Biotechnology Progress

2022

DOI: 10.1002/btpr.3290

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Expressing antigen binding fragments with high titers in a targeted integration CHO host by optimizing expression vector gene copy numbers and position: A case study

Danming Tang¹,

Jane Gunson²,

Eric Tran³

et al.

Abstract: Antigen binding fragments (Fab) are a promising class of therapeutics as they maintain high potency while having significantly smaller size relative to full-length antibodies. Because Fab molecules are aglycosylated, many expression platforms, including prokaryotic, yeast, and mammalian cells, have been developed for their expression, with Escherichia coli being the most commonly used Fab expression system. In this study, we have examined production of a difficult to express Fab molecule in a targeted integrat… Show more

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Cited by 2 publications

References 28 publications

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Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

Liu,

Chen,

Yin

et al. 2024

Epigenetics

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The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.

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Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

Liu,

Chen,

Yin

et al. 2024

Epigenetics

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A Highly Efficient Workflow for Detecting and Identifying Sequence Variants in Therapeutic Proteins with a High Resolution LC-MS/MS Method

Cadang¹,

Tam²,

Moore³

et al. 2023

Molecules

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Large molecule protein therapeutics have steadily grown and now represent a significant portion of the overall pharmaceutical market. These complex therapies are commonly manufactured using cell culture technology. Sequence variants (SVs) are undesired minor variants that may arise from the cell culture biomanufacturing process that can potentially affect the safety and efficacy of a protein therapeutic. SVs have unintended amino acid substitutions and can come from genetic mutations or translation errors. These SVs can either be detected using genetic screening methods or by mass spectrometry (MS). Recent advances in Next-generation Sequencing (NGS) technology have made genetic testing cheaper, faster, and more convenient compared to time-consuming low-resolution tandem MS and Mascot Error Tolerant Search (ETS)-based workflows which often require ~6 to 8 weeks data turnaround time. However, NGS still cannot detect non-genetic derived SVs while MS analysis can do both. Here, we report a highly efficient Sequence Variant Analysis (SVA) workflow using high-resolution MS and tandem mass spectrometry combined with improved software to greatly reduce the time and resource cost associated with MS SVA workflows. Method development was performed to optimize the high-resolution tandem MS and software score cutoff for both SV identification and quantitation. We discovered that a feature of the Fusion Lumos caused significant relative under-quantitation of low-level peptides and turned it off. A comparison of common Orbitrap platforms showed that similar quantitation values were obtained on a spiked-in sample. With this new workflow, the amount of false positive SVs was decreased by up to 93%, and SVA turnaround time by LC-MS/MS was shortened to 2 weeks, comparable to NGS analysis speed and making LC-MS/MS the top choice for SVA workflow.

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Expressing antigen binding fragments with high titers in a targeted integration CHO host by optimizing expression vector gene copy numbers and position: A case study

Cited by 2 publications

References 28 publications

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

A Highly Efficient Workflow for Detecting and Identifying Sequence Variants in Therapeutic Proteins with a High Resolution LC-MS/MS Method

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