「Quality control of biotherapeutics using Octet systems」

Quality control of biotherapeutics using Octet systems Bio-Layer Interferometry (BLI) has been rapidly adopted as an important analytical tool in laboratories that work with biological molecules, either as drug products, vaccines, or diagnostic reagents. Widely-used and accepted in research and assay development, BLI assays are now also utilized for quality control (QC) in biologics development workflows for ligand binding and product quantitation during lot release and in-process testing. Octet® systems combine real-time binding analysis with a • High Precision Streptavidin 2.0 biosensors (SAX2) qualified for simple Dip and Read™ approach for rapid assay and method seamless use across multiple lots of biosensors development. For example, the 8-channel Octet RED96e system • Support service for customer-run software validation quantitates 96 mAb samples in only 30 minutes, compared to >6 hours by ELISA1 or as long as 10 hours via HPLC, resulting in Is the Octet platform recommended for use significant project cost and time savings. Optimizing a generic potency assay takes approximately half the time on Octet systems in regulated environments? as it would with SPR or on manual ELISA. Regulatory bodies such as the FDA, EMEA, NMPA and others do not typically recommend or refer to specific product, brand, Octet systems also offer several other advantages over or vendors in their guidance on analytical technologies. While comparative analytical technologies. The microplate-based there may be references to certain technologies in some USP sample format is ideal for Design of Experiments (DoEs), enabling chapters, these are references based on general concepts rapid, high-throughput method optimization. Disposable, ready- around the application in question. Label-free methods such as to-use or customizable biosensors are simply dipped into samples BLI and SPR are simplified immunoassays based on the ELISA- for analysis – no priming needed. This lack of microfluidics results type binding assay principle, so their use for measurement of in a more robust and flexible platform that can also accommodate potency or product quantity is based on decades of established direct analysis of crude samples. and accepted measurement principles. Binding kinetics Is the Octet platform GxP ready? constant measurements using label-free methods are also well established, and BLI technology has been demonstrated All Octet instrument models are GxP ready. ForteBio offers GxP through use in thousands of laboratories world-wide and in users an assortment of products and services that ensure full peer-reviewed publications. In fact, best practice in the drug confidence in data integrity. They include: development industry employs orthogonal or alternative platforms that are fit for purpose. The key factor here is that • 21 CFR Part 11-compliant software that can be used on all Octet the sponsor sets appropriate acceptance criteria and that the models including Octet K2, RED96e, RED384, QKe and HTX technology in use enables the development of suitable methods • Instrument Installation and Operational Qualification (IQOQ) Kits that are robust and meet the criteria for quality control for the • Performance Qualification (PQ) Kit validated for the Octet specific product. RED96e instrument

Which Octet biosensors are suitable for What if I have a different Octet model in QC methods? development than the one in QC – is Octet The selection of biosensor surface chemistry depends on data comparable? the application. All Octet biosensors can be used to develop Analytical method changes are a normal process in the product QC methods. However, during assay method development, life cycle and can be triggered by a variety of factors such as repeatability, intermediate precision and reproducibility studies the need to better understand product quality or to replace that include biosensor lot to lot assessment should be done legacy methods, and other reasons. In any case, regulatory to determine assay robustness in line with recommendations bodies require that bridging studies be done when a method from the relevant regulatory bodies. High Precision Streptavidin change is necessitated and that the new method be validated. 2.0 biosensors (SAX2) (Figure 1), intended for use with Although bridging studies are a requirement, the data obtained biotinylated ligands, have been developed to ensure minimal on any Octet system – from the Octet HTX to the RED384 or lot-to-lot variations in ligand immobilization. This biosensor the 8-channel RED96e – should be comparable so long as the is recommended for use with any assay where high ligand acquisition parameters are consistent across the experiments. immobilization reproducibility is critical. The SAX2 biosensor is suitable for both ligand binding kinetics assays and for custom quantitation assays (through pre-coating of a capture ligand on the biosensor). A B Loading Association Dissociation Global fitting for kinetics 2.0 1.8 1.0 1.6 1.4 0.8 1.2 1.0 0.6 0.8 0.6 0.4 0.4 0.2 0.2 0 0 0 400 800 1200 1600 2000 2400 2800 0 200 400 600 800 1000 1200 1400 1600 1800 Time (sec) Time (sec) Figure 1: (A) Kinetic analysis of the interaction between a ligand biotin-Fcγ Receptor (~55 kDa) and analyte hIgG (150 kDa) with SAX2 biosensor, overlaying three lots of SAX2 and raw data aligned at the ligand loading step. (B) Data was processed and curve fitted using a 1:1 binding model. Yellow lines represent fitted curves; other colors represent raw curves. See the SAX2 biosensor datasheet for ligand loading specifications. Binding (nm) Binding (nm)

