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1 Continuous Processing TECHNOLOGIES Using Technology to Overcome Bioprocessing Complexity Advanced Concentration and Analytical Technologies Accelerate Development and Manufacture of mabs, Vaccines, and Biosimilars Engin Ayturk, PhD, and Jeannette Marshall Unlike chemically synthesized drugs, whose structure is known and reproducible, biological drugs are derived from living cells and are sensitive, complex mixtures requiring cuttingedge biological technologies for their production. The growing importance of biosimilars in recent years is reflected in a corresponding rise in market value. The value of the global biologic therapeutic drug market reached approximately US$230 billion in 2014 and, according to BCC Research, will increase to nearly $390 billion by the end of This corresponds to a compound annual growth rate (CAGR) of just over %. BCC Research also estimates that biosimilars accounted for nearly $2.0 billion of the biologic therapeutic drug market in 2014 and that this segment is growing at a CAGR of 15%. Key drivers for the growth of biologic therapeutic drugs include the aging of the global population and the worldwide increase in chronic diseases; biologic drugs are very effective for treating arthritis, diabetes and heart diseases. Rising wealth and improving healthcare systems in emerging regions are also having a positive impact on the demand for biopharmaceuticals. The higher growth rate for biosimilars is due to the fact that numerous blockbuster monoclonal antibody (mab)-based drugs will be off patent by Despite this strong growth, competition in the biopharmaceutical market is fierce. Governments, insurance carriers, and patients are all looking for new drugs with demonstrated value in terms of both cost and efficacy. To gain real market share, being first to market is imperative whether for branded products or biosimilars. There is, consequently, tremendous pressure to reduce development times and costs while still providing safe drugs. Targeted therapies such as antibody drug conjugates that include large- and small-molecule components, although highly potent, can create safety and handling challenges. For a biosimilar, it is necessary to demonstrate similarity to its branded biologic. This requires extensive characterization of the drug candidate during development and requisite controls during the manufacturing process. Biopharmaceutical manufacturers are therefore seeking new processes as well as analytical technologies and relevant combinations that can help reduce complexity and increase productivity and efficiency. In particular, there is a need for processing equipment and analytical tools/ techniques that facilitate continuous processing and enable the seamless integration of different upstream and downstream bioprocess steps. Optimizing Processes with Single-Pass Concentration During concentration applications, there is potential for aggregation, particularly with sensitive biologics even when they are only slightly Figure 1: Cadence Inline Concentrator setup; F = flow meter, P = pressure Permeate stressed. Therefore, an effective concentration system for continuous processing must operate efficiently under mild conditions while at the same time being flexible enough to integrate readily with different downstream operations. Today, tangential-flow filtration (TFF) is the most widely used technique for the concentration of biologic drugs, but the technique can be challenging for shear-sensitive molecules because it involves recirculation of the process fluid. Single-pass TFF (SPTFF) provides a more attractive alternative. Eliminating the recirculation loop by using a unique flow path design and cassettes staged in a serial and parallel configuration, SPTFF is a viable option even for shear-sensitive proteins. Yields are also often higher because of improved product recovery and lower system working and holdup volumes. The Cadence Inline Concentrator (ILC) from Pall is specifically designed to allow volume reduction before or after different types of downstream processing steps (Figure 1, see Patents and Technologies box). 20 BioProcess International 14(6)s June 2016 Sponsored Supplement tank F P to break tank or inline to next unit operation

