Technical Note「Residual Protein A Detection Kit」

Residual Protein A Detection Kit Ready BLI Kit for simple, sensitive, and accurate quantitation of leached Protein A in bioprocess samples. Technical Note -Scop --- e What is BLI? Assay principle and workflow - Important notes Assay protocol - Data analysis Precision and accuracy The Residual Protein A Detection Kit is intended for the detection and quantitation of recombinant Protein A or other Protein A constructs such as MabSelect SuRe™. It has been developed as a simpler, faster alternative to ELISA method with reduced hands-on time for customers who require a sensitive and robust assay for measuring small amounts of leached Protein A in antibody or Fc-fusion protein samples. This kit is for research and manufacturing use only and is not intended for diagnostic use in humans or animals.

Introduction What is BLI? Detection of residual Protein A (RPA) in product purified Bio-Layer Interferometry (BLI) is a technology based on from Protein A purification columns is a critical quality control measuring interference patterns of white light applied to step in manufacturing of antibody therapeutics. During the the tip of a disposable fiber-optic biosensor in order to de- purification process, Protein A can leach off of the chroma- tect molecular binding events (Figure 1). The family of BLI tography matrix and co-elute with the drug substance. To instruments provides an extremely versatile and easy-to- prevent potentially dangerous adverse reactions in patients, use platform that can replace or complement a variety Protein A contamination must be detected and minimized. of analytical functions – from rapid quantitation and titer Testing for leached Protein A is performed in several stages assays to determining affinity constants and binding activity of biologic development and production including purification of antibodies or receptors to their targets. Systems are process development, manufacturing, and finished product widely utilized in the biopharmaceutical industry from early release testing. discovery and characterization through bioprocess devel- opment and quality control. With BLI, measurements that The Residual Protein A Detection Kit provides sensitivity to ordinarily require high sample volumes, complex setup, or accurately measure down to 100 pg/mL of contaminating excessive time and experimenter intervention can be run Protein A in samples containing up to 5 mg/mL of antibody. efficiently and with minimal effort to achieve fast, accurate It is designed for use with both native and unnatural results. Protein A constructs, including MabSelect SuRe™. The kit is compatible with all Octet® systems with the exception of the Octet® R2 system. 1.0 Incident BLI signal processing white 0.8 Therapeutic Purified by Protein A light antibody column 0.6 Biocompatible surface 0.4 Bound molecule 0.2 Unbound molecules Leached ProA co-elutes have no effect with the product Wavelength (nm) Figure 1: BLI measurement using Dip and Read biosensors. BLI is an Purification of product with impurity optical analytical technique that analyzes the interference pattern of testing at different stages white light reflected from two surfaces. Changes in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that is measured in real time. 2 Relative Intensity

Assay Principle and Workflow Assay Optimization The Residual Protein A Detection Kit applies the principles Depending on the instrument used, either all or part of the of BLI to enable sensitive and accurate measurement of RPA assay is automated. On the Octet® RH16 instrument, leached Protein A in bioprocess samples while maintaining the initial capture step is performed on a Octet® AS shaker a simple workflow. The kit utilizes a validated sample treat- and the biosensors are transferred to the Octet® instrument ment method for dissociating Protein A from antibodies for automated detection steps. Using the Octet® RH96 without boiling, neutralization, or centrifugation steps. instrument, all steps are automated after sample treatment After sample treatment, Dip and Read RPA biosensors with no further intervention required. Table 1 lists perfor- with pre-immobilized chicken anti-Protein A antibody are mance and workflow information for each instrument for- sequentially dipped into treated samples, buffers, and mat, as compared to a typical Protein A ELISA assay. Com- detection antibody arrayed in 96-well plates to create an parison to ELISA requirements demonstrates significant immunoassay on the biosensor tip. Signal detection occurs time savings and less hands-on intervention with BLI detec- in real time at the secondary antibody step without need tion. for conjugates or enzymatic reactions (Figure 2). 1 2 3 Pre-treat samples to Incubate samples with Quantify bound Protein A using separate Protein A RPA Biosensors to capture Protein A Protein A Detection Reagent from IgG 10 min 60 min 5 min – 120 min Figure 2: Illustration of RPA detection with BLI. 3

