9403 GE Pre Dictor Plates Handbook
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GE Healthcare ÄKTA, ÄKTA avant, AxiChrom, BioProcess, Capto, Deep Purple, Drop design, HiScreen, HiTrap, MabSelect, MabSelect SuRe, MabSelect Xtra, PreDictor, ReadyToProcess, Sepharose, and Tricorn are trademarks of GE Healthcare companies. Capto ViralQ: Separating viral particles with Capto Q products may require a license under United States patent number 6,537,793 B2 and equivalent patents and patent applications in other countries owned by Centelion SAS. Such a license is not included with the purchase of Capto Q but is included with the purchase of Capto ViralQ products. With the purchase of Capto ViralQ the customer is granted a free limited license under US patent 6,537,793 B2 and equivalent patents and patent applications in other countries owned by Centelion SAS to separate viral particles solely through use of the product purchased. Deep Purple Total Protein Stain: Deep Purple Total Protein Stain is exclusively licensed to GE Healthcare from Fluorotechnics Pty Ltd.Deep Purple Total Protein Stain may only be used for applications in life science research. Deep Purple is covered under a granted patent in New Zealand entitled “Fluorescent Compounds”, patent number 522291 and equivalent patents and patent applications in other countries. Tricorn Columns: The Tricorn column and components are protected by US design patents USD500856, USD506261, USD500555, USD495060 and their equivalents in other countries. All third party trademarks are the property of their respective owners. © 2009 General Electric Company – All rights reserved. First published Dec. 2009. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare that supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ 08855-1327, USA GE Healthcare Europe GmbH Munzinger Strasse 5 D-79111 Freiburg, Germany GE Healthcare Japan Corporation Sanken Bldg. 3-25-1 Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan For local office contact information, please visit www.gelifesciences.com/contact High-throughput Process Development with PreDictor Plates – Principles and Methods GE, imagination at work, and GE monogram are trademarks of General Electric Company. High-throughput Process Development with PreDictor™ Plates Principles and Methods www.gelifesciences.com/predictor GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden imagination at work imagination at work 28-9403-58 AA 12/2009 Handbooks from GE Healthcare GST Gene Fusion System Handbook 18-1157-58 Gel Filtration Principles and Methods 18-1022-18 Affinity Chromatography Principles and Methods 18-1022-29 Recombinant Protein Purification Handbook Principles and Methods 18-1142-75 Antibody Purification Handbook 18-1037-46 Protein Purification Handbook 18-1132-29 Ion Exchange Chromatography and Chromatofocusing Principles and Methods 11-0004-21 Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods 11-0012-69 Cell Separation Media Methodology and Applications 18-1115-69 2-D Electrophoresis using immobilized pH gradients Principles and Methods 80-6429-60 Purifying Challenging Proteins Principles and Methods 28-9095-31 Isolation of mononuclear cells Methodology and Applications 18-1152-69 High-throughput Process Development with PreDictor Plates Principles and Methods 28-9403-58 Microcarrier Cell Culture Principles and Methods 18-1140-62 Nucleic Acid Sample Preparation for Downstream Analyses Principles and Methods 28-9624-00 High-throughput Process Development with PreDictor Plates Principles and Methods 28-9403-58 AA 1 Contents Introduction............................................................................................................................................3 1. Process development in PreDictor 96-well plates...................................................................5 1.1 Why use PreDictor 96-well plates as part of a process development workflow?.......... 5 1.2 Screening experiments in PreDictor plates vs small-scale columns..................................... 5 1.3 What types of studies can be performed?........................................................................................ 6 1.4 How PreDictor plates work and how screening experiments are performed.................. 6 2. Batch experiments using PreDictor plates...............................................................................7 2.1 Batch experiments using PreDictor plates vs column chromatography............................ 7 2.2 The batch experiment................................................................................................................................. 7 2.3 Capacity determinations........................................................................................................................... 8 2.4 The adsorption isotherm............................................................................................................................ 9 2.5 Incubation time............................................................................................................................................10 2.6 Phase ratio......................................................................................................................................................11 2.7 Sample concentration and media volume......................................................................................11 2.8 Mixing................................................................................................................................................................12 2.9 Types of studies............................................................................................................................................12 3. Practical considerations.............................................................................................................17 4. Application examples..................................................................................................................19 4.1 Screening binding conditions on Capto S using PreDictor plates........................................19 4.2 Effect of incubation time, pH, and ionic strength on the binding of amyloglucosidase to Capto DEAE.......................................................................................................20 4.3 Optimization of the wash step for MabSelect SuRe....................................................................22 4.4 Screening elution pH on MabSelect SuRe........................................................................................24 4.5 Rapid development of CIP protocols for affinity media.............................................................26 4.6 Adsorption isotherms with PreDictor plates...................................................................................28 5. Quality aspects of the PreDictor plate workflow...................................................................31 5.1 Functional testing of reproducibility...................................................................................................31 5.2 Increasing the robustness of experiments......................................................................................31 5.3 Method variability vs chromatography media volume variability........................................32 6. Assist software.............................................................................................................................35 6.1 Assist software workflow.........................................................................................................................35 7. PreDictor plate selection............................................................................................................39 7.1 PreDictor Plate selection guide.............................................................................................................39 7.2 Available plates............................................................................................................................................41 8. Experimental designs/setups....................................................................................................43 9. Steps in process development following screening..............................................................45 9.1 HiScreen and HiTrap columns...............................................................................................................45 9.2 ÄKTA chromatography systems and UNICORN control