「Octet Systems: Modernize biopharmaceutical QC testing to increase efficiency」

Octet Systems: Modernize biopharmaceutical QC testing to increase efficiency Quality control of biological products to support clinical trials Quantitation assays and post market assay activities require the evaluation of multiple critical product attributes. The evaluation can include The simple Dip and Read™ sample analysis approach that allows functional assessment for specificity to a target receptor, titer for the use of 96- or 384-well plate formats enables streamlined and other quality attributes such as glycosylation, Fc receptor workflows and rapid quantitation of as many as 96 samples in molecule binding, stability assessment to check for the forma- as little as 2 minutes depending on the instrument. In a typical tion of aggregates, etc. quantitation assay, biosensors coated with capture molecules that bind specifically to the analyte are simply dipped into The Octet® platform provides a comprehensive screening and analyte samples placed in a microtiter plate. The resulting characterization tool across a diverse range of applications binding is later analyzed using either the rate of the initial slope during drug development and is especially versatile in antibody of binding or the equilibrium of binding; both dependent on and protein quantitation, contaminant testing and general prod- sample concentration. To quantify samples, a standard curve is uct characterization. This platform circumvents the limitations of generated using the binding rates of known concentrations of ELISA and HPLC platforms, enabling informed decisions to be the same analyte as the unknown. The unknown sample con- made earlier in bioprocess development. centrations can then be extrapolated from the standard curve. Key benefits VALIDATED FAB QUANTITATION ASSAY - BOEHRINGER INGELHEIM (BI) • Enhances productivity by increasing capacity to run 20X–40X and 8X–16X more QC potency samples testing/day than A key advantage of the Octet instrument’s Dip and Read assay ELISA and SPR* respectively format is the ability to rapidly develop methods. The combina- tion of real-time monitoring of assay response with the ability to • Automated assay operation allows for 10X less analyst vary conditions in a sample plate and the instrument’s multiple hands-on time than ELISA, resulting in FTE cost savings of read heads allow for faster assay development when compared > $40,000†. to ELISA or SPR. In one such example, the analytical group at • Robust instrument resulting in significant maintenance cost Boehringer Ingelheim, Fremont, USA were able to develop an savings and low downtime compared to SPR; no fluidics active Fab quantitation assay for in-process testing as well as means no clogging of samples and less instrument downtime. stability and lot release testing in less than one week. The as- You will not need a second backup instrument to support say, which required the immobilization of a small molecule onto uninterrupted operation. Streptavidin biosensors resulting in the binding of only active • Easy to use platform that is approximately 2X faster than Fab molecules1, was subsequently qualified for use in QC in less ELISA and SPR* in method validation for ligand binding and than a month. Compared to their ELISA protocol which required potency assays overnight incubation, the Octet Fab assay processed a 96-well * Compares OctetRED96e and OctetRED384 with a 4-channel SPR system † Please contact ForteBio staff for breakdown

A B C 500 y = 1.005x ± 0.0204 R2 = 0.9989 Theoretical Octet assay % Theoretical Octet assay % (µg/mL) (µg/mL) %CV Accuracy (µg/mL) (µg/mL) %CV Accuracy 400 2000 682 14.3  34% 400 400.2 3.8 100% 1000 702 12.2  70% 200 200.1 4.2 100% 300 500 526 11.3 105% 100 99.9 3.2 100% 250 265 2.3 106% 50 49.9 3.7 100% 200 125 128 1.3 102% 25 25.5 4.2 102% 62.5 64 2.1 102% 12.5 12.0 3.8  96% 100 31.3 30 2.7  96% 6.25 5.8 5.9  93% 15.6 13 2.7  83% 3.13 3.1 2.6  99% 0 0 100 200 300 400 500 Theoretical Concentration (µg/mL) Figure 1: Accuracy assessment for the Fab quantitation evaluated in the range 15.6–2000 µg/mL (A) and 3.13 – 400 µg/mL (B). Linearity of the Fab assay in the range 3.13 – 400 µg/mL (C). Data in blue on Figure 1A indicate sample concentrations that fall out of specifications for accuracy or % CV. plate in one hour, which included sample preparation time. In LEAD MOLECULE CHARACTERIZATION another