Enhancing efficiency and economics in process development and manufacturing of biotherapeutics

APPLICATION NOTE 11 Enhancing efficiency and economics in process development and manufacturing of biotherapeutics Rashi Takkar, Applications Scientist; Sriram Kumaraswamy, Director, Marketing Field Applications Introduction to improve the efficiency and economics in all stages of devel- opment. These key drivers have fueled the search for innovative Analytical techniques that measure protein quantity and quality analytical techniques that provide improved performance and are used in nearly all stages of research, process development speed without increasing costs. Biopharmaceutical companies and manufacturing of biotherapeutics. UV spectroscopy, ELISA have enthusiastically adopted ForteBio’s Octet® systems due to and HPLC have been in use for decades for protein quantitation their broad utility in protein quantitation and functional character- in physiological and process samples, and continue to be the ization combined with enhanced throughput, decreased sample workhorses despite their many limitations. To characterize the preparation requirements, and low cost of operation. This white functional activity of proteins during biotherapeutic development, paper describes the use of Octet instruments for protein quan- label-free biosensor-based binding assays are increasingly being titation, particularly in the areas of process development and utilized. The high cost and lengthy times associated with drug quality control. discovery and development have forced biopharm companies Target ID & Lead screening Lead optimization Preclinical Process QC & validation & selection & characterization development development manufacturing Mechanism Screening A‚nity Pharmacokinetics Cell line develop.– of action – for binders maturation – (PK) titer and growth Activity assay biomolecular (hybridoma, phage binding kinetics media assessment interactions or lysates) Pharmacodynamics FcRn binding Fc engineering/ (PD) Chromatography assay ELISA assay A‚nity/ on-rate/ humanization – conditions development o„-rate ranking FcRn binding optimize – DBC, of clones binding, wash, Immunogenicity Binding elution and CIP kinetics Epitope mapping Epitope binning Contaminant testing – insulin, HCP and residual Mammalian Protein A clone selection for scale-up Loading concentration for Quantitation small-scale specific purification Figure 1: Applications of Octet instruments in the drug research and development process. 1

Protein quantitation of 70 complex samples Octet systems Sample and instrument prep Sample analysis 30 min 10 min 60 min total assay time 40 min operator hands-on time Sample run time 20 min HPLC Sample and instrument prep Sample injection and run time Sample analysis 4 hr 18 hr 50 min 23 hr total assay time 5 hr operator hands-on time ELISA Bind capture Sample and antigen to plate instrument prep Sample run time Sample analysis Overnight 1 hr 4 hr 30 min 21.5 hr total assay time 5.5 hr operator hands-on time Figure 2: Comparison of protein quantitation in complex matrices using Octet systems and alternative methods. Advantages over ELISA and HPLC analyte concentration in crude matrices such as cell culture supernatant, cell lysate and serum. Octet concentration assays The principles of concentration measurement with an Octet system are similar to established immunoassays such as ELISA are complemented by the platform’s ability to measure func- and HPLC. However, protein quantitation protocols on the tional activity. For example, titer for a monoclonal antibody Octet platform provide several advantages. The Octet plat- (mAb) can be determined using biosensors coated with Protein form monitors binding of proteins from solution to a biosensor A, while the functional activity of the mAb can be assessed in surface in real time, without need for labels or other detection a second assay step involving binding to its specific antigen. reagents. This real-time monitoring of binding interactions In contrast, HPLC and A280 spectroscopy can determine enables clear discrimination between specific and non-specific only the total protein concentration of a sample, and separate binding signals, which can shorten assay development times assays must be used to measure biological activity. dramatically. Octet assays are also much faster: quantitation of a 96-well plate requires 15–30 minutes, or 60 minutes for a 384-well plate, depending on the instrument model. Fig- Bio-Layer Interferometry technology (BLI) ure 2 provides a comparison of analysis times. Analysis of 70 BLI technology monitors and analyzes the interference pattern samples on an Octet RED96 system requires as little as 55 generated from the reflection of white light from two different minutes including operator hands-on time, whereas ELISA or surfaces: a layer of immobilized protein on the biosensor tip and HPLC assays require at least 22 hours including several hours an internal reference layer (Figure 3). Any increase or decrease of analyst involvement. Samples run on Octet systems are also in the number of binding molecules to the biosensor surface recoverable, so that researchers may save and reuse precious produces a change in optical thickness that causes a shift in the sample for other experiments. In addition, Octet assays are not affected by absorption interferences in colored samples or by interference pattern. Unbound molecules in complex matrices light scattering with turbid samples, enabling measurement of and changes in the refractive index of the surrounding medium have minimal effect on the interference pattern. BLI technology 2