software........................................46 9.3 BioProcess media . .....................................................................................................................................46 10. References.....................................................................................................................................47 11. Nomenclature...............................................................................................................................49 12. Ordering information..................................................................................................................51 PreDictor plates........................................................................................................................................................51 Software.......................................................................................................................................................................52 Related products......................................................................................................................................................52 Related literature.....................................................................................................................................................53 Introduction Time-to-clinic and time-to-market are two key factors for successful biopharmaceutical development. Efficient development of the manufacturing process is a crucial component of the overall project plan. However, screening optimal process conditions can be time-consuming and tedious. In addition, steadily increasing demands from regulatory authorities for better understanding and control of manufacturing processes put even more pressure on development work. Employing high-throughput tools for process development helps address these challenges. High-throughput process development (HTPD) shortens development time at the same time as it increases the amount of information available during early process development. Evaluating chromatographic conditions can be performed in parallel using 96-well filter plates. As a result, a large number of experimental conditions can be evaluated simultaneously. This allows a large experimental space to be characterized and supports the definition of a well-established process design space where process parameters that need to be monitored and controlled are understood (Fig 1). This handbook focuses on process development work in PreDictor 96-well filter plates, which are prefilled with chromatography media (resins). The handbook describes how results obtained using PreDictor plates can be used to define process conditions for large-scale chromatography. Applications of PreDictor plates are provided, as well as practical hints and tips. Assist software, dedicated to allow high-throughput methodology using PreDictor plates, is also described. 28-9403-58 AA 3 4 28-9403-58 AA 1. Process development in PreDictor 96-well plates 1.1 Why use PreDictor 96-well plates as part of a process development workflow? In the past, process development was either performed using tedious and time-consuming column experiments, or such experiments were simply not performed. This resulted in a less well understood and less well optimized process. With PreDictor plates, many types of experiments can be performed in parallel, which result in significant time-savings and lower consumption of sample and other reagents. The high-throughput format allows rapid screening of a wide range of conditions and thus allows generation of large amounts of valuable process data. The workflow in a PreDictor experiment comprises the same steps as any column experiment. When using PreDictor plates, the fundamental interactions between the chromatography medium and the target molecule are the same as in chromatography columns. Basic concepts such as mass balance, rate of uptake, and adsorption isotherms are the same for PreDictor plates and chromatography columns. Successful application of PreDictor experiments does not require in-depth knowledge of these concepts nor of the equations presented later in this handbook. The applications presented show that data obtained in PreDictor experiments are relevant for full-scale process design and characterization. 1.2 Screening experiments in PreDictor plates vs small-scale columns High-throughput tools such as PreDictor plates and Assist software are suitable for early chromatographic screening experiments. They can be used for the initial screening of process conditions, or for a more thorough investigation of a defined space as a basis for detailed process understanding and/or robustness studies. After scouting and screening with PreDictor plates, verification and fine-tuning are still performed with ÄKTA™ design systems such as ÄKTA avant™ 25 at a smaller scale than the expected manufacturing process, see Section 9. Prepacked formats such as HiScreen™ 10 cm bed height columns are recommended, or HiTrap™ 1 ml and 5 ml columns if sample volumes are low, see Ordering information. Figure 1 shows a conceptual workflow for process development with PreDictor plates as the starting point. Total characterization space Detailed studies to define design space Area for further optimization Scale-up PreDictor 96-well plates and Assist software for screening of condition ÄKTA avant 25, UNICORN™ 6, HiScreen, and HiTrap columns for optimization ReadyToProcess™/ AxiChrom™ columns for scale-up and production Fig 1. Conceptual workflow for process development. 28-9403-58 AA 5 1.3 What types of studies can be performed? PreDictor plates are used to: • Screen chromatographic conditions. This includes all parts of the chromatographic cycle: Binding, wash, elution, and cleaning-in-place. Screening can be performed for different media simultaneously, or for a single selected medium. • Determine adsorption isotherms. Briefly, the adsorption isotherm describes the relationship between the concentrations of protein(s) in the liquid and solid phases at equilibrium under a given set of experimental conditions. This helps understand and correctly describe what happens during protein uptake under different conditions. It also helps to determine optimal phase ratios (ratio between sample volume and volume of chromatography medium) for capacity, wash, or elution studies. Typical applications for PreDictor 96-well plates are shown in Table 1. Table 1. Studies that can be performed with PreDictor plates Study Factors Comments Binding conditions Protein concentration, conditions (salt, pH, etc.), incubation time Qualitative and quantitative analyses of capacity Wash conditions Protein concentration, conditions (salt, pH, additives, etc.) Intermediate wash step(s) can improve purity of the next step, i.e., elution Elution conditions Conditions (salt, pH, additives, etc.) Conditions for step elution and also gradient elution can be studied. Small systematic differences in conditions can be used to mimic a chromatography gradient elution. Cleaning-in-Place (CIP) Conditions (salt, pH, additives, etc.), time Effectiveness of different CIP solutions can be tested Adsorption isotherms Conditions (salt, pH, additives, etc.) Utilizes the convenience of the 96-well plate format for constructing adsorption isotherms Determination of optimum phase ratio for capacity/wash/elution studies 1.4 How PreDictor plates work and how screening experiments are performed Each well in a PreDictor plate represents a batch. The main difference between a batch and a column is that in the batch, each separation only occurs once. In the column, we see a cascade of stages, sometimes referred to as theoretical plates. The batch system can be used to investigate the distribution of target protein between the chromatography medium and the liquid phase. The batch experiment involves the same steps of equilibration, sample addition, wash, and elution as a column chromatographic separation. Experimental setups using high-throughput PreDictor 96-well plates allow simultaneous testing of many different conditions (factors) such as pH, salt, etc. Experimental setups such as Design of Experiments (DoE), which uses statistics to identify and define factors having the greatest impact on the process or product, facilitate complex screening of conditions using PreDictor plates. Other experimental setups can, however, also be used. In summary, applying high-throughput techniques means that future-generation processes can be developed quicker and more thoroughly. 