Table 1: Overview of workflow and requirements for Residual Protein A Detection Kit using different Octet® systems. Octet® Quantitation range (ng/mL) Minimum Assay time per plate Workflow | Throughput system | assay sample hands-on steps per day format Protein A MabSelect required Hands-on Octet® Total (plates) Standard SuRe™ instrument assay time Octet® RH96 0.1 – 25 0.25 – 25 60 µL 30 min 1 h 11 min < 2 h Sample treatment 7 (96-channel detection) Place on instrument Octet® RH96 0.1 – 25 0.1 – 25 60 µL 30 min 2 hs 3 min 2.5 h Sample treatment 5 (16-channel detection) Place on instrument Octet® RH16 + 0.1 – 25 0.1 – 25 60 µL 1.5 h (with 36 min 2.25 h Sample treatment 4 Octet® AS intermission) Octet® AS incubation (1h) Octet® AS wash Place on instrument Octet® R8 + 0.1 – 25 0.1 – 25 130 µL 1.5 h (with 1 h 12 min < 3 h Sample treatment 2-3 Octet® AS intermission) Octet® AS incubation (1h) Octet® AS wash Place on instrument ELISA Varies by Varies by 50 µL 5 h (with n/a 5 h + O/N Sample treatment 3 manufacturer manufacturer intermission) incubation Incubation of samples (1h) 4 manual washes Incubate with antibody (1h) 4 manual washes Incubate with conjugate (30 min) 4 manual washes Add substrate/ Read plate 4

Materials Included - 5.0 1 tray of 96 Residual Protein A (RPA) biosensors - Pre-immobilized with chicken antibody and preserved - Protein A MabSelect SuRe™ Sample Dilution Buffer (2 x 50 mL) Phosphate buffered saline with detergent, BSA and preservative Denaturing Buffer (1 x 20 mL) 0.5 Citrate buffer with detergent and preservative - (Caution: Acidic) Detection Diluent – Protein A (1 x 50 mL) Phosphate buffered saline with PEG, detergent, BSA and - preservative Protein A Detection Reagent (1 mg/mL, 1 x 450 μL) - Chicken antibody in Phosphate buffer with preservative 0.1 Protein A Standard (1 mg/mL, 1 x 100 μL) 0.05 0.5 5 50 Recombinant Protein A in phosphate buffered saline Log (ng/mL) Figure 3: Comparison of standard curves for provided Protein A Standard and MabSelect SuRe.™ (Components are not available separately for purchase) Additional Materials Required Important Procedural Notes for - Optimal Performance Octet® system with Octet® BLI Discovery Software and Octet® Analysis Studio Software software -- version 9.0 or later 1. Selection of Protein A Standard: The Protein A standard Octet® AS shaker (not required with Octet® RH96 system) provided in the kit is unconjugated Pierce™ Recombinant Black polypropylene 96-well, flat bottom microplates Protein A (Thermo Scientific #21184). It functions similarly (Greiner Bio-One # 655209) to native Protein A and can be used to accurately quantitate similar recombinant Protein A constructs. Optional: Black polypropylene 384-well microplates An important exception is MabSelect SuRe™ from GE (Greiner Bio-One # 781209) Healthcare. MabSelect SuRe™ has significantly modified Optional: Black polypropylene 384-well Tilted Bottom structure compared to more natural recombinant Protein microplates (# 18-5080) A constructs, and is recognized differently by Protein A Optional: MabSelect SuRe™ protein antibodies. These differences can result in quantitation (GE Healthcare Life Sciences # 28-4018-60) inaccuracies against the included Protein A Standard (Figure 3). For accurate quantitation of Protein A in samples purified Storage and Stability with MabSelect SuRe™, we recommend using uncon- - jugated MabSelect SuRe™ ligand to generate the standard Protein A Standard can be stored at 2°C to 8°C for up to curve. MabSelect SuRe™ can be ordered directly from - 1 month. For longer-term storage, place at -20°C. GE Healthcare Life Sciences, # 28-4018-60. - All other reagents should be stored at 2°C to 8°C. RPA biosensors should be stored at room temperature in 2. Assay Interference: Samples with concentrations of the provided re-sealable bag with desiccant packet away antibody of up to 5 mg/mL can be quantitated accurately - from direct sunlight. Do not refrigerate or freeze biosensors. using this assay. However, some antibodies can interfere The kit is stable under recommended storage conditions with Protein A quantitation and lead to inaccuracies. To until the date printed on the kit and biosensor package. minimize interference, we recommend diluting samples to antibody concentration of 1 mg/mL or below, keeping Protein A within the quantitation range of the assay. Samples should only be diluted in the provided Sample Dilution Buffer. 5 Log (Binding Rate)