1.0 Incident BLI signal white processing 0.8 light 0.6 Biocompatible surface 0.4 Bound molecule 0.2 Unbound molecules have no effect Wavelength (nm) Figure 3: Bio-Layer Interferometry is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces. Changes in the number of molecules bound to the biosensor causes a shift in the interference pattern that is measured in real time. Biosensors Standard Reference Unknown 0 Time (sec) 120 Concentration Figure 4: A typical quantitation assay setup. Biosensors dip into standards run in duplicate to obtain binding (nm shift) vs. time (sec) curves. The initial slope of the interaction is used to create the standard curve of the binding rate vs. con- centration. The concentration of an unknown sample is then interpolated from the standard curve. simplifies protein quantitation by enabling specific measure- or antibody in a sample is determined via a direct binding or ment in complex samples. The one-step Dip and Read™ assay sandwich assay. Biosensors coated with a capture molecule, format uses native proteins, without need for labels or second- called the ligand, are dipped into solutions containing the ana- ary reagents. lyte in a highly parallel, automated method to measure binding Concentration measurement interactions. In a typical quantitation assay, a standard curve is generated using known amounts of the protein analyte, and Accurate determination of biologically relevant protein concen- unknown sample concentrations are interpolated from the stan- trations is essential to several areas in the biopharmaceutical dard curve (Figure 4). Concentration can be calculated from the industry including research, bioprocessing, quality control and manufacturing. The Octet platform uses a simple Dip and initial binding rate of the interaction which is based on the initial Read approach for rapid analysis of samples in 96 and 384- slope or from the binding rate at equilibrium. well microplate formats. The concentration of the target protein 3 Relative Intensity Binding Rate Binding (nm)

Quantitation applications for drug Titer assessment and growth media optimization development using the Octet platform RESEARCH AND EARLY BIOPROCESS DEVELOPMENT The Octet platform is a useful tool for cost-effective protein expression screening in research and early bioprocess devel- Pe-adapted opment with several significant benefits. Host Cells Vector Octet platform advantages Transfections • Antibody and protein concentrationst can be determined in crude matrices, such as cell lysates or hybridoma superna- 96-well Plates tants, saving time and resources when processing a large Range: 1–300 µg/mL Throughput: 1000s clones number of samples. • Octet assays have a dynamic range of greater than two or- Octet system ders of magnitude, enabling a single quantitation assay to be 24-, 12- and 6-well Plates utilized across all development stages – from early cell culture Range: 1–500 µg/mL Throughput: 200–500 clones to production bioreactors. • Octet systems perform rapid quantitation with minimal user in- Octet system volvement. 96 samples are analyzed in as little as 20 minutes, and 384 samples in 70 minutes. With additional plate handling T-flasks automation, Octet 384 systems can process more than 1200 Range: 1–500 µg/mL samples per day. Throughput: 100–150 clones • Samples are analyzed in a non-destructive method and are fully recoverable, which is advantageous when working with low sample volumes and precious samples. Octet system • Octet systems are easy to learn and operate. Multiple analysts can operate the instrument with minimal training, allowing Shake Flasks rapid integration of these systems into laboratory workflows. Range: 1–700 µg/mL Throughput: 50–100 clones Early clone selection In clone selection, thousands of hybridoma or phage clones are screened to determine positive binding clones and their Octet system protein secretion levels. Titer measurements are used to select high-producing clones and to normalize the functional activ- Fed-batch Shake Flasks ity of these clones in crude matrices. Integration of an Octet Range: 1–700 µg/mL system into the antibody discovery workflow affords increased Throughput: 12–20 clones screening throughput. With Octet RED384 and Octet QK384 instruments, automated plate handling can also be added to achieve even higher throughput. Octet quantitation assays Octet system are also used to determine loading levels of chromatography columns for small-scale purification. Cell line development 1–3 L Seed Bioreactors Harvest samples are screened on Octet systems to select Range: 1–4 g/L Throughput: 2–5 clones high-expressing clones during various scale-up procedures involving 96-, 48-, 24- and 6-well plates, fed batch shake flasks, and bioreactors (Figure 5). Octet assays also are used to de- termine protein levels during media development for fed-batch and bioreactor processes (Figure 5). This is performed by com- Figure 5: Protein titer assessment and growth media optimization using paring protein secretion levels following variations in feeding the Octet system at different stages of cell line development. regimes, strategies and concentrations. Data acquisition and 4