2 Figure 2: Scalability of ILC units with (left) Delta kda and (right) Delta 30 kda regenerated cellulose membrane for T01 (0.065 m 2 ) and T06 (3.5 m 2 ) modules during processing with 2, 5, and g/l bovine IgG in PBS over the feed pressure operational window of psig (1); PBS = phosphate buffered saline, J = flux in L/m 2 /h, and VCF = volumetric concentration factor for ILC Delta -kda T01 module (0.065 m 2 ) 0 VCF for / Fluxes for VCF J RETENTATE J FEED T01 vs T06 for ILC Delta 30-kDA T01 module (0.065 m 2 ) 0 VCF for / Fluxes for VCF J RETENTATE J FEED T01 T vs for ILC Delta -kda T06 module (3.5 m 2 ) for ILC Delta 30-kDA T06 module (3.5 m 2 ) With this module, biopharmaceutical manufacturers can achieve typical concentration factors of 2 4 (or more) and optimize downstream processing by performing product concentration at various process points. Continuous processing is also made possible because the Cadence ILC can be coupled with many different downstream processes. Some of the potential applications of the Cadence ILC module in addition to its use as an alternative to conventional TFF include in-line volume reduction, concentration before capture, and concentration between different chromatographic purification steps. Different module sizes are available for use at all phases in the drug development cycle. Pall has demonstrated that similar results are achieved when scaling up from one size to another, which is important for accurately predicting processing times, membrane areas and tank sizes (Figure 2) (1). Regardless of where the Cadence ILC is installed in a biopharmaceutical process, one of its major benefits compared with conventional TFF is the potential minimization of damage and aggregation of therapeutic proteins due to the reduced residence time and shear exposure that are achieved with only one pass through the pump and module (2). The technology also provides for smaller working and holdup volumes, leading to enhanced product recovery and the reduction in size of intermediate storage tanks Sponsored Supplement Figure 3: Biolayer Interferometry for measuring biomolecular interactions through detection of wavelength shifts Incident white light Reflected Beams BLI Signal Processing Incident white light Reflected Beams Wavelength (nm) Wavelength (nm) Wavelength (nm) required for in-process volume reduction. In addition, the Cadence ILC is easier to install than TFF: The feed port is connected to a pressurized feed source (such as a pump or pressure vessel), the retentate port is connected to product collection, the permeate port is connected to waste, and then the single-use module can be put into operation. Concentration Determination and Much More In addition to concentration, which is monitored and analyzed for both upstream and downstream operations, accurate antibody quantitation is another important parameter necessary for the selection of appropriate cell lines for development and optimization of bioreactor titers in production. Accurate antibody quantitation is also necessary for determining breakthrough concentrations when establishing dynamic binding capacities of Protein A sorbents to achieve the most efficient affinity purification of mabs using Protein A capture chromatography. Determining protein concentration using conventional analytical methods BLI Signal Processing Figure 4: Correlation of BLI (Octet platform) and HPLC data for human IgG concentrations over a broad dynamic range Octet ( 0 μg/ml) HPLC ( 0 μg/ml) can, however, create a bottleneck during process development or manufacturing. Traditional measurement methods including ELISA, HPLC, nephelometry, and densitometry suffer from long analysis times, lack of specificity, laborintensive protocols, and variable imprecision. Measuring protein concentration without the need for labeling the protein is a fast and effective alternative to those methods. Among such label-free assays, the most popular is biolayer interferometry (BLI). June (6)s BioProcess International 21 λ y = x R 2 =

3 BLI is an optical, label-free technology for measuring biomolecular interactions. It analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on a biosensor tip and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real time (Figure 3). Most important, only molecules binding to or dissociating from the biosensor can shift the interference pattern and generate a response profile. Unbound molecules, changes in the refractive index of the surrounding medium, and changes in flow rate do not affect the interference pattern, which makes it possible to analyze crude samples. The BLI-based Octet family of instruments from Pall ForteBio provides label-free, real-time results on drug-ligand binding interactions as well as drug product titer and activity, and thus it can support each step of bioprocessing for streamlined and accelerated workflows. Significantly easier, faster, and better characterization of drug candidates is also possible, providing greater value in drug development applications for which existing methods may be limited in throughput, performance, and