Some antibodies or other proteins can interfere with the 6. Overall assay signal will vary from lot to lot. This assay even at low concentrations. Certain sample matrices variation does not impact results or assay quantitation can cause interference as well. It is important to test for range. The Binding Rate Separation value on the assay interference as part of end user validation by one enclosed Certificate of Analysis will indicate approxi- o- f two methods: mate assay signal range to expect for the associated lot. Quantitate several 2-fold dilutions of antibody sample that fall within the Protein A quantitation range of the 7. Do not mix and match kits and biosensor trays. Kit assay and assess dilution linearity. Interference will reagents are paired with a specific lot of biosensors - cause non-linearity in quantitation of sample dilutions. to perform optimally. Lot numbers of individual Use Protein A Standard or MabSelect SuRe™ to per- components of a kit are printed on the Certificate form a spike-recovery experiment in antibody sample of Analysis. that contains very low to undetectable levels of Protein A. Recoveries that are not within 20% of expected 8. Minimum volume required in microplate well for values indicate assay interference. biosensor dipping is 195 μL in a 96-well microplate or In most cases protein and/or matrix interference can be 80 μL in a 384-well microplate. These minimum volumes eliminated by further dilution of the sample in Sample apply for treated samples, buffers and reagents. Dilution Buffer. For assistance with quantitation in problematic matrices, please contact Technical Support. 9. Using 384-well Tilted Bottom microplates (# 18-5080) instead of standard 384-well microplates can reduce 3. Octet® AS Shaker: Use of the Octet® AS shaker is rec- noise and increase sensitivity when running the assay ommended for this assay as it decreases total assay on the Octet® QK384 system or the Octet® RH96 time and ensures precise measurements. The Octet® AS system in 96-Channel Detection mode. Minimum is designed to mimic sensor incubation in the Octet® sys- volume required in these plates is 40 μL. tem at precise shaking speed and temperature. Since the Protein A quantitation assay has been validated on the Octet® AS shaker we cannot guarantee assay preci- Assay protocol sion, accuracy or robustness with other sample incuba- tion methods. Note that only 96-well plates can be used on the shaker. Sample pre-treatment A Octet® AS shaker is not required when running this -- Equilibrate all samples and reagents to room temperature. assay on the Octet® RH96 system, since all steps can be - Be sure to process all samples identically. performed on the instrument. Ensure that the Octet® instrument is turned on and allowed to warm up for at least 1 hour before starting the 4. Re-use of detection reagent: Assay plate wells containing Detection Reagent at working concentration can be - assay. Before starting, open the appropriate Residual Protein A reused in a single experiment for multiple biosensor dips advanced quantitation template in the Octet® BLI in a 96-well plate. For example, on the Octet® R8 a single Discovery software software. In the Plate Definition tab, 8-well column of Detection Reagent is used to measure define the well location, Sample ID, concentration, all 96 samples – resulting in 12 biosensor dips per well. dilution factor and replicate group information for each Over-dipping in Detection Reagent wells can cause data of your standards and samples. This will serve as a map or a- rtifacts or trending. To maintain accuracy: work list for sample plate preparation. - Detection reagent in a 96-well plate (200 μL volume) should not be dipped into more than 12 times. Detection reagent in a 384-well plate (80 μL volume) should be dipped into only once. 5. Run a standard curve for each assay. Standards and samples should be assayed at least in duplicate. 6