Polishing Cell culture Anity chromatography Viral Ultrafiltration harvest chromatography (at least 2 steps) filtration diafiltration Optimizing dynamic binding Optimizing dynamic binding Optimize different capacity of column resins capacity of resins formulations based on: • Detect protein breakthrough • Bind and elute mode: determine • Protein concentration in flow-through in crude protein breakthrough point required for stability matrix • Flow-through mode: determine • Protein activity/binding Optimizing binding, wash and final fraction when target protein elution conditions detectable • Protein binding/activity Optimizing binding, elution, and • Residual Protein A wash conditions • HCP (general & specific) • Protein recovery • Protein recovery • Protein binding/activity • Residual protein A • HCP Figure 6: Use of Octet systems in the downstream purification process of proteins and antibodies. subsequent data analysis can be performed rapidly for hun- containing a known concentration of target protein and moni- dreds of samples, bypassing traditional processing bottlenecks. toring this protein in the flow-through fractions. Quick determi- Please see ForteBio Application Note 13, Fc-Fusion Protein nation of DBC using HPLC or A280 spectroscopy is hampered Quantitation in Cell Culture Supernatants, for more information. by the presence of large amounts of host cell proteins in the flow-through fractions. Specific detection of the protein of inter- DOWNSTREAM PROCESS DEVELOPMENT est among contaminants is straightforward with Octet systems, reducing the time required to optimize purification conditions Efficient development of manufacturing processes for anti- (Figure 6). bodies and recombinant proteins is a critical need for bio- pharmaceutical companies. Increasingly stringent regulatory requirements targeting better understanding and control of Binding, wash and elution conditions manufacturing processes are expected to impact product qual- Numerous chromatography binding and elution conditions are ity and performance. The Octet platform can quickly determine tested during optimization studies, including different buffer the impact of multiple process variables at different stages of compositions, salt, pH, operating temperature and sample the purification process, and help identify optimal conditions injection volume. High-throughput tools, such as mini columns that provide protein product with the desired yield, binding and 96-well filter plates, often are used to screen these process specificity and potency (Figure 6). Pre-configured reagents variables. The impact of different conditions on product titer and protocols are available for rapid quantitation of protein and quality can be analyzed rapidly and effectively on Octet products, host cell proteins (HCP), and residual Protein A levels systems, speeding identification of optimal chromatography during purification processes. conditions (Figure 6). Octet platform advantages Contaminant testing • One Octet instrument can be used to measure protein titer, Downstream purification processes must remove host cell pro- host cell proteins and residual Protein A contaminant levels. teins, residual Protein A and residual DNA impurities. According • Octet assays are faster to develop and run than ELISA and to guidance from regulatory authorities, host cell proteins in a HPLC assays. drug substance should be below detectable levels using a high- ly sensitive analytical method, and as a rule this level should not • Octet assays can be automated with robotic and liquid han- exceed 100 ppm. The type of assay required for HCP determi- dling systems for complete, walk-away screening. nations depends on the phase of clinical studies for which the material is produced. For earlier clinical phases, a generic assay Dynamic binding capacity (DBC) of chromatography columns may be sufficient. However, a process-specific HCP assay gen- Affinity chromatography often is the first major purification pro- erally is required for phase 3 and later studies. Leached Protein cedure performed on harvested cell culture samples in down- A is another contaminant of concern in process development. stream bioprocessing. The dynamic binding capacity (DBC) of The elution of antibodies during Protein A chromatography an affinity chromatography column is defined as the amount requires acidic conditions, which in turn can accelerate leaching of protein that will bind to the column resin under a defined of Protein A from the column. Residual Protein A levels should condition. DBC is determined by continuously loading a sample not exceed 10 ppm in the final drug product. 5

Customer highlight: GlaxoSmithKline Benefits of automated Octet CHO HCP assay compared to manual ELISA The analytical lab at GlaxoSmithKline incorporated a generic HCP assay on the Octet QK384 system to streamline their Benefit Details workflow in process development. The automated Octet HCP Precision Liquid handling robot reduces pipetting variation assay required minimal analyst intervention and provided inherent in manual pipetting more accurate and precise results than their manual ELISA assay (Figure 7). Hands-on time for preparation and process- Reliability Method performed exactly the same each time ing of 1–3 assay plates was reduced to 30 minutes from the Streamlined Worklist drives robotic method and creates sample previous 2.5 hours with manual ELISA, and antibody consump- process plate importation files Robotic method automatically creates and executes tion decreased by 40%. Octet method file More information on the development of the HCP assay on Walk away No analyst intervention needed to complete method Octet systems can be found in Technical Note 24, Host Cell after instrument loaded and diluent volumes are checked Protein Dectection on the ForteBio website. Washing No washing steps needed and plate washer Process development assays for residual Protein A and prod- steps integration not required uct titer can be fully automated on Octet 384 systems using Analysts Automated Octet ~30 minutes for 1–3 assay plates external liquid handling platforms. The Octet assay for leached involvement Manual ELISA ~2.5 hours per assay plate Protein A is highly sensitive with a LLOQ of 0.20 ppm, has >2.5 Throughput 3 assay plates can be run in ~5 hours logs of dynamic range, and is faster than competing methods. 38 samples/plate in duplicate wells > 108 samples in A residual Protein A assay on the Octet RED384 system can 3 plates be completed in 1 hour and 45 minutes per plate with minimal Antibody Re-use of coating antibody can significantly reduce analyst involvement, compared to a minimum of 3.5 hours for consumed consumption over multiple assay plates ELISA (including significant analyst hands-on time). For more information on the Octet residual Protein A quantitation proto- Figure 7: Benefits of automated Octet CHO HCP assay compared to manu- al ELISA summarized by GSK. col, see Technical Note 18, Dip and Read Residual Protein A Detection Kit on the ForteBio website. QUALITY CONTROL Octet platform advantages Octet systems provide robust and highly reproducible assays • Octet systems are designed for GLP/GMP environments, and for protein concentration and functional activity, and are suit- provide 21 CFR Part 11 compliance tools. able for operation in quality control and manufacturing environ- ments. Protein activity and various kinetic assays are used to • Octet assays provide detailed information about the binding support in-process testing, drug potency, lot-to-lot variability behavior of protein products, and reveal subtle differences in and stability studies. binding activity between production lots. Activity assay Quantitation assay Streptavidin Biotin Fab Ligand — binds Fab Antibody Fc Protein A — binds Fc Figure 8: An activity assay can be developed on the Octet platform by immobilizing a specific biotinylated ligand on the biosensor and then detecting binding of an analyte, FAb or protein. In the quantitation assay, mAb titer is determined using Protein A-loaded biosensors, which does not measure mAb activity towards its target. 6