study, the Octet system was used as an alternative to Once a drug candidate has been purified, functionality studies HPLC in measuring Fab product concentration in fermentation including receptor binding analysis follow. With three different broth2. In these studies, the Octet system was determined to options: the 8-channel Octet RED96e system, the 16-channel be 20X and 12X faster than HPLC and ELISA respectively with Octet RED384 system and the 96-channel Octet HTX system, far fewer assay steps (2, 4 and 7 respectively for Octet assays, the Octet platform is optimally designed to enable the rapid HPLC and ELISA). screening of thousands of monoclonal antibody clones for the quick selection of the lead candidate. The technology allows Binding assays for characterization of effector functions, complement binding For binding analysis, a ligand molecule can be captured onto and potency assessment amongst other applications through the desired biosensor surface based on the chemistry of the target binding assays. It further facilitates cross blocking stud- biosensor. The choice of the ligand molecule is dependent on ies to enable the grouping of selected clones into bins based the assay of interest; for example, a biotinylated lectin specific on competition for the target antigen’s binding epitopes. to a given glycan could be immobilized onto a Streptavidin Further downstream, the Octet system is highly versatile in Fc biosensor and used to screen for the presence of the glycan receptor binding analysis for selected candidates; a variety on the molecule of interest 3. A chaperonin protein could also of off the shelf biosensor chemistries allow for a more flexible be immobilized in a similar manner and used to screen for the assay design. presence of hydrophobic populations in a biological sample The interactions of therapeutic antibodies with fragment crys- to evaluate its stability. Moreover, for antibody:antigen binding tallizable Fc receptors and neonatal Fc receptors (FcRn) are assays, off the shelf biosensors including Anti-Human Capture measured in vitro as indicators of antibody functional perfor- (AHC) and Anti-Mouse Capture (AMC) biosensors allow for the mance4. A primary consideration when developing assays for immobilization of antibodies without the need for purification, Fc receptor-IgG kinetic assays is the format. The consideration and are typically cheaper than comparative sensor chips from depends in part on reagent availability. Since Fc receptor-IgG other label-free technologies. interactions are often relatively low affinity, concentrations of analyte for association may need to be quite high, often in the micromolar range. ForteBio provides users with multiple options for off-the shelf and ready to use biosensor chemis- 2 Octet concentration (µg/mL)

A B 0.30 0.8 KD=1.33E-6 KD=1.95E-6 0.6 0.20 0.4 0.10 0.2 0 0 0 20 40 60 80 100 0 20 40 60 80 Time (sec) Time (sec) Figure 2: Flexibility in assay design for IgG binding to Fc-gamma IIIa. (A) Anti-Human Fab biosensors used to immobilize the antibody while the receptor molecule is titrated. (B) His-tagged receptor molecule was first immobilized onto Anti-His antibody biosensors followed by a titration of the antibody 5. KD (M) increase with H2O2 treatment tries to fit purpose and circumstances. These include Ni-NTA, 1.4E-06 antibody-based anti-histidine (Anti-HIS) biosensors (both for his-tagged ligands; commonly available with most vendors of Fc receptor molecules), High Precision Streptavidin biosensors 1.2E-06 and Anti-Human Fab-CH1 biosensors among others. It is also important to note that compared to fluidics-based 1.0E-06 technologies, the Octet platform’s design enables it to be better suited for FcRn binding studies where the variable pH conditions required for the different steps of the assay may be 8.0E-07 difficult to design. y = 2E-07x + 5E-07 R2 = 0.99007 OCTET BIOSENSORS FOR QC ASSAYS 6.0E-07 The selection of biosensor surface chemistry depends on the application. All Octet biosensors can be used to develop QC methods. However, during assay method development, 4.0E-07 0 1 2 3 4 5 6 repeatability, intermediate precision and reproducibility studies H2O2 Treatment Time (hr) that include biosensor lot to lot assessment should be done to Figure 3: Impact of methionine oxidation on the binding of Herceptin to FcRn. determine assay robustness in line with recommendations from Human IgG1 was incubated