1-. Prepare dilutions of standards and samples 3. P If MabSelect SuRe™ resin was used for purification, use MabSelect SuRe™ protein as the standard to ensure - - repare Wash Plate and Detection Plate Wash plate: Add 200 µL Detection Diluent into each well of a black, flat-bottom 96-well microplate corre- accurate results. All samples and standards should be diluted in Sample - Dilution Buffer. - sponding to biosensors in the incubating microplate. Detection plate: Add 200 µL Detection Diluent into Column 1, wells A through H, of a black, flat-bottom To cover the entire assay range, suggested concentrations 96-well microplate. Add 200 µL of the diluted Protein for Protein A Standard curve are 25, 10, 5, 2.5, 1, 0.5, 0.25, A Detection Reagent into Column 2, wells A through 0.1, 0.05, zero ng/mL. Alternatively, prepare initial dilution H, of the same microplate (Figure 4A). of 20 ng/mL Protein A and perform 2-fold serial dilutions down to 78 pg/mL. Include a zero ng/mL standard as a 4. W negative control. - ash the RPA biosensors - - When Sample Plate incubation is complete, remove To minimize interference, we recommend diluting the tray of RPA biosensors and the Sample Plate from samples so that antibody concentration is 1 mg/mL or the shaker. lower, and Protein A falls within the assay quantitation - Place the Wash plate on the shaker. range. Lower the tray of RPA biosensors onto the Wash Plate so that the tips of the biosensors are soaking in the 2. Transfer 130 µL diluted standards and samples to desig- Detection Diluent. Shake for 5 minutes at 30°C at nated wells of 96-well black, flat-bottom microplate 1000rpm. (Sample Plate). If using 384-well microplate on the Octet® RH96 instrument, transfer 60 µL of standards 5. R-- un Detection steps on Octet® R8 instrument and samples. When the wash is complete, remove the tray of RPA biosensors and the Wash Plate from the shaker. 3. Pre-treat standards and samples in the 96-well Sample Place the Wash Plate into the blue biosensor tray Plate by adding 65 µL Denaturing Buffer to each well holder for the RPA biosensor tray, and re-assemble and mix thoroughly by pipetting up and down gently the tray apparatus by placing the RPA biosensor tray 15–20 times. For 384-well microplate add only 30 µL on top so that the biosensors tips are soaking in the Denaturing Buffer. - Detection Diluent. - Place the Detection Plate and the RPA biosensor 4. Incubate on the benchtop for 10 minutes tray assembly in the Octet® instrument in designated positions. - In Octet® BLI Discovery Software, open the advanced Assay protocol for Octet® R8 quantitation template ResidualProteinA_8CH_96W.fmf. instrument In the Plate Definition tab, define well location, Sample ID, concentration, dilution factor and replicate group 1. Capture Protein A onto Residual Protein A biosensors information for each of the standards and samples if u- sing the Octet® AS shaker - - this was not done previously. - Place the Sample Plate onto the shaker. Ensure the ‘Delay Start Time’ box is checked in the Run Lower the green tray of RPA biosensors onto the Experiment tab. The 300 second delay is required to Sample Plate so that the tips of the biosensors are - enable the Detection Plate to equilibrate to 30°C. soaking in the samples. Start the run. A full 96-well plate of samples will take Lock the plate and shake for 1 hour at 30°C with about 75 minutes to complete. shaking at 1000 rpm. 2. P- repare working-strength Protein A Detection Reagent Dilute Protein A Detection Reagent 20-fold by adding 90 µL to 1710 µL of Detection Diluent (This volume will fill one column of a 96-well plate, which is adequate for measuring 96 samples) 7

