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Application Guide February 1, 2022 Keywords or phrases: Octet, upstream analytics, titer screening, glycan screening, detection of residual contamination, residual protein A detection Octet® Systems in Bioprocessing: Easy-to-Use and Cost-Effective Tools for Multiple Applications David Apiyo, Sartorius, Fremont, CA Correspondence Email: octet@sartorius.com Abstract Optimization of upstream processes and cell culture conditions are leading to increased production yields of biologics. Along with these improvements, advances in analytical technologies are also required to facilitate titer determination and assessment of critical quality attributes early in the discovery process. This application guide describes the Octet® platform, biosensors, and assay kits that offers intermediate and high throughput capabilities for titer, host cell protein analysis, residual protein A detection and sialic acid content detection and the associated time and cost savings. Find out more: www.sartorius.com

Advances in the optimization of upstream bioprocessing in recent years - primarily, improved cell culturing conditions - - Complete hands-off, walk-away HCP analysis on the -- Octet® RH96 system have led to higher production of target biologics. This leads High precision assays with 5–10% CVs to amplified production of process related attributes as well1. Detection sensitivity as low as 0.5 ng/mL for HCP assays The selection and optimization of bioprocessing therefore, - and 0.1 ng/ml for residual Protein A requires the integration of analytical technologies that facili- No heating or centrifugation steps required for residual tate both titer determination as well as critical quality Protein A analysis attributes assessment of these biologics early on in the dis- covery and optimization stages. One platform that is an Titer and Glycan Screening industry accepted workhorse for multiple analytical applica- tions in bioprocessing is the Octet® platform. The Octet® The Octet® platform is routinely used for titer determina- instrument comes with biosensors and assay kits that offers tion, especially with monoclonal antibodies in both users both intermediate and high throughput capabilities for upstream and downstream bioprocessing. The Octet® titer, host cell protein analysis, residual protein detection and RH96 instrument is capable of analyzing as many as sialic acid content detection. The fluidics-free system allows 96-samples in just two minutes in a simple Dip and Read users to screen for these properties without the need for format, where biosensors pre-coated with Protein A or G or purification, resulting in significant time and cost savings; it is other antibody binding proteins are dipped into IgG sam- estimated that with the Octet® platform, as much as 12X FTE ples for specific binding response measurement. In addition costs can be saved when compared to ELISA in IgG titer. In to titer, the Octet® platform is also compatible for use in gly- addition, the Octet® RH16 and RH96 systems are automa- can screening. For example several groups used this tion-ready, allowing for extended walk-away assay times. method for different glycans screening 2. A common - approach for the screening of glycans on the Octet® system Automation ready platforms suitable for multiple applica- involves the immobilization of sugar-specific lectins onto - the biosensor surface followed by dipping the coated bio- sensor into the sample under certain buffer conditions. - tions (Octet® RH16 and RH96 systems) Real time label-free data acquisition enabling rapid assay optimization Sartorius has recently released a kit (Part No. 18-5135) for Sample plate format allowing for the use of crude and the screening of sialic acid content in biologics which can - non-purified samples be used in combination with titer determination to deter- Combine titer and sialic acid analysis from the same mine sialic content per mg of IgG Figure 1. sample. Analyze 1000 samples in one day on the Octet® RH96 system Titer analysis Sialic acid content vs. titer Time (seconds) Quantitation Quantitation biosensor Crude or Octet® purified Analysis protein Studio Octet® Relative glycan GlyS Kit screening Glycan screening Protein titer Time (seconds) Figure 1: Titer and sialic acid work-flow on the Octet® system. Sialic acid versus normalized titer can be used to select the best conditions for bioprocessing. 2 Binding Binding Sialic acid level