cost. For instance, protein quantitation of 96 samples using BLI can be completed in as little as two minutes a task that can take from four hours to overnight using traditional ELISA. Proteins also can be assayed in crude mixtures, such as clarified cell cultures, cell lysates or hybridoma supernatants, glycerol, or in up to % DMSO, which dramatically minimizes sample preparation. The results obtained using BLI correlate well with results obtained using ELISA and HPLC (Figure 4). With full-plate antibody quantitation in as little as two minutes, BLI-based instruments provide labelfree quantitation of antibodies and other proteins that are critical to early cell culture screening, cell line selection for optimizing antibody production, chromatography optimization, and biologicals manufacturing monitoring. A multistep assay format is used to achieve high-sensitivity for sub-ng/ml protein quantitation of residual Protein A and host-cell protein contaminants in bioprocessing. Equally important, BLI can be used for rapid analysis of binding kinetics, screening, and epitope Figure 5a: Comparing time and effort involved in protein quantitation using Octet platform (a) and alternative methods (b, c) preparation: 30 minutes Sample run time: minutes minutes minutes total assay time 40 minutes operator hands-on Octet system Figure 5b: Comparing time and effort involved in protein quantitation using Octet platform (a) and alternative methods (b, c) HPLC preparation: 30 minutes Sample injection and run time: 18 hours 50 minutes 23 hours total, with 5 h operator hands-on binning/mapping assays when combined with multiplexed biosensor formats. Association rates (k a ), dissociation rates (k d ), affinity constants (KD), and affinity rankings and epitope binning/mapping data all can be obtained. For instance, using biosensor analysis on the Octet platform, the binding affinities of Fc-gamma receptors to monoclonal antibodies can be determined in a high throughput and highly sensitive format. Thus, the Octet platform facilitates a critical application in many stages of biopharmaceutical development, including comparability studies and qualified or validated CGMP lot release and stability studies. The Octet platform includes instruments, biosensors, reagents, and assay kits for analysis of biomolecular interactions in 96- and 384-well microplates. Six different instruments enable end-users to meet their specific throughput, sensitivity, and budgeting needs. The most popular Octet RED96 system enables highly sensitive detection of large and small molecules and allows for simultaneous evaluation of eight samples in a 96-well plate. Users who need high sensitivity and very high throughput turn to the Octet HTX system (96-well simultaneous read-out), whereas low-throughput users prefer the Octet K2, an economical two-channel system with high sensitivity. Other systems are also available offering different throughputs and lower sensitivity (only for larger molecules >5 kda). All of these instruments rely on the Dip and Read method (ForteBio), which allows simultaneous, automated monitoring of large numbers of interactions. Anti-hIgG, antimurine and Protein A biosensors are ready to use in quantitating a variety of IgG isotypes. Streptavidin-coated and amine-reactive biosensors expand quantitation, making it possible to Figure 5c: Comparing time and effort involved in protein quantitation using Octet platform (a) and alternative methods (b, c) Bind and capture antigen to plate overnight preparation: 60 minutes Sample run time: 4 hours 30 minutes ELISA 21.5 hours total assay time, with 5.5 hours of operator hands-on 22 BioProcess International 14(6)s June 2016 Sponsored Supplement

4 Figure 6: Cadence ILC postharvest, precapture concentration study (left) feed and retentate flow rate profiles and (right) quantitation of feed and retentate mab concentrations using the ForteBio Octet platform Flow Rate (ml/min) ~ Time (minutes) Concentration (g/l) Time (minutes) ~4 develop custom protein assays that are easier to process than ELISAs, while still providing rapid and accurate results. Furthermore, the microplate format combined with Dip and Read biosensors enables highly parallel processing on the Octet system in sample volumes as low as 40 μl. In fact, the Octet system delivers much higher throughput than ELISA and HPLC, with faster time to results, minimal sample consumption, and little to no instrument maintenance. Reagents in microplate wells also can be reused throughout an assay. The Octet platform is also cost effective. A comparison of the time and effort involved in generating results using the Octet BLI-based platform, ELISA, and HPLC