Assay protocol for Octet® RH16 instrument - In the Plate Definition tab, define well location, Sample ID, concentration, dilution factor and replicate group information for each of the standards and samples if 1. Capture Protein A onto Residual Protein A biosensors u- sing the Octet® AS shaker - - Place the Sample Plate onto the shaker. Lower the green tray of RPA biosensors onto the Sample Plate so that the tips of the biosensors are - - this was not done previously. Ensure the ‘Delay Start Time’ box is checked in the Run Experiment tab. The 300 second delay is required to enable the Detection Plate to equilibrate to 30°C. Start the run. A full 96-well plate of samples will take about 36 minutes to complete. soaking in the samples. Lock the plate and shake for 1 hour at 30°C with shaking at 1000 rpm. A. 2. P- repare working-strength Protein A Detection Reagent Dilute Protein A Detection Reagent 20-fold by adding 180 µL to 3420 µL of Detection Diluent. (This volume will fill two columns of a 96-well plate, which is adequate for measuring 96 sample wells) 3 . P- repare Wash Plate and Detection Plate Wash plate: Add 200 µL Detection Diluent into each well of a black, flat-bottom 96-well microplate corresponding to biosensors in the incubating - microplate. Detection plate: Add 200 µL Detection Diluent into Columns 1 and 2, wells A through H, of a black, flat-bottom 96-well microplate. Add 200 µL of diluted Protein A Detection Reagent into Columns 3 and 4, B. wells A through H, of the same microplate (Figure 4B). 4. W- ash the RPA biosensors When Sample Plate incubation is complete, remove the tray of RPA biosensors and the Sample Plate from - shaker. - the Place the Wash Plate on the shaker. Lower the tray of RPA biosensors onto the Wash Plate so that the tips of the biosensors are soaking in the Detection Diluent. Shake for 5 minutes at 30°C at 1000 rpm. 5. R-- un Detection steps on the Octet® RH16 instrument After wash is complete, remove the RPA biosensors and Wash Plate from the shaker. B = Buffer (Detection Diluent) Place the Wash Plate in the Octet® instrument in D = Detection (Protein A Detection Reagent, working strength) the biosensor tray position. Position the green Figure 4: Detection plate loading guide for Octet® RH16 RPA Biosensor tray on top of the plate so that the - biosensors are soaking in the Detection Diluent. Place the Detection Plate in the instrument in the Reagent Plate position. Do not place in the other - plate position! In Octet® BLI Discovery Software, open the advanced quantitation template ResidualProteinA_16CH_96W.fmf. 8

- In the Plate Definition tab, define well location, Sample Assay protocol for Octet® RH96 ID, concentration, dilution factor and replicate group instrument information for each of the standards and samples if - this was not done previously. - Ensure the ‘Delay Start Time’ box is checked in the Run 1. Select assay format for the Octet® RH96 system. Three Experiment tab. The 300 second delay is required to possible assay configurations can be run depending on enable the Detection Plate to equilibrate to 30°C. throughput and sensitivity needs: Start the run. A full 96-well plate of samples will take about 36 minutes to complete. a. 9- 6-Channel Detection (Higher Throughput) - All Protein A standards, samples, and detection reagents are combined in a single 384-well A. microplate (Figure 5). Each step of the assay is performed on 96 biosen- sors simultaneously, providing the fastest and - simplest quantitation assay. B = Buffer (Detection Diluent) Note: Using 96-channel mode for detection results D = Detection (Protein A Detection Reagent, working strength) in noisier signal than the alternative 16-channel mode. Expect slightly lowered precision and Figure 5: Plate loading guide for Octet® RH96 96-Channel Detection format. All reagents are loaded into a single 384-well plate. reduced sensitivity at the bottom of the assay quantitation range when using this format. b. 1-6 -Channel Detection (Higher Sensitivity) Standards and samples are prepared in one 96-well plate (Sample Plate), while Detection reagents are added to a second 96-well plate (Detection Plate) - (Figure 6A, 6C). Sample incubation is performed on 96 biosensors B. simultaneously. Detection steps are performed on 16 biosensors at a time for greater sensitivity.This assay configuration takes more time but provides more precision at the low end of the quantitation range than 96-Channel Detection format. c. 1-6 -Channel Detection, low sample volume This configuration is identical to 16-Channel Detection, however a 384-well microplate is used for the Sample Plate in order to conserve sample volume. (Figure 6B, 6C) 2. Perform sample pre-treatment. After selecting your Octet® RH96 assay format, prepare Sample Plate and B = Buffer (Detection Diluent) perform sample pre-treatment steps as described in the D = Detection (Protein A Detection Reagent, working strength) “Sample pre-treatment” section. Figure 4: Detection plate loading guide for Octet® RH16 9