6 28-9403-58 AA 2. Batch experiments using PreDictor plates 2.1 Batch experiments using PreDictor plates vs column chromatography In a typical adsorption process, both the mass transfer mechanism (responsible for protein transport) and ligand selectivity are independent of the mode of operation, that is, the adsorption process is the same whether it occurs in a batch system or in a packed column. In a column however, the adsorption process occurs continuously during the transport of protein through the column. This repeated adsorption process is described as a cascade of stages (column theoretical plates) where adsorption occurs. One well in a PreDictor 96-well plate can be regarded as a single stage in such a cascade. In a chromatography column, any separation taking place in a single stage is further magnified by the next stage in series. However, as long as a difference in adsorption capacities/rates for different constituents of a sample can be quantitated in a single well, the results obtained using PreDictor plates can be used to describe the same separation occurring in a column. The workflow in a batch experiment comprises the same steps as any column experiment equilibration of the medium with the desired buffer, sample application, wash, and elution (Fig 2) - and therefore the same types of study can be performed on both formats (Table 1). Medium in well Wash/ Equilibration Sample addition Wash 1–3 times Elution 1–3 times Incubation Mixing Vacuum filtration or centrifugation Mixing Mixing Vacuum filtration or centrifugation Waste Analysis Fig 2. Schematic illustration of the workflow of a batch experiment in the wells of a PreDictor plate. The same steps would be employed in a column experiment, i.e., equilibration, sample addition, wash, and elution. The gray color in the wells is chromatography medium, red shades (red and pink) describe different concentrations of protein solution, brown is medium with bound sample. 2.2 The batch experiment Batch experiments investigate the distribution of target protein(s) between the solid phase (chromatography medium) and the liquid phase. In a finite bath (batch) system, the amount of target protein is constant and is either free in solution or bound to the medium in the well. Initially when sample is added, none of the target protein is bound to the medium. After a certain incubation time, however, it becomes distributed between the liquid and solid phases (Fig 3). 28-9403-58 AA 7 Mass added = mass unbound + mass bound Filter-plate well Filter-plate well Phase separation Collection plate well t start t final Incubation time Fig 3. Schematic illustration of the mass balance in a single well. At tstart, all protein is in liquid phase. At tfinal, the protein is distributed between the solid and liquid phases. Two different approaches can be used to determine the distribution of a protein between the phases: 1). Calculate the amount of protein bound to the solid phase (mbound) as the difference between the amount added to the well (madded) and the amount remaining in the liquid phase (munbound): mbound = madded − munbound (1) 2). Perform a number of consecutive wash and elution steps to elute the protein. The amount of eluted protein (meluate) is the amount of protein bound (mbound) to the solid phase: mbound = meluate (2) When performing a full experiment with all steps, the full mass balance can be obtained by collecting and analyzing all fractions (flowthrough [FT], wash, elution). The amount added should be found either as unbound or bound protein: madded = mbound + munbound ↔ madded = meluate + mFT + mwash (3) The recovery can be calculated from the amount bound, unbound, and added: Recovery (%) = 100 × mbound + munbound madded (4) Yield can be calculated from the amount of protein eluted from the solid phase and the amount bound: m Yield (%) = 100 × m eluate bound (5) 2.3 Capacity determinations The two different approaches described above for determining the amount of protein bound to the medium can be used to calculate the capacity of a medium under a given set of experimental conditions. 8 28-9403-58 AA Equation 1 can also be expressed in terms of concentration (Eq. 6): mbound = madded – munbound ↔ Vmedium × q = Vliq × (co – cunbound) (6) where q is the binding capacity of the medium under given conditions at termination of incubation, Vmedium is the volume of medium in the well, Vliq is the volume of liquid in the well, co is the concentration of target protein at the start of the experiment (t=0), and cunbound is the concentration of target protein found in the flowthrough (liquid phase) at termination of incubation. Binding capacity can also be calculated from the protein concentrations found in the eluate(s): i q= 1 Veluate, i × celuate, i Vmedium (7) where i = each individual elution operation Always calculate capacities using flowthrough data unless the experimental conditions are such that all of the bound material is recovered in the eluate fractions. For most accurate results in the calculations, the volume of retained liquid in a well (Vr) has to be accounted for. Vacuum filtration and centrifugation do not entirely eliminate all liquid from PreDictor plate wells; liquid always remains both in the pores of the chromatography medium and in the filters of the plate wells after these procedures. With PreDictor plates, measurements have shown that the volume of liquid retained by the medium is approximately 60% of the medium volume (Vmedium), and that about 6 µl of liquid is retained in the filter. The retained liquid volume, Vr is therefore a function of the medium volume (Vmedium) in the well: Vr = 6 µl + 0.6 Vmedium (8) When accounting for retained volume in, for example, Equation 6, use co = csample × Vsample Vsample + Vr (9) instead of co = csample (10) Vliq = Vsample + Vr (11) and instead of Vliq = Vsample (12) 2.4 The adsorption isotherm A good understanding of thermodynamic and kinetic effects is needed to correctly describe a chromatographic separation. The thermodynamics of protein adsorption is described by the adsorption isotherm, which describes the relationship between concentration of protein in the liquid and solid phases at equilibrium under a given set of experimental conditions. Among many types of adsorption isotherms, the Langmuir isotherm is frequently used to describe protein adsorption. It is the only type of isotherm discussed in this handbook. Equation 13 describes the Langmuir isotherm for a single-component system. q= qmax × ceq Kd + ceq (13) 28-9403-58 AA 9 where qmax is the maximum saturation capacity, Kd is the equilibrium dissociation constant, ceg is the concentration of target protein in the liquid phase at equilibrium, and q is the binding capacity of the medium (concentration of target protein in the solid phase). The isotherm thus describes how q changes with ceq as illustrated in Figure 4. Binding capacity (solid phase), q (g/l) The isotherm provides information about the maximum capacity (qmax) of the medium for a given target molecule under given conditions. It also provides information on the binding strength (Kd). With good binding conditions (low Kd), the isotherm is relatively ”rectangular”, that is, it has a large plateau in which the capacity is relatively independent of the equilibrium concentration. The linear part of the isotherm is reached at low equilibrium concentrations. In this region, capacity is proportional to the equilibrium concentration (Fig 4). Plateau region 120 qmax 100 80 60 40 Kd = concentration at which q = 20 qmax 2 0 0 1 2 3 4 Conc. of target protein at equilibrium (liquid phase), ceq 5 Fig 4. A Langmuir isotherm where the maximum binding capacity, qmax=100 g/l and dissociation constant, Kd=0.1 g/l are both indicated by red lines. The plateau region where capacity is relatively independent of equilibrium concentration is indicated. 