Figure 2 and Tables 1 and 2 show exemplary kinetics characterization data for PSA antigen binding to its antibody as derived from the three different Octet models. The data suggests insignificant differences in response signals for both ligand immobilization and antigen binding, resulting in very low variability in the extracted affinity constants. This data demonstrates that Octet systems deliver consistent performance across the platform, regardless if researchers are using multiple, same-model instruments, or different Octet instruments. A B 5 0.6 4 0.4 3 2 0.2 1 0 0 0 20 40 60 80 100 120 140 160 0 200 400 600 800 1000 1200 Time (sec) Time (sec) C D 0.6 0.6 0.4 0.4 0.2 0.2 0 0 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 Time (sec) Time (sec) Figure 2: Octet comparability kinetics studies showing side by side kinetics data for biotinylated anti-PSA antibody immobilized on two different lots of SAX2 biosensors binding to PSA antigen. (A) An overlay of the immobilization data from three instruments where PSA antigen was used at 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM using the Octet RED96e (B), Octet RED384 (C) and Octet HTX (D) systems. Data was acquired in duplicates for each biosensor lot. Only one lot was used on the Octet HTX system. Experiments were run at 25°C using the 8-channel read head mode for all three Octet models. Binding (nm) Binding (nm) Binding (nm) Binding (nm)

Mean Range, nm Model response (nm) (high-low) % CV RED96e 1.50 1.59–1.42 3.16 RED384 1.67 1.73–1.61 2.04 HTX 1.68 1.74 –1.64 1.78 Table 1: Comparison of biotinylated anti-PSA ligand response on different Octet models. Data was obtained from two lots of SAX2 biosensors in duplicate (see Figure 2A). Loading response is reported at 150 seconds. Biosensor Biosensor Mean, Octet model lot 1 KD (M) lot 2; KD (M) KD (M) % CV RED96e 6.91E-09 5.79E-09 6.35E-09 12.5 RED384 7.46E-09 7.13E-09 7.29E-09 3.25 HTX 7.71E-09 n.d. 7.71E-09 n.d. Inter-instruments (mean) 7.36E-09 6.4565E-09 7.12E-09 9.79 Table 2: Octet kinetics comparability study data shows kinetics parameters for biotinylated anti-PSA antibody immobilized to two different lots of SAX2 biosensors binding to PSA antigen. Characterization was performed in duplicate for each biosensor lot and on all three Octet instruments using the 8-channel read head. Experiments were run at 25°C. Reference 1 Biolayer Interferometry as an Alternative to HPLC for Measuring Product Concentration in Fermentation Broth, Anurag S. et al., LCGC, 35(12) 870–877. ForteBio ForteBio Analytics (Shanghai) Co., Ltd. Molecular Devices (UK) Ltd. Molecular Devices (Germany) GmbH 47661 Fremont Boulevard No. 88 Shang Ke Road 660-665 Eskdale Sauerbruchstr. 50 Fremont, CA 94538 Zhangjiang Hi-tech Park Winnersh Triangle 81377 München 888.OCTET-75 or 650.322.1360 Shanghai, China 201210 Wokingham, Berkshire Germany www.fortebio.com fortebio.info@moldev.com fortebiosh@moldev.com RG41 5TS, United Kingdom + 00800 665 32860 +44 118 944 8000 uk@moldev.com ©2019 Molecular Devices, LLC. All trademarks used herein are the property of Molecular Devices, LLC. Specifications subject to change without notice. Patents: www.moleculardevices.com/product patents. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. FB_4006 Rev A