3. Pre-- hydrate RPA biosensors Sample plate Add 200 µL Sample Dilution Buffer into each well of a black, flat-bottom 96-well microplate A. corresponding to biosensors in the incubating - microplate (Hydration Plate). Place the Hydration Plate in the instrument in the biosensor tray position. Position the green RPA Biosensor tray on top of the plate so that - the biosensors are soaking in the buffer. Pre-hydrate biosensors for a minimum of 10 minutes. 4. Pre-p are working-strength Protein A Detection Reagent For 96-Channel Detection: Dilute Protein A Detec- tion Reagent 20-fold by adding 400 µL to 7.6 mL Detection Diluent. This volume will fill 96 wells of a - 384-well plate. Sample plate – low sample volume For 16-Channel Detection: Dilute Protein A Detec- tion Reagent 20-fold by adding 180 µL to 3420 µL B. of Detection Diluent. This volume will fill two columns of a 96-well plate, which is adequate for measuring 96 sample wells. 5. Pre-p are Detection Plate For 96-Channel Detection: Add 80 µL treated samples/standards, Detection Diluent, and working strength Protein A Detection Reagent into wells of - a single 384-well plate (Figure 4). For 16-Channel Detection: Add 200 µL Detection Diluent into Column 1, wells A through H, of a black, flat-bottom 96-well microplate. Add 200 µL of the diluted Protein A Detection Reagent into Column 2, wells A through H, of the same microplate (Figure 5). Detection Plate 6. -- Place assay plate(s) in the instrument C. For 96-Channel Detection: Place the combined Sample/Detection Plate in the instrument in the Plate 1 position. For 16-Channel Detection: Place the Sample Plate in the Plate 1 position. Place the Detection Plate in the Plate 2 position. B = Buffer (Detection Diluent) D = Detection (Protein A Detection Reagent, working strength) Figure 6: Plate loading guide for Octet® RH96 16-Channel Detection format. A) Sample Plate (suggested layout) using 96-well plate, B) Sample Plate (suggested layout) using low sample volume option in 384-well plate, C) Detection Plate layout for both formats 10

7. In Octet® BLI Discovery Software, open the appropriate Example data and performance adv-a nced quantitation template. qualification In the Plate Definition tab, define well location, Sample ID, concentration, dilution factor and The Residual Protein A Detection Kit has been qualified replicate group information for each of the for performance using standard criteria described below. standards and samples if this was not done We recommend the user perform their own specific - previously. qualification and validation to assess kit performance with Ensure the ‘Delay Start Time’ box is checked in internal samples and specific Protein A construct. the Run Experiment tab. The 300 second delay is The performance data shown demonstrate the kit’s ability required to enable the Sample/Detection Plate(s) to detect Protein A in the presence of up to 0.5 mg/ml hIgG - to equilibrate to 30°C. in Sample Dilution buffer and also determination of preci- -S tart the run. sion, accuracy, linearity, and sensitivity. A full plate of 96 samples will take 1 hour 11 minutes - to complete. For 16-Channel Detection: A full plate of samples Example data will take 2 hours 3 minutes to complete. Standard curve was generated using Protein A Standard provided in the kit. Samples were run on the Octet® RH96 Data analysis system using 16-Channel Detection. mode. Curve fitting was performed using weighted 4 parameter logistic model. 1. After the run is complete, open the Octet® BLI Analysis Figure 7 shows sample raw data traces and plotted standard Software. In the Data Selection tab, load the experimen- curve. tal folder to be analyzed. Multiple data sets can be selected at once for analysis. All selected data sets will Protein A Standard curve (n=8) be analyzed as a group. 25 ng/mL 10 ng/mL 2. In the Results tab, select the appropriate Standard 3.5 5 ng/mL 2.5 ng/mL Curve Equation. 1 ng/mL 3.0 0.5 ng/mL 0.25 ng/mL 0.1 ng/mL 3. Important: Select ‘R Equilibrium’ as the binding rate 2.5 equation. 2.0 1.5 4. Click the Calculate Binding Rate! Button. This will determine binding rate equilibrium signal for each 1.0 data trace. Binding rates and calculated results are 0.5 presented in the data table. 0 3900 3950 4000 4050 4100 4150 4200 5. If desired, utilize the Octet® BLI Analysis Software Time (sec) features to group data or data sets, display individual graphs, set sample threshold alerts, and view data statistics. Fitting curve Standard 6. Click the Save Report button to generate a complete 100 Microsoft® Excel formatted report. 10-1 10-1 100 101 Concentration (ng/ml) Figure 7: Standard curve made with Protein A Standard included in the kit. 11 Binding (nm) Binding (nm)