Residual Contaminant Detection Contaminants are any molecules that may elute with the target drug product during purification. They can adversely affect the efficacy and immunoreactivity of the drug prod- uct and should therefore be cleared from the product Optical layer through further purifications. The easy to use BLI technolo- BLI biosensor Biocompatible tip surface matrix gy has comparable throughput to manual ELISA but with better precision in contaminant detection. In addition, the Immobilized molecules platform shows data in real time allowing for a rapid optimi- zation of assays. Metal DAB E Transfer Your ELISA Host Cell Protein (HCP) Detection Anti-FITC: HRP Assay to the Octet® Platform Anti-CHO: The clearance of host cell proteins (HCPs) that co-express fluorescein with biologics is important since high concentrations can adversely affect the safety and efficacy of the biologics. CHO Sartorius’ HCP kit comes with all the reagents required to CHO HCP biosensor convert a manual HCP ELISA assay into a better controlled automated assay with lower variability (Table 1) and where data can be observed in real time. Unlike ELISA, real time Figure 2: Host cell protein (HCP) detection lay-out on the Octet® plat- analysis techniques allow assay developers to monitor form. The biosensors come pre-immobilized with anti-CHO antibody. every step of the assay enabling the fast detection of areas that need further optimization. The kit comes with biosen- sors already coated with a Cygnus capture antibody, a purified antigen for the development of the reference Residual Protein A (RPA) Detection curve and the detection reagents (Figure 2). The Octet® RH96 system can be used to screen > 1000 samples in A common challenge in bioprocessing is the copurifica- one day making it highly suitable for screening for these tion of antibody-based biologics with Protein A leaching process impurities in a high throughput manner. off purification columns. Similar to HCPs, these proteins can affect the efficacy of the drug molecule and need to Assay performance Cygnus 3G Sartorius-Cygnus Anti-CHO be detected and cleared. Sartorius’ residual Protein A ELISA Kit HCP Detection Kit detection kit has a highly simplified workflow compared to traditional methods. The commonly used heat denatur- Time to result 210 min 62 min on Octet® RH96 system ation and sample centrifugation steps which can result 75 min on Octet® RH16 system into high process variability have been removed resulting with Octet® AS instrument into a significantly reduced assay time (Figure 3). This 90 min on Octet® R8 system combined with the throughput and the automation capa- with Octet® AS instrument bilities of the Octet® instruments especially the Octet® Dynamic range 1–100 0.5–200 ng/mL RH96 and the RH16 systems, results into a rapid assay; ng/mL 96-samples can be analyzed in under 2 hours on the Precision (CV) 15–25% 5–10% Octet® RH96 system. The Sartorius kit can be adopted for Table 1: Comparison of overall assay performance for HCP analysis on the detection of leached Protein A from a resin that utiliz- Octet® systems and ELISA for 96 samples. es either recombinant Protein A or MabSelect Sure. 3

1 2 3 Incubate samples with Quantify bound Protein A using Pre-treat samples to RPA Biosensors to capture Protein A Protein A Detection Reagent separate Protein A (Sidekick™ or Octet®) (Octet®) from IgG 10 min 60 min 5 min – 120 min Figure 3: Residual Protein A workflow on the Octet® platform, no heating step is required. 1) Samples are treated with X and no heating step is required. 2) Separated samples are then incubated with RPA biosensors for 60 mins to capture free protein A. 3) A secondary Ab is then used to quantify the concentration of residual protein A from the sample. References 1. Trends in Upstream and Downstream Process Development for Antibody Manufacturing, Gronemeyer P, Ditz R and Strube J, Bioengineering, 1(4):188-212, 2014. 2. High-Throughput Sialylation Measurement Using Lectins on an Octet® Platform for Clone Screening, Jonnalagadda KN, et al., Analytical Methods, 8(39):7193-8, 2016. Germany USA Sartorius Lab Instruments GmbH & Co. KG Sartorius Corporation Otto-Brenner-Strasse 20 565 Johnson Avenue 37079 Goettingen Bohemia, NY 11716 Phone +49 551 308 0 Phone +1 888 OCTET 75 Or +1 650 322 1360 F or further contacts, visit www.sartorius.com/octet-support Specifications subject to change without notice. Copyright Sartorius Lab Instruments GmbH & Co. KG. For Research Use Only. 4017 Rev B