2.5 Incubation time Incubation time (also known as contact time) describes how long the target protein is in contact with the medium. Note that capacity varies with incubation time. For long incubation times, capacity will approach the equilibrium dictated by the isotherm (Fig 4). For shorter times, it will depend on the kinetics of uptake (Fig 5). If these kinetics are fast, the capacity difference between the two incubation times will be small. If slow, this difference will be large. If plate incubation times are compared with column residence times, incubation times are generally longer. This difference relates to differences in the techniques. Incubation time corresponds better to the total loading time in columns since it reflects the total time chromatography media beads are in contact with the sample. An incubation time of 60 min in PreDictor plate experiments will generally give a good estimate of the potential binding capacity. Shorter times may suffice, depending on the uptake kinetics. Binding capacity, q (g/l) For adsorption isotherm studies, longer incubation times (e.g., 3 h) are preferable. Some systems will nevertheless give an adequate estimation of the isotherm with an incubation time of just 60 min. Kinetics of uptake 160 140 30 120 60 15 100 80 60 40 10 5 2 20 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Incubation time (min) Fig 5. Curve demonstrating the kinetics of uptake, with binding capacity as a function of incubation time (2, 5, 10, 15, 30, and 60 min). 10 28-9403-58 AA 2.6 Phase ratio An important aspect to consider when setting up an experiment is the phase ratio ( ), which is the ratio between liquid volume and media volume. The mass balance equation (Eq. 6) can be rewritten with the phase ratio. q = co – c (14) where = Vliq Vmedium Binding capacity, q (g/L) For a given system, and co are constants, and q is therefore a linear function of the concentration of target protein in the liquid phase, c. The line described by the equation is called the operating line (Fig 6). Any measurement with the given system will be found along this line. 160 140 60 Adsorption isotherm 30 15 120 10 100 80 60 40 5 2 20 0 0 c = co 1 2 3 4 Conc. of target protein (liquid phase), c (g/l) Fig 6. A graphical illustration of capacity vs concentration. The adsorption isotherm, the operating line and incubation times along the operating line are shown. The operating line (red) is given by the experimental setup (Vliq, Vmedium, co). The operating line starts at c=co and ends at q defined by the isotherm. The slope of the operating line is equal to the negative of the phase ratio in the system, - = -Vliq/Vmedium. The operating line starts at c = co (time 0) and has a slope that equals - . During the experiment, the capacity will increase along this line. For short incubations, the capacity obtained will be located far down on the operating line, while for very long incubations (where equilibrium is approached), the capacity will approach the capacity dictated by the isotherm. If the phase ratio is low (i.e., large volume of medium per well or small liquid volume), the potential capacity of the medium is not being explored, because the intercept between the operating line and the underlying isotherm is not in the plateau region of the isotherm. In simple terms, there is too much medium to reach the maximum capacity of the medium, qmax. 1) The type of study will determine the medium volume to use. 2) Use small volumes for capacity studies. 3) Use larger volumes for wash and elution studies. Several different types of PreDictor plates have been developed for different applications. To select the relevant plate type, see Section 7. 2.7 Sample concentration and media volume The underlying adsorption isotherm strongly influences the capacity of the medium. For capacity studies, operations should take place in the plateau region of the isotherm, that is, where capacity is relatively independent of the concentration of target protein in the liquid phase. Set up the system so that the final concentration in the liquid phase corresponds to the capacities in the plateau region (refer to the operating line in Fig 6). Once the favorable binding conditions 28-9403-58 AA 11 have been found, the isotherm is relatively rectangular; only at very low concentrations will the capacity be dramatically affected by the concentration. For capacity experiments, ensure that an excess of target protein is added to the well so that the chromatography medium is overloaded (see the example in Section 2.9.1). For wash and elution studies, a somewhat larger medium volume in the wells is often advantageous to ensure that sufficient amounts of protein and/or impurities for detection and analysis are obtained. When larger volumes are used, multiple loads can be required to reach the desired amount bound (see Section 4.3). Understanding the effects of the phase ratio and the isotherm on capacity facilitates study planning. 2.8 Mixing In batch experiments, agitation is essential. Without sufficient agitation, the rate-limiting mass transfer will be different in wells compared with a chromatography column. Many factors affect mixing efficiency, for example, well diameter, liquid volume, liquid viscosity/density, particle density, and orbital amplitude of the shaking table. Figure 7 illustrates the effects of liquid volume and agitation speed. Use an agitation speed of 1100 rpm with a 3 mm circular centripetal movement and a liquid volume of 100 to 300 µl in the wells. Mix in all steps (equilibration, loading, wash, and elution)! A B Liquid volume decreased Volume too large. Mixing too slow C Stirring speed increased Mixing speed and volume optimized Volume correct. Mixing too slow Fig 7. Schematic illustration of the effect of liquid volume and agitation speed on mixing efficiency. The situation before (red) and during (blue) mixing is shown. A) Liquid volume too large, B) liquid volume and agitation speed optimized, C) agitation speed too low. 2.9 Types of studies The examples below refer to studies performed in PreDictor plates. 2.9.1 Binding studies Binding studies can be performed in single-medium plates, screening plates, or adsorption isotherm plates (see Section 7). With single-medium plates, 96 wells per plate are available for the experiment, while with screening plates, 24 or 32 wells are available per chromatography medium and plate. If isotherm plates are used, the response per condition is a whole isotherm and not just a capacity value. With isotherm plates, a maximum of 16 different conditions can be studied per plate. Use low-volume plates (2 or 6 µl) for binding studies in single-medium plates or screening plates. Low media volumes are easier to saturate, that is, load to maximum capacity. 12 28-9403-58 AA When planning the binding study, make sure to load enough protein. At termination of the incubation, the sample concentration should not decrease to less than 50% of the initial concentration. This ensures working in the plateau region of the adsorption isotherm and achieves the highest precision in analysis and calculations. Decreasing to 80% of initial concentration is, however, acceptable when still working in the plateau region of the adsorption isotherm. Here, capacity is relatively independent of equilibrium concentration. By rearranging Equation 6, the excess of protein used in the experiment can be calculated: q × Vmedium c 100 g/l × 2 µl ___ = 1– ________ = 1– ___________ = 0.75 Vliq × co co 200 µl × 4 g/l (15) assuming a binding capacity of 100 g/l, feed concentration of 4 g/l, 200 µl of liquid volume, and 2 µl of chromatography medium. Use an incubation time of 60 min. Shorter times may suffice if the uptake kinetics are fast. See the application example in Section 4.1. 2.9.2 Binding studies – time-dependent In the batch system, the chromatography medium is incubated for different periods of time in the presence of the feed/sample (Fig 8). A) B) Waste Sample Sample Start Time Elution Sample Vacuum filtration Flowthrough × Nconditions Medium Simultaneous separation of phases and subsequent concentration measurements Wash/Equilibration 1 2 3 4 5 6 7 8 9 10 11 12 A 2.5 min 2.5 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min 60 min 60 min B 2.5 min 2.5 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min 60 min 60 min C 2.5 min 2.5 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min 60 min 60 min D 2.5 min 2.5 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min 60 min 60 min E 2.5 min 2.5 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min 60 min 60 min F 2.5 min 2.5 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min 60 min 60 min G 2.5 min 2.5 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min 60 min 60 min H 2.5 min 2.5 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min 60 min 60 min End End Fig 8. A) Outline of experimental protocol for the high-throughput batch uptake method with variable incubation time. B) Example of a PreDictor 96-well plate layout that can be used. Four different conditions (indicated by green, yellow, orange, and blue shading) with six different incubation times (2.5, 5, 10, 15, 30, and 60 min) in quadruplicate are studied. Recommended incubation times (in minutes) are shown in each well. Studying dynamic binding capacity (DBC) as a function of process conditions (pH, conductivity, residence time, feed concentration, etc.) is typically one of the first investigations of downstream development. The aim is to optimize conditions with respect to DBC and robustness, yet the work, when performed using columns, is laborious and requires a substantial amount of valuable sample. PreDictor plates can be used for time-dependent studies and as outlined in Figure 8, the chromatography medium is incubated for different periods of time in the presence of the feed/ sample. Low-volume, single-medium plates (2 or 6 µl) work best (see Plate selection guide, Section 7.1). Using PreDictor plates to investigate time-dependent protein uptake on chromatography media (Fig 8A) allows prediction of DBC data as a function of residence time in a chromatography column. Studies can either be quantitative or qualitative, that is, enable relative ranking of rate of uptake. Qualitative study: Using different incubation times in the batch uptake system (e.g., 5 and 60 min) provides valuable information that can indicate the effect of residence time in a column, as schematically illustrated in Figure 9. 