in 0.3% H2O2 for the indicated 0, 1, 3 and 5 hours respectively before binding kinetics was assessed on an Octet instrument. the relevant regulatory bodies. High Precision Streptavidin 2.0 Data indicates clear inactivation of the human IgG over-time 7. biosensors (SAX2), intended for use with biotinylated ligands, have been developed to ensure minimal lot-to-lot variations in ligand immobilization. This biosensor is recommended for use with any assay where high ligand immobilization reproducibility is critical. The SAX2 biosensor is suitable for both ligand bind- ing kinetics assays and for custom quantitation assays (through pre-coating of a capture ligand on the biosensor). 3 Binding (nm) KD (M) Binding (nm)

FORMULATION AND STABILITY ASSESSMENT different stresses including elevated temperatures, acidic pH, Formulation development is the critical last step in downstream and addition of guanidine HCl6. In another study, the Octet bioprocessing. Production and storage media play a critical platform was used to rapidly evaluate the effect of oxidation on role in the activity and stability of biological therapeutic drugs. the functional activity of Herceptin through the binding charac- Unlike traditional tools like dynamic light scattering (DLS) and terization of the drug product against an FcRn molecule. In this Circular Dichroism (CD) that mainly measure stability param- study, the Dip and Read plate format allows for the incubation eters such as onset of aggregation or melting temperatures, of the drug product in peroxide (H2O2)in the sample plate in a the Octet platform can be used to evaluate both stability and time-staggered manner followed by the analysis of drug activity functional characteristics of these biological molecules. A at these various time points (Figure 3). group at Kansas University Medical Center (KUMC) developed an automated method using biotinylated GroEL-streptavidin GxP-ready solutions biosensors with Bio-Layer Interferometry (GroEL-BLI) to detect Octet platforms come with a complete GxP package that in- the formation of transiently formed, pre-aggregate species in clude instrument installation and qualification (IQOQ) kits, per- various pharmaceutically relevant monoclonal antibody (mAb) formance qualification (PQ) kits and 21 CFR Part 11-compliant samples. The relative aggregation propensity of various IgG1 software. In addition, ForteBio provides users with software and IgG4 mAbs was rank ordered using the GroEL-BLI biosen- and biosensor validation support that facilitates compliance sor method, and the least stable IgG4 mAb was subjected to with GMP requirements. References 1 ForteBio Application Note 12: Validated Quantitation and Activity Assay of 5 ForteBio Application note 17: Analysis of Fc-gamma Receptor-IgG interac- Antibody Fragment Molecule (Fab). tions on the Octet platform. 2 LCGC North America: Biolayer Interferometry as an Alternative to HPLC for 6 The Use of a GroEL-BLI Biosensor to Rapidly Assess Pre-aggregate Popula- measuring Product Concentration in Fermentation Broth; Anurag S. Rathore, tions for Antibody Solutions Exhibiting Different Stability Profiles; Samantha Deepak Kumar, Jyoti Batra, Ira Krull E. Pace, Sangeeta B. Joshi, Reza Esfandiary, Robert Stadelman, Steven M. 3 Biotechnol Prog : Lectin bio-layer interferometry for assessing product qual- Bishop, C.R. Middaugh, Mark T. Fisher, David B. Volkin, J Pharm Sci. 2018 ity of Fc- glycosylated immunoglobulin G Wallner J, Sissolak B, Sommereg- 107(2):559-570. doi: 10.1016/j.xphs.2017.10.010. ger W, Lingg N, Striedner G, Vorauer-Uhl K, 2019 35(5):e2864. doi: 10.1002/ 7 Sangeeta B. Joshi, Reza Esfandiary, Robert Stadelman, Steven M. Bishop, btpr.2864. C.R. Middaugh, Mark T. Fisher, David B. Volkin, J Pharm Sci. 2018 107(2):559- 4 Febs Open Bio: Rapid screening of IgG quality attributes – effects on Fc 570. doi: 10.1016/j.xphs.2017.10.010. receptor binding; Karin P. M. Geuijen, Cindy Oppers-Tiemissen, David F. Egging , Peter J. Simons, Louis Boon , Richard B. M. Schasfoort and Michel H. M. Eppink, 2017 https://doi.org/10.1002/2211-5463.12283 FOR SALES & SERVICE VISIT: fortebio.com/contact-us +1 650 322 1360 | info@fortebio.com © 2020 Sartorius BioAnalytical Instruments, Inc. All rights reserved. Fortebio and Octet are trademarks of Sartorius AG and/or any of its affiliated entities. Specifications subject to change without notice. For Research Use Only. FB_4021 Rev B