clearly showed that the BLI approach afforded a lower cost per sample (Figure 5). Octet systems have optional IQ/ OQ tools and FDA 21 CFR Part 11 software to operate in CGMP laboratories. Enabling Advanced Biopharmaceutical Technologies As the biopharmaceutical industry continues to evolve, advanced technologies such as the Cadence ILC and Octet BLI platforms enable continuous improvement of bioprocessing across the development life cycle by providing improved efficiency, productivity, and quality assurance. Such technologies facilitate development and implementation of continuous bioprocesses for further gains in efficiency and process/product consistency. Sponsored Supplement Case Study: At-Line Analytics via ILC/Octet Coupling The Cadence ILC was used to process a clarified harvest material during an integrated continuous bioprocessing run with the clarification and Protein A capture chromatography steps linked together. A 4 postharvest volume reduction (or precapture concentration) was targeted. To this end, the Octet RED96 system was used to determine the mab concentrations sampled from surge vessels placed postharvest and precapture, which corresponded to the Cadence ILC feed and retentate streams, respectively. Clarified feedstock samples at ~1g/L mab concentration were collected at timed intervals following the continuous clarification step, which involved the Cadence Acoustic Separator (CAS) followed by depth and sterile filtration (see the Patents and Technologies box). samples at ~4 g/l mab concentration were collected at the same intervals from the Cadence ILC retentate stream, which provided a constant loading onto the Cadence BioSMB PD system used for the Protein A capture step (see the Patents and Technologies box). The Cadence ILC was operated at flow rates (Figure 6a) targeted to deliver a 4 volumetric concentration factor. The stable performance evidenced by the stable flow rate trends shown in Figure 6a was confirmed by the ForteBio mab quantitation assay (Figure 6b). All samples collected during the ILC-enabled process integration run were diluted to a concentration of 0.2 g/l with a diluent buffer (1 PBS, Technology Patents and Licensing SPF technology patents are owned by SPF Innovations LLC: 7,384,549B2; 7,682,511B2; 7,967,987B2; 8,157,999B2. In 2009, Pall Pall Corporation acquired an exclusive license from SPF Innovations LLC to manufacture, market, and sell SPF technology for biopharmaceutical applications. On 15 June 2015, Pall announced the exclusive licensing agreement with FloDesign Sonics for acoustic wave separation, a disruptive technology for cell culture clarification for both fedbatch and perfusion applications. Pall announced the acquisition of the BioSMB technology platform from Tarpon Biosystems on 31 March % Tween-20, 1 g/l BSA, ph 7.4). In addition, an in-house mab standard at 4.2 g/l was diluted with the diluent buffer followed by a 2 serial dilution. The Protein A coated disposable biosensors used in the Octet were soaked in diluent buffer for at least minutes before each analysis. The six feed and six retentate samples (200 µl each) were plated in triplicate in a 96-well plate, along with serially diluted standards, regeneration buffer ( mm glycine, 0.02% Tween- 20, ph 1.5), and diluents. The plate was then placed in the Octet, and the analysis was run for 30 minutes. Octet analysis of the samples revealed a consistent and expected concentration of both the feed and retentate samples, as illustrated in the plotted data shown in Figure 6b. It is June (6)s BioProcess International 23

5 also noteworthy to mention that the flow-based volumetric concentration factors monitored continuously throughout the testing were in good agreement with the concentration profiles measured using the Octet system, which performed as an effective and essential at-line analytical tool. References 1 Casey C, Ayturk E. Scalability of Cadence Inline Concentrator Modules for Bovine IgG Processing. Pall BioPharm Applications R&D Note USD3004; www. pall.com/pdfs/biopharmaceuticals/ USD3004_Scalability_Cadence_ILC_ BovineIgG_AN.pdf. 2 Casey C., et al. Cadence Single-Pass TFF Coupled with Chromatography Steps Enables Continuous Bioprocessing While Reducing Processing Times and Volumes. Pall BioPharm Applications R&D NoteUSD3003; pall.com/pdfs/biopharmaceuticals/usd3003_ Cadence_SPTFF_ChromSteps_AN.pdf. Engin Ayturk, PhD, is senior R&D manager, biopharm applications, and Jeannette Marshall is an R&D applications engineer II; Featured Services Service and Support Leverage Customized Services from Pall for Optimized Performance and Increased Speed to Market Innovative separation and purification technologies offered