28-9403-58 AA 13 Binding capacity (solid phase), q Dynamic binding capacity (DBC) Short Incubation time Long Residence time Fig 9. Batch experiments (left), performed with a short and a long incubation time, give qualitative information on trends that can be observed in column experiments (right). Quantitative study: DBC versus residence time can be predicted based on data from a batch system, using a combination of modeling and experimental approaches (1–5). The rate of uptake of the target protein (and other components if desired) onto the chromatography medium is measured and subsequently described in a mathematical model. A similar model is then used to describe protein adsorption in a chromatography column and the DBC versus residence time relationship is simulated. The method is schematically illustrated in Figure 10. An application example that predicts the DBC of human IgG on MabSelect SuRe™ is described in Reference 6. Batch model Model parameters Batch correlations Incubation time Column model Column correlations Binding capacity (solid phase), DBC Binding capacity (solid phase), q In addition to determining DBC data, the method can be used to study the effect of incubation time on the purity of the target protein by monitoring changes in contaminant concentration with time. Residence time Fig 10. Outline of data and information flow for a quantitative method for determining the effect of residence time on DBC. The dashed line represents an optional method expansion. Batch experiments are shown on the left and the predicted DBC on the right. 2.9.3 Flowthrough studies (non-binding mode) Studies of chromatographic steps using flowthrough mode are performed where, instead of maximizing (or optimizing) the binding of the target protein, the focus is on maximizing contaminant binding while maintaining a high yield of the target protein in the flowthrough fraction. 2.9.4 Wash and elution studies Wash and elution studies are performed to optimize the purity and/or yield of the target protein. When the effect of load can influence the results, use similar loads in the plate and in the column experiments. Based on the specific application, the desired column load (in terms of percent of DBC) should be defined. In PreDictor plates (batch mode), this load is obtained from binding experiments in lowvolume plates (2 or 6 µl). Note that the phase ratio changes when scaling up to recommended plate volumes of 20 µl (first choice) or 50 µl (second choice) for wash and elution studies. Multiple loadings may be required to obtain the desired load with 20 or 50 µl plates as the maximum volume per load is 300 µl. An example using multiple loads is shown in Section 4.3. For some studies, the load is not necessarily critical. Section 4.4 shows such an elution study example. 14 28-9403-58 AA 2.9.5 Cleaning-in-Place (CIP) studies Traditionally, small-scale columns are used for CIP studies. However, such studies are labor intensive and time- and sample-consuming. In addition, small-scale columns are limited to evaluation of one CIP condition at a time. By using PreDictor plates, several cleaning protocols can be evaluated in parallel. Section 4.5 shows the development of a CIP protocol for a protein A affinity medium in PreDictor plates. 2.9.6 Adsorption isotherm studies Adsorption isotherms give information about binding strength (Kd) and potential binding capacity (qmax) under the conditions studied. With PreDictor plates, different approaches can be used to obtain adsorption isotherms. For example: 1) sample concentration is kept constant while the phase ratio is varied, 2) phase ratio is kept constant and the sample concentration is varied, and 3) combination of approaches 1 and 2. A) Binding capacity (solid phase), q The use of adsorption isotherm plates (Approach 1) simplifies the experimental protocol as no sample manipulation is necessary. The adsorption isotherm plates contain variable amounts of medium in the wells (Fig 11A). The same sample (same concentration and volume) is applied to all wells. Approach 2 uses single-volume plates and the initial concentration of sample is varied while the phase ratio is kept constant (Fig 11B). Thus, if single-volume plates are used, dilution of the sample is necessary in order to obtain different initial concentrations. For both approaches, varying the sample volume added to the wells is also possible. Note, however, that as the recommended Vsample is limited to between 100 and 300 µl, this technique must be used in combination with Approach 1 or 2 in order to cover the whole isotherm. An application example is given in Section 4.6. A B C D E F G H -β B) Binding capacity (solid phase), q Conc. of target protein (liquid phase), c A B C D E F G H Medium volume A B C D E F G H -β Conc. of target protein (liquid phase), c Sample conc. A B C D E F G H Medium volume Sample conc. Fig 11. Illustration of two methods to determine adsorption isotherms in a batch system. A) Constant initial concentration (co) and varying phase ratio ( ), B) Constant and varied initial concentration, co. Colored bars show variables and constants in the different methods to determine adsorption isotherms (see text for details). 28-9403-58 AA 15 2.9.7 Chromatography media screening PreDictor plates can be used to compare different chromatography media. PreDictor anion exchange (AIEX) and cation exchange (CIEX) media screening plates are available (see PreDictor plate selection, Section 7). Each type of screening plate is available either in low-volume (2 µl/6 µl) or high-volume formats (20 µl). The workflow used is the same as with the single-medium plates, except that 24 or 32 wells are available for each medium instead of 96 (Fig 12). PreDictor AIEX media screening plates 1 2 3 4 5 6 7 8 9 10 PreDictor CIEX media screening plates 11 12 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Capto™ Q 2 µl Capto DEAE 2 µl Q Sepharose™ Fast Flow 6 µl Capto adhere 6 µl Capto S 2 µl SP Sepharose Fast Flow 6 µl Capto MMC 6 µl A B C D E F G H Capto Q 20 µl Capto DEAE 20 µl Q Sepharose Fast Flow 20 µl Capto adhere 20 µl Capto S 20 µl SP Sepharose Fast Flow 20 µl Capto MMC 20 µl Fig 12. Plate layout of PreDictor AIEX and CIEX media screening plates. Low-volume plates contain 2 µl of medium/well (high-capacity Capto media) and 6 µl of medium/well (other media). High-volume plates contain 20 µl of medium/well for each of the media shown. Note that a multimodal ion exchanger (Capto adhere and Capto MMC) is included in each type of plate. Because of their special properties (7, 8), Capto adhere and Capto MMC can have a different optimal operation window than traditional ion exchangers. Capto adhere, for example, has been shown to bind the target protein at pH values significantly below the isoelectric point (Fig 13). The optimal conductivity can also differ from that of traditional ion exchangers. Traditional anion exchanger Capto adhere Net charge + Net charge pI + pI pH - pH Binding pH Binding pH Fig 13. Schematic illustration of a titration curve for a protein. Different binding pH should be expected on traditional ion exchangers and Capto adhere. In most cases, Capto adhere will bind the protein at a pH significantly below the isoelectric point (pI). 