Customer highlight: Aragen Biosciences • Octet quantitation assays provide a direct measure of the biological activity of the analyte(s) (Figure 8). Aragen Biosciences created a stable and scalable CHO cell line, purification platform and manufacturing process for a • Octet assays can be easily transferred to manufacturing oper- particular product in a GMP environment. They developed ations. an Octet assay to compare the activity and quality of a new product lot (Lot 2) with a reference lot (Lot 1) throughout Activity assays their bioprocess and manufacturing processes. The as- An activity assay is generally utilized during process develop- say involved loading a biotinylated ligand on Streptavidin ment, QC and manufacturing to compare various prepared lots biosensors, and measuring binding interaction of the ligand of the drug molecule, as well as its stability. Activity assays are with the protein analyte. As seen in Figure 9, Lot 2 contained critical because they differentiate active protein from inactive a large second peak that was absent in the Lot 1 reference or clipped variants, as those species will not bind the ligand. material. The second peak in Lot 2 exhibited a slower on-rate Active protein concentration can be determined using a binding and much faster off-rate, indicative of a less-active fraction assay on the Octet platform by immobilizing a specific ligand (Figure 10). Octet system activity data results were confirmed against the target analyte onto the biosensor, and then measur- with a cell-based assay, and Aragen was able to modify their ing its binding interaction with the analyte as shown in Figure 8. production conditions to significantly reduce this second peak fraction. Lot 1 Lot 2 High Specific Low Specific Binding Activity Binding Activity Peak 2 Presence of large Peak 1 second peak Peak 1 correlated with reduced specific Peak 2 binding activity 30 35 30 35 Figure 9: HPLC spectra of Lot 1 and Lot 2 of a drug molecule. Lot 2 was made by Aragen Biosciences and had an additional peak (Peak 2) compared to the reference lot (Lot 1) provided by their customer. Data provided courtesy of Aragen Biosciences. Peak 2 Peak 1 30 35 A lower on-rate and an increased o -rate was clearly indicative of a less-active fraction Figure 10: The Octet binding kinetics or functional assay demonstrated that Peak 1 was the active fraction. Peak 2 was the less-active fraction, with a lower on-rate and a much faster off-rate in a binding experiment. Data provided courtesy of Aragen Biosciences. 7

Conclusion Octet systems deliver comprehensive characterization of the high throughput needed to screen through large libraries of biotherapeutics, as well as rapid and reproducible determi- candidate drug molecules. In later stages of process develop- nation of protein concentrations during different stages of the ment and manufacturing, Octet systems provide the required development process. Titer and functional activity assays on reliability, robustness and measurement accuracy. The broad Octet systems are useful for a broad array of applications in utility of this single platform makes the Octet instrument unique target identification, lead selection, process development, in its ability to deliver high value across a wide range of appli- formulation development, quality control, and manufacturing. cation needs in biopharmaceutical discovery, development and In early stages of drug development, Octet systems provide manufacturing processes. 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 Bismarckring 39 Fremont, CA 94538 Zhangjiang Hi-tech Park Winnersh Triangle 88400 Biberach an der Riss 888.OCTET-75 or 650.322.1360 Shanghai, China 201210 Wokingham, Berkshire Germany www.fortebio.com fortebio.info@moldev.com salesops.china@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. AN-4011 Rev B