by Pall Life Sciences are designed to optimize bioprocess development and manufacturing for both batch and continuous bioprocessing. Pall also provides extensive support services that help customers optimize those technologies. Our extensive technical and applications support services include process development, technology transfer and scale-up, assistance with equipment selection, operator training, validation, and instrumentation support. Our integrated network of industry scientists, accomplished applications engineers, technology/validation consultants, and knowledgeable laboratory staff work together to deliver consistent, high-quality support for batch and continuous operations to customers around the globe. With over 40 years of experience serving the biopharmaceutical industry, Pall s experts in biochemical engineering, biochemistry, chemistry, microbiology, physics, engineering, automation, and medical technology are used to investigate and solve complex problems. Our team is available for on-site guidance for larger projects and to partner at one of our global center-of-excellence customer support laboratories featuring four modern BL2/3 cell culture and process development laboratories or through off-site solutions. Every program can be tailored to continuously improve bioprocesses, regardless of the size of the project or stage of development. Accelerating Development: Our process development (PD) services group provides bespoke high-performance solutions that improve process economics with streamlined paths to implementation using the most modern methods available. Clear timelines and process transfer support allow clients to successfully meet aggressive internal timelines while enabling them to achieve more with their existing resources. For upstream applications, we offer full process support for both robust and sensitive cell lines, including adherent and suspension growth forms. We also support a wide range of expression systems, viral vaccine and viral vector production, and cell therapy manufacturing processes. Downstream batch and continuous operations are supported by an extensive team of application, R&D, and field-based scientists and engineers with critical understanding in harvesting, filtration, chromatographic purification, and integration/automation of unit operations. Training, Validation, and More: Our extensive validation and operator training support programs deliver comprehensive solutions from drug development through to commercial manufacturing operations. A comprehensive range of instrument services also is offered to customers who want a reliable way to ensure worry-free instrument operation with minimal downtime. Our validation services cover extractables and leachables, compatibility testing, sterilizing filter-capsule validation, bacterial viability/flush testing and challenge studies, evaluation of filter adsorptive effects, product wet-filter integrity test parameters, and bacterial retention studies with process isolates. Our technical training course topics include filter sterilization, integrity testing and certification, sterile filtration, filter validation and regulatory requirements, chromatography sorbent packing and handling, tangential-flow filtration (TFF) optimization, single-pass TFF optimization and implementation, and single-use systems validation. Our instrument services include instrument qualification and repair, preventive maintenance, calibration, instrument training, technical support, service contracts, spare parts, documentation, and instrumentation upgrades. Scientific Knowledge At Your Service: Our bioprocess expertise in filtration and purification enables us to deliver highlevel technical support and services for a range of challenging applications, from complex continuous biopharmaceutical processes through active ingredient purification. Regardless of location, our team can examine unique product and process characteristics and aid in selection of the correct test methods and conditions to validate individual processes. Field-based staff provides on-site support for batch and continuous harvest, filtration, chromatographic purification, and aseptic processing applications in process development and optimization, training, equipment selection, preinspection reviews, and process troubleshooting. We base our success on your success and aim to be a life-long, global partner with each customer. See how our scientists and engineers can help your team substantially reduce process development times and make your batch and continuous bioprocess unit operations more efficient and cost effective. 24 BioProcess International 14(6)s June 2016 Sponsored Supplement