16 28-9403-58 AA 3. Practical considerations Sample prerequisite: Samples should be clarified prior to application using normal-flow filtration since unclarified samples can clog the wells. Analytics: The precision and sensitivity of the analytical method used can influence what can be achieved with a given experimental setup. Capacities can, for example, be calculated using flowthrough data or from elution data, as discussed in Section 2.3. If the amount bound is measured via the mass balance equation (Eq. 6), the analytical method must quantitate c = co – c. If the amount bound is measured from eluted fraction(s), the concentration in the eluate fractions must be quantifiable. A requirement for estimating capacity from eluted material is that close to 100% of the bound amount is eluted with the chosen elution conditions. For further discussion on the quality of data, see Section 5. The 96-well plate format allows a large number of conditions to be studied in parallel, which puts pressure on the analytical methods used and resources available. With this in mind, a sequential approach to the experimental setup can be considered. This can be a funnel approach, where the initial broad screening is performed on responses with rapid analytical methods. From this initial screening, conditions can be selected for responses that require more advanced (or laborious) analytical methods. Incubation time vs residence time: Incubation time (contact time) is not quantitatively comparable to residence time in columns. Incubation time better corresponds to the total loading time in columns since this reflects the total time the medium particles are in contact with the sample. If long incubation times are used, the measured capacity better reflects the equilibrium capacity of the medium. For short incubation times, the rate of mass transfer will affect the measured capacity (Fig 5). Pipetting, buffer preparation, and sample preparation: Buffer and sample preparation can be relatively time-consuming with a manual workflow. If a robotic system is available, buffer preparation can be set up on the robot, thus simplifying this labor-intensive activity. Pipetting should be performed with multipipettes (8- or 12-tip) or by a robot. For practical transfer in a reasonable time, buffers and samples should be prepared in and dispensed from deep-well plates (4, 8, 12, 48 wells) according to the experimental plate layout. Minimizing leakage from the plate during operation: To minimize risk of leakage through the bottom filter, it is important to avoid direct contact between the PreDictor plate outlets (the drips on the bottom) and any surface. Repeated loadings and long incubation times using buffers or samples containing certain detergents can induce leakage through the filter. Recommendations for minimizing leakage when working with detergents are provided in the PreDictor plate Instructions (Instructions 28-9258-34). Mixing during incubation: Adequate mixing must be used during all incubation steps in order to keep the chromatographic medium suspended in the liquid phase (see Section 2.8 and Reference 6). UV as quantitation method: If UV absorbance is used to determine concentrations, ensure that the calibration curve is linear and covers the range of concentrations to be measured; extrapolation is not recommended. Use standard procedures to compensate for potential light scattering effects, blank absorbance, and pathlength. Transferring experiments from low- to high-medium volume plates: When transferring experiments from low-volume plates to high-volume plates, the capacity obtained on the high-volume plate can be much lower than on the low-volume plate. The reason for this is the 28-9403-58 AA 17 different phase ratios obtained with the different plates containing different media volumes. Multiple loading will possibly have to be performed in order to reach the desired load (Fig 17). Evaporation and sealant: When longer incubation times are used (more than 60 min) as in, for example, adsorption isotherm studies, consider sealing the plates to prevent evaporation. GE Healthcare microplate foils (96 well), code number BR-1005-78 can be used. These microplate foils are transparent, self-adhesive plastic films where the adhesive does not cover the well. (avoids contact between the sample and the adhesive). Liquid compatibility: PreDictor plates and the chromatography media with which they are prefilled are compatible with all solutions commonly used in the purification of biopharmaceuticals, see Instructions 28-9258-34 for details. Plate re-use: PreDictor plates are disposable items. Repeated use can cause leakage as well as cross-contamination between experiments. 18 28-9403-58 AA 4. Application examples 4.1 Screening binding conditions on Capto S using PreDictor plates This application presents a binding study performed with a 60 min incubation. The binding capacity of Capto S for two proteins, conalbumin and α-chymotrypsin, was investigated at different ionic strengths and pH. The data obtained were compared to results obtained with column experiments (9). Experimental: PreDictor plate Plate: PreDictor Capto S, 2 µl Sample: 3.5 g/l conalbumin or α-chymotrypsin in each equilibration buffer Equilibration/loading buffers: Fifteen different buffers comprising 20 mM sodium acetate at three pH levels (4.25, 4.75, 5.25) supplemented with sodium chloride to obtain the desired ionic strengths (0.02, 0.05, 0.08, 0.12, 0.15) Steps: Equilibration 1–3: 200 µl equilibration buffer, 1 min incubation Sample loading: 200 µl sample solution, 60 min incubation Mixing was performed on an orbital shaker at 1100 rpm during all incubation steps. Liquids were removed by vacuum filtration. The concentration of unbound protein was quantitated in the flowthrough from the loading step by reading UV absorbance. The amount bound was determined from the mass balance equation (Eq. 6). Experimental design and plate layout is shown in Figure 14. Conalbumin α-Chymotrypsin Fig 14. Plate layout for the experiment described in section 4.1. The 15 different conditions are shown in different colors. Six wells (empty cells) were not used. Experimental: Column chromatography Column: 2 ml of Capto S packed in a Tricorn™ 5/100 column Sample: Approx. 4 g/l conalbumin or α-chymotrypsin in each equilibration buffer Equilibration/loading buffers: 15 different buffers. 20 mM sodium acetate at three pH levels (4.25, 4.75, 5.25) supplemented with sodium chloride to obtain the desired ionic strengths (0.02, 0.05, 0.08, 0.12, 0.15) Residence time: 2 min 28-9403-58 AA 19 By recording the UV-signal during sample load the dynamic binding capacity at 10% breakthrough was calculated. Results and discussion The two proteins behave differently with respect to ionic strength. α-Chymotrypsin behaves in a traditional manner with the highest capacity obtained at low conductivity (Fig 15A and C). Conalbumin, on the other hand, behaves in a non-traditional manner (Fig 15B and D) (9, 10, 11). The trends observed with PreDictor plates (Fig 15A and B) are essentially the same as those obtained in column experiments (Fig 15C and D). Binding capacities in plates obtained with a 60 min incubation correspond very well to dynamic binding capacities obtained in columns with a residence time of 2 min. This is because incubation time better corresponds to the total loading time in columns since it reflects the total time the media particles are in contact with the sample. B) 250 pH 4.25 180 pH 4.75 160 200 pH 5.25 Binding capacity (mg/ml) Binding capacity (mg/ml) A) 150 100 50 pH 4.25 pH 4.75 140 pH 5.25 120 100 80 60 40 20 0 0 0 0.05 0.10 Ionic strength (M) 0.15 0 0.20 C) 0.05 0.10 Ionic strength (M) 0.15 0.20 D) 160 pH 4.25 Dynamic binding capacity (mg/ml) Dynamic binding capacity (mg/ml) 250 pH 4.75 pH 5.25 200 150 100 50 pH 4.25 140 pH 4.75 pH 5.25 120 100 80 60 40 20 0 0 0 0.05 0.10 Ionic strength (M) 0.15 0.20 0 0.05 0.10 Ionic strength (M) 0.15 0.20 Fig 15. Determination of loading conditions for α-chymotrypsin and conalbumin on Capto S. Binding capacities of α-chymotrypsin A) and conalbumin B) after 60 min incubation in PreDictor Capto S, 2 μl plates. Dynamic binding capacities at 10% breakthrough for α-chymotrypsin C) and conalbumin D). The residence time was 2 min and the column was a Tricorn 5/100 (CV 2 ml). 