Precision and accuracy Inter-assay precision was determined using 3 different preparations of sample in 3 independent assays. Protein A Precision (%CV where %CV = Standard Deviation X 100 / Standard or MabSelect SuRe™ were spiked into Sample Di- Average) and Accuracy (% Recovery where % Recovery = lution Buffer containing 0.5 mg/mL human IgG. Data Calculated Conc X 100 / Expected Conc) were calculated shown in Table 3. for data generated on the Octet® RH96 system using 16-channel detection mode. Table 2 shows results for stan- dard curves generated with Protein A Standard included in the kit and with MabSelect SuRe™. Table 2: Intra-assay precision and accuracy for Protein A Standard and MabSelect SuRe™. Protein A Standard (included) n = 8 MabSelect SuRe™ n = 8 Target (ng/mL) % CV Recovery Target (ng/mL) % CV Recovery 10 3.6% 105% 10 3.7% 99% 2.5 1.6% 98% 2.5 3.5% 105% 0.5 4.4% 101% 0.5 2.5% 92% 0.1 12.5% 100% 0.1 11.7% 100% Table 3: Inter Assay precision data for samples containing 0.5 mg/mL human IgG. Protein A Standard (included) MabSelect SuRe™ No. tests (n=3) Target (ng/mL) % CV No. tests (n=3) Target (ng/mL) % CV 3 5 6.4% 3 5 7.3% 3 2.5 6.6% 3 2.5 7.8% 3 0.5 4.8% 3 0.5 7.0% 12

Dilution linearity Dilution linearity Linearity of dilution was established by comparing expect- 100 ed concentration to calculated concentration. The chart shown in Figure 8 shows excellent correlation of calculated y = 1.0001x + 0.0164 concentrations in the presence of human IgG throughout R2 = 0.99997 the range of the assay. 10 Sensitivity 1 Limit of detection (LOD) is defined as the minimum con- centration at which an analyte’s presence can be detected by a given assay, whereas Lower Limit of Quantitation (LLOQ) is defined as the minimum concentration at which 0.1 0.1 1 10 100 an analyte can be reliably quantified. Here LOD was deter- Expected Protein A (ng/mL) mined for various assay formats as the concentration corre- sponding to binding rate signal three standard deviations above the mean of the zero standard. LLOQ was deter- mined based on the lowest concentration where recovery is within 20% of the expected/theoretical value and precision (%CV) is below 20%. LOD and LLOQ values for both Pro- tein A Standard and MabSelect SuRe™ can be found in Table 4. LOD (ng/mL) LLOQ (ng/mL) Octet® instrument Protein A Standard MabSelect SuRe™ Protein A Standard MabSelect SuRe™ Octet® RH96 (96-channel detection) 0.07 0.08 0.1 0.25 Octet® RH96 (16-channel detection) 0.03 0.06 0.1 0.1 Octet® RH16 0.03 0.06 0.1 0.1 Octet® R4 0.07 0.08 0.1 0.25 Table 4: LOD and LLOQ values for Protein A Standard and MabSelect SuRe™ for samples containing 0.5 mg/mL human IgG. Ordering information and technical support Part no. UOM Description MabSelect SuRe™ is a trademark of GE Healthcare. 18-5128 Kit Residual Protein A Detection Kit. Contains 1 tray of 96 Residual Protein A biosensors and reagents for analysis of 96 samples. 13 Tested Protein A (ng/mL)

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 information, visit www.sartorius.com/octet-support Specifications subject to change without notice. Copyright Sartorius Lab Instruments GmbH & Co. KG. For Research Use Only. 41-0247-TN Rev D