Accelerating Antibody Discovery by Monitoring Titer and Glycosylation With the Octet Platform

Application Guide September 3, 2021 Keywords or phrases: Octet®, Titer, Glycosylation, Screening, Cell Line Development Cell Line Development: Accelerating Antibody Discovery by Monitoring Titer and Glycosylation With the Octet® Platform Hongshan Li, Fremont, CA Correspondence Email: octet@sartorius.com Abstract Cell line development involves multiple processes. Large numbers of clones are screened and selected on the basis of productivity and stability. Octet® systems have been an established platform for rapid titer of antibody clones to enable quick selection of high-producing clones. Combined with the Octet® Sialic Acid (GlyS) and Octet® Mannose (GlyM) kit assays, cell line development scientists can also screen for the relative terminal sialic acid content in crude or purified samples to better select optimal clones that are both high producers and have desirable sialic acid content. Find out more: www.sartorius.com

Key Features quantification or for kinetic analysis. This technology essen- - tially eliminates any sample preparation beyond an optional Significantly reduce time to develop antibodies by dilution step. - combining titer screening and glycan characterization Save FTE costs and perform more projects using the BLI measures only what’s captured on biosensor chemistries, high-throughput Octet® RH16 or RH96 systems making it specific when measuring in complex matrices such respectively. system as crude supernatant. High-throughput Octet® models can process up to 96 samples simultaneously. Enhance your Cell line development typically includes the screening of confidence in clone selection during clone screening and thousands of clones in an effort to find the few that are process optimization of biotherapeutics by measuring stable, grow as expected, and produce high yields of the protein titer and sialylation in crude cell culture supernatant bioproduct. The time it takes from engineering an optimal using rapid assays on the Octet® RH96 system. cell line to the production of the target biologic can be prohibitive and may differ from molecule to molecule. Titer Measurements While expression level analysis like titer screening is carried out early, other critical quality attributes such as glycan Octet® instruments offer cell line development scientists a characterization are often assessed only later in the platform for the rapid titer of antibody clones that enables a development process due to a lack of appropriate and quick selection of optimal clones, allowing for reduced time high-throughput analytical techniques that can be used to drug development. With ready-to-use biosensor surfaces, to perform quick screens (Figure 1). such as Protein A and G, combined with the automa- tion-ready Octet® RH16 instrument or high-throughput Octet® RH96 instrument, organizations can effect significant FTE cost savings over comparative technologies such as ELISA and HPLC. Moreover, the time to results on the Octet® platform should allow for many more projects run annually than when using either HPLC or ELISA for titer (Table 1). ELISA HPLC Octet® R8 System1 FTE labor costs 15X 3X X Figure 1: Automated Octet® platform for enhanced productivity. Time to results (hrs) 625 1040 52 # projects/year 3 2 40 Commonly used methods for antibody quantitation require Table 1: Comparison of Octet® R8 system, ELISA and HPLC for mAb either specialized instrumentation and skilled personnel screening. The comparison table refers only to the titer segment of the (HPLC) or are time-consuming (ELISA). In contrast, the cell line development work-flow. A project in this case is defined as the titer determination of a total of 10,000 mAb clones. The data in the table Octet® platform (Figure 2) uses Bio-Layer Interferometry assumes an analysis labor time of 0.2 hours, 0.5 hours and 3 hours for 96 (BLI) to detect real-time binding of molecules as a means of samples on the Octet®, HPLC and ELISA platforms respectively.1 Screening of Subcloning of Shake flasks stable pools top stable pools Top clones (Fed-batch) Mini-bioreactor 3 L bioreactor 50 L bioreactor Growth and Growth, titer, Titer screening titer screening and PQ Growth MAYBE PQ MAYBE PQ (glycan, stability, Titer and glycan Titer and Expression screening characterization glycan screening (glycan) screening (glycan) screening etc.) screening ~1000 clones ~200–500 clones ~100–200 clones ~12–48 clones ~3–6 clones characterization Figure 2: Typical clone selection and optimization workflow with typical screens/tests performed at each stage. Expression and titer are screened earlier in the workflow, while product quality (PQ) attributes are assessed later due to screening limitations. 2

Relative Glycan Screening and Titer A combination of the Octet® GlyS and GlyM kits and Octet® ProA Biosensors, or any of the Sartorius quantitation Drug product glycosylation is a critical quality attribute biosensors can be used to perform titer and sialic acid (CQA) due to its potential impact on pharmacokinetics content screening on the same samples using Octet® properties and stability of the product. The Sartorius systems. Octet® Analysis Studio Software allows titer data to Octet® GlyS and GlyM kits enable high-throughput relative be combined with sialic acid and mannose content data screening of sialic acid and manose contents respectively (Figure 4). The ability to view and choose from desired titer in crude and purified samples (Figure 3). There is no need and sialylation and mannose content levels at the same time for sample purification or glycan digestion steps. 1000 provides more in-depth knowledge that facilitates more clones can be screened in just under 10 hours on the informed decisions. The software produces data and reports Octet® RH96 system. with these combined CQAs that can be used directly for further reporting. Octet® GlyS/GlyM Biosensor Binding Selective Secondary pre-immobilized amplification amplification with lectin Pre-immobilized lectin Glycosylated human IgG/Fc-fusion protein Anti-human IgG detection antibody Terminal sialic acid Glycosylated host-cell protein Glycan detection mix Figure 3: Example assay workflow for human IgG or human Fc-fusion proteins. Selective amplification of signal is from the protein of interest and not from host-cell proteins (HCP). Refer to the Octet® GlyS and GlyM user guides for additional protocols and assay guidelines. Sialic acid content vs. titer Titer analysis Time (seconds) Titer/ Quantitation Quantitation biosensor* Crude or Octet® Protein titer purified Analysis protein Studio Mannose content vs. titer Octet® Relative glycan GlyS Kit screening Glycan screening Octet® GlyM Kit Time (seconds) Protein titer Figure 4: Workflow of titer analysis and glycan screening on the Octet® platform. Reference 1. Biolayer Interferometry as an Alternative to HPLC for Measuring Product Concentration in Fermentation Broth, Anurag S. et al., LCGC, Volume 35, Issue 12, 870–877. 3 Binding Binding Mannose level Sialic acid level

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