4.2 Effect of incubation time, pH, and ionic strength on the binding of amyloglucosidase to Capto DEAE Taking a time-dependent study all the way from plate data to predicted dynamic binding capacity (DBC) values in a column is not always necessary. As shown below, valuable qualitative information can be obtained from an incubation time study without predicting DBC. An incubation time of 60 min is generally recommended for capacity studies. Shorter and longer incubation times provide more data on how optimal conditions change over time. 20 28-9403-58 AA Experimental: PreDictor plate Plate: PreDictor Capto DEAE, 2 µl Sample: 4 g/l amyloglycosidase in each equilibration buffer Equilibration/loading buffers: 30 mM buffer, Tris pH 8 and 9, or bis-Tris pH 6 and 7, and 12 levels of total ionic strengths between 0.02 and 0.5 M at each pH. Sodium chloride was used to obtain the desired ionic strengths Steps: Equilibration 1–3: 200 µl equilibration buffer, 1 min incubation Sample loading: 150 µl sample solution at three different incubation times; 2 min, 60 min, or 20 h. Mixing was performed on an orbital shaker at 1100 rpm during all incubation steps. Liquids were removed by vacuum filtration at -300 mbar for 5 s during all steps. The filtrate fraction from the loading was collected in a UV-readable multiwell plate and absorbance at 280 nm was read. The absorbance was used to calculate protein concentration by applying a standard curve prepared using protein stock solutions. Binding capacities were calculated from the mass balance equation (Eq. 6). Results and discussion Contour maps describing the effects of ionic strength, pH, and incubation time on capacity were generated directly from raw data (i.e., no modeling or fitting was involved). The contour maps show how optimal conditions for capacity vary with incubation time. In this study, the pH optimum (pH 7) was the same for all incubation times, but the optimal ionic strength varied from 0.07 at 2 min, through 0.04 at 60 min, to 0.02 at 20 h (Fig 16). This is an important result from a process development perspective as the shift in optimal conditions observed can be expected as an effect of column residence time. The maps shown in Figure 16, generated from SigmaPlot™ v8.0 software (Systat Software Inc.), are characteristic for ion exchange situations where changes in ionic strength and pH have opposite effects on intraparticle mass transfer rates and on equilibrium capacity. The reduced equilibrium capacity caused by an increase in ionic strength is balanced by improved intraparticle mass transfer resulting in a binding capacity maximum at intermediate ionic strength. The equilibrium capacity effect dominates at longer incubation times, shifting the maximum to lower ionic strengths. This type of non-traditional ion exchange behavior (9, 10, 11) can also be seen for conalbumin in Figure 15. For further details of this study, see Reference 6. A) B) C) 0 mg/ml 50 mg/ml 100 mg/ml 150 mg/ml 200 mg/ml 250 mg/ml Ionic strength (mM) 0.14 0.12 0.10 0.80 0.06 0.04 0.02 6.5 7.0 7.5 pH 8.0 8.5 6.5 7.0 7.5 pH 8.0 8.5 6.5 7.0 7.5 8.0 8.5 pH Fig 16. Contour maps showing the effect of ionic strength and pH on binding capacity of Capto DEAE for amyloglucosidase over A) 2 min, B) 60 min, and C) 20 h. PreDictor plates filled with 2 µl of chromatography medium per well were used for the experiment. 28-9403-58 AA 21 4.3 Optimization of the wash step for MabSelect SuRe The possibility of reducing host cell protein (HCP) levels in the elution pool from a protein A chromatographic step by introducing an intermediate wash step was investigated. The effect of 17 different intermediate wash buffers (variations in pH, NaCl concentration, and n-propanol concentration), as well as the effect on MAb yield was evaluated. The study was performed both in PreDictor plates and by column chromatography to check the correlation between the results obtained with the two formats. Experimental: PreDictor plate Plate: PreDictor MabSelect SuRe, 50 µl Sample: MAb feed with a MAb concentration of 1.3 g/l Equilibration buffer: 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 Intermediate wash buffers: 17 different buffers (variations in pH, NaCl and n-propanol) Elution buffer: 20 mM sodium citrate, pH 3.6 Steps: Equilibration 1–3: 300 µl equilibration buffer, 1 min incubation Sample loading1: To reach a capacity of 23 g/l as used in the chromatography step described later, multiple loadings were needed; 3 × 300 µl of MAb feed was loaded each time and incubated for 20 min Wash 1 and 2: 200 µl equilibration buffer, 1 min incubation Wash 3 and 4: 200 µl intermediate wash buffer, 1 min incubation Wash 5 and 6: As Wash 1 and 2, 1 min incubation Elution 1–3: 200 µl elution buffer. The medium was incubated with elution buffer for 2 min before drainage. The three consecutive eluates were collected into three different UV-readable plates for analysis. Liquid was removed by centrifugation and incubation was performed on a shaker (1100 rpm) in all steps. A) B) 23 0 0.5 1.0 1.5 2.0 Conc. of the target protein (liquid phase), c Binding capacity (solid phase), q Multiple loadings: As discussed above, the underlying isotherm and phase ratio control the capacity achieved. In this application, an incubation time of 20 min was enough for the capacity to approach the isotherm capacity. Because of the relatively low phase ratio (6 = 300/50) and the relatively low feed concentration of the MAb, only about 1/3 of the desired load was reached by loading once. This was overcome by loading three times to reach a capacity or load of 23 g/l (Fig 17A). In this particular experiment, it would have been better to use a 20 µl PreDictor plate. If such a plate was used, the phase ratio would have increased from 6 to 15 (300/20) and the desired load (capacity) would have been attained in only one load (Fig 17B). Other options to reach the desired load are to change the phase ratio by using a different liquid phase volume (Vliq), and/or using a different initial concentration, Co . Binding capacity (solid phase), q 1 23 0 0.5 1.0 1.5 2.0 Conc. of the target protein (liquid phase), c Fig 17. Schematic illustration of how to reach the desired load of 23 g/l. Multiple loadings on a 50 µl PreDictor plate A) or a single load on a 20 µl plate B). Circles represent the initial starting point (blue) and final point (purple) for each incubation. MAb concentration (UV absorbance) and HCP concentration (ELISA) were analyzed in the eluates. 22 28-9403-58 AA Experimental: Column chromatography Column: HiTrap MabSelect SuRe, 1 ml The steps employed are described in detail here: Step Column . volume (CV) Flow (cm/h) Description Equilibration 10 250 20 mM sodium phosphate, 150 mM NaCl, pH 7.4. Sample load 18 63 23 g/l medium (18 ml 1.3 g/l clarified MAb feed). 2.4 min residence time Wash 6 250 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 Intermediate wash 2 250 Intermediate wash buffer Wash 2 250 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 Elution - 100 Fractionation with watch function based on UV levels (fractionation started at UV = 500 mAU and stopped at UV = 400 mAU) Regeneration 4 250 100 mM sodium citrate, pH 3.0 CIP 10 100 0.5 M NaOH, 15 min contact time Re-equilibration 10 250 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 MAb concentration (UV absorbance) and HCP concentration (ELISA) were analyzed in the collected eluate. Results and discussion Compared with the reference run using a standard wash in a column, none of the intermediate washes improved the wash procedure with respect to HCP levels in the eluate fractions (Fig 18). Four of the intermediate wash buffers (expts. 1, 2, 10, and 11 in Fig 18) even proved detrimental in the column experiments as HCP levels higher than the reference were obtained. The plate experiments gave the same results except with wash buffer 2, where a detrimental effect was not observed. With respect to yield, some of the intermediate washes (expts. 2, 7, 14, and 16 in Fig 18) proved detrimental, while for others, yield was in the same range as the reference runs. Correlation between the yields obtained with the two methods was good. 8000 Plate Column 7000 HCP in eluate (ppm) 6000 5000 4000 3000 2000 1000 0 1 2 3 4 5 6 7 8 9 10 11 12 Intermediate wash buffer 13 14 15 16 17 Ref. 100 Plate Column 90 MAb yield in eluate (%) 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 Intermediate wash buffer 13 14 15 16 17 Ref. Fig 18. Effect of different wash buffers on the host cell protein (HCP) levels and monoclonal antibody yield in the elution pool from MabSelect SuRe. Results from plate experiments using PreDictor MabSelect SuRe, 50 µl (red) are compared with column experiments using HiTrap MabSelect SuRe, 1 ml (blue). 28-9403-58 AA 23 4.4 Screening elution pH on MabSelect SuRe A PreDictor MabSelect SuRe 50 µl plate was used to screen elution pH for five monoclonal antibodies. A simulated pH gradient was set up in the plates and the yield at different pH was measured. Results obtained with PreDictor plates were compared with results obtained with pH gradient elution in a Tricorn 5/100 column packed with 2 ml of MabSelect SuRe. Experimental: PreDictor plate Plate: Equilibration buffer: Final wash buffer: Elution buffers: Samples: PreDictor MabSelect SuRe, 50 µl 20 mM sodium phosphate, 150 M NaCl, pH 7.4 20 mM sodium citrate, pH 6.0 20 mM sodium citrate, pH 5.8–3.0 1.3 g/l of five different MAb’s, MAb no. 1, 3, 4, and 5 in equilbration buffer, MAb no. 2 in unpurified clarified NSO-cell supernatant Steps: Equilibration 1–3: Sample loading: Wash 1 and 2: Wash 3: Elution 1–3: 200 µl equilibration buffer, 1 min incubation 300 µl MAb sample solution incubated for 20 min 200 µl equilibration buffer, 1 min incubation 200 µl final wash buffer, 1 min incubation 200 µl of the different elution buffers, 1 min incubation Liquid was removed by centrifugation and incubation was done on a shaker (1100 rpm) in all steps. The plate layout is shown in Figure 19. All fractions were collected in UV-readable plates and the concentration of MAb in the different fractions was determined from absorbance measurements at 280 nm. Control/ No MAb added MAb 1 MAb 2 MAb 3 MAb 4 MAb 5 pH 5.8 4.4 5.8 4.4 5.8 4.4 5.6 4.2 5.6 4.2 5.6 4.2 5.4 4.0 5.4 4.0 5.4 4.0 5.2 3.8 5.2 3.8 5.2 3.8 5.0 3.6 5.0 3.6 5.0 4.8 3.4 4.8 3.4 4.6 3.2 4.6 3.2 4.4 3.0 4.4 3.0 5.8 4.4 5.8 4.4 5.8 4.4 5.6 4.2 5.4 4.0 5.6 4.2 5.6 4.2 5.4 4.0 5.4 5.2 4.0 3.8 5.2 3.8 5.2 3.8 3.6 5.0 3.6 5.0 3.6 5.0 3.6 4.8 4.6 3.4 4.8 3.4 4.8 3.4 4.8 3.4 3.2 4.6 3.2 4.6 3.2 4.6 4.4 3.2 3.0 4.4 3.0 4.4 3.0 4.4 3.0 Fig 19. Plate design for the elution study using PreDictor MabSelect SuRe, 50 µl plate, a simulated pH gradient elution evaluation. Experimental: Column chromatography Column: Samples: Equilibration/ loading buffer: Wash buffer 1: Wash buffer 2: Elution buffer: 2 ml of MabSelect SuRe packed in Tricorn 5/100 column 1.3 g/l of five different MAb’s, MAb nos. 1, 3, 4, and 5 in equilibration buffer, MAb no. 2 in unpurified clarified NSO-cell supernatant were loaded to 0.7 mg MAb/ml chromatography medium. 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 20 mM sodium citrate, pH 6.0 A linear pH-gradient during 15 column volumes decreasing from pH 6 to 3 Absorbance at 280 nm was used for tracing elution. 24 28-9403-58 AA Results Figure 20 shows the MAb recovery in the elution fractions from the PreDictor plates together with the corresponding chromatogram from the column experiment. Good correlation between the formats was obtained. Generic elution at pH 3.6 was shown with four of the five MAbs on both formats. The fifth MAb behaved differently. In the plates it started to elute as early as pH 5.8, although most of it eluted at pH 4.8. In the column, this MAb eluted in a broad peak at pH 4.8. For further details, see Reference 12. MAb MAb 1 1 PreDictor plate 100 Abs 280 nm (mAU) Recovery (%) 80 60 40 20 0 5.8 Chromatography column 1990 1490 990 490 –10 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 pH 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pH 4.2 4.0 3.8 3.6 3.4 3.2 3.0 3.6 3.4 3.2 3.0 3.6 3.4 3.2 3.0 3.6 3.4 3.2 3.0 MAb MAb 2 2 PreDictor plate 100 Abs 280 nm (mAU) Recovery (%) 80 60 40 20 0 5.8 Chromatography column 2000 1500 1000 500 0 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 pH 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pH 4.2 4.0 3.8 MAb MAb 3 3 PreDictor plate 100 Abs 280 nm (mAU) Recovery (%) 80 60 40 20 0 5.8 Chromatography column 2000 1500 1000 500 0 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 pH 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pH 4.2 4.0 3.8 MAb MAb 4 4 PreDictor plate Recovery (%) 80 60 40 20 0 5.8 Chromatography column 2000 Abs 280 nm (mAU) 100 1500 1000 500 0 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 pH 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pH 4.2 4.0 3.8 MAb MAb 5 5 PreDictor plate Recovery (%) 80 60 40 20 0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 pH Chromatography column 2000 Abs 280 nm (mAU) 100 1500 1000 500 0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 pH 4.2 4.0 3.8 3.6 3.4 3.2 3.0 Fig 20. Comparison of elution patterns on MabSelect SuRe for PreDictor MabSelect SuRe, 50 µl relative to column chromatography with 2 ml of the medium packed in a Tricorn 5/100 column. The bars in the histograms correspond to the cumulative relative amount of MAb recovered after elution at different pH for each MAb. The corresponding chromatograms are shown adjacent to the histograms. 28-9403-58 AA 25 Comments Note the low loads used in these experiments. In the column experiment, an analytical load (0.7 mg MAb/ml medium) was used. For simplicity, the same sample was used in the plate, which gave a load of 8 mg/ml medium. If load can affect the results, use the same load in the plates as in the column experiments. See Sections 2.6, 2.7, and 4.3 for discussion on choosing phase ratio and initial concentration in order to reach a desired load. 4.5 Rapid development of CIP protocols for affinity media Commonly used protein-based affinity ligands such as protein A are very stable, which can allow several hundred monoclonal antibody purification cycles starting from mammalian cell culture. Critical to maximum media lifetime is the use of efficient and media-compatible cleaning-in-place (CIP) solutions. CIP protocols for MabSelect SuRe and MabSelect developed using PreDictor plates (13) are presented below. Note that even though CIP studies show good results, the plates should not be reused as repeated loading of protein can cause leakage. Screening cleaning efficiency Fouling in a protein A column can only appear after several purification cycles. Therefore, artificial fouling of the medium in 96-well filter plates was performed in order to evaluate the cleaning efficiency of various CIP conditions in a high-throughput manner (Fig 21). Medium in well 1. 2. Equilibration Feed application 3. Wash 4. Fouling agent incubation 5. Wash 6. Elution 7. Equilibration incubation Fig 21. Schematic description of artificial fouling of media. Media in all wells were mixed at every step and liquid was removed by centrifugation between each step. Steps 1, 3, 5, and 7 show wash or equilibration with PBS, pH 7.4. Step 2 is E. coli lysate spiked with polyclonal human IgG (3 g/l) and added to MabSelect SuRe (PreDictor MabSelect SuRe, 50 µl medium/well). Incubation was 45 min. Step 4 shows the addition of fouling agent (2.9 M ammonium sulfate, 0.6 M phosphoric acid, pH 2.5). Incubation was overnight. Plates were covered with adhesive film to prevent evaporation. Step 6 shows sodium citrate, pH 3.0, which was applied to elute most of the bound protein. Several potential CIP agents were evaluated for cleaning efficiency of the artificially fouled medium. The agents were added to the wells after step 7. Following a 15 min contact time, the cleaning solutions were removed and the wells washed several times with PBS and water. Medium samples were removed and boiled with SDS sample buffer for analysis by SDS-PAGE (Fig 22). MabSelect SuRe, agarose-based protein A medium 1. PBS (control) 2. 0.1 M NaOH 3. 0.2 M NaOH 4. 0.5 M NaOH 5. 0.1 M NaOH, 30% isopropanol 6. 0.1% commercial detergent 7. 0.1 M NaOH, 1 M NaCl 8. 0.1 M NaOH followed by 0.1 M citric acid 9. 0.1% commercial detergent followed by 0.1 M NaOH 10. Blank (control) 1 2 3 4 5 6 7 8 9 10 Fig 22. Proteins remaining on the MabSelect SuRe medium after cleaning with different agents. SDS-PAGE (Deep Purple™ stained) was used to evaluate cleaning efficiency. NaOH proved to be very effective. 26 28-9403-58 AA Screening protein A media stability Media stability was determined in a separate study by measuring functionality (i.e., IgG binding capacity) before and after exposure to cleaning chemicals. Determination of IgG binding capacity PreDictor 96-well filter plates: prefilled with MabSelect SuRe or MabSelect™ (6 µl medium/well) were used to determine IgG binding capacity. The binding capacity was determined by saturating the media with 200 µl of IgG at a concentration of 1.5 mg/ml. After incubating for 60 min on an orbital shaker (1100 rpm), unbound protein was collected and analyzed for IgG concentration. Binding buffer (PBS, pH 7.4) was used as wash and the bound protein was eluted with 0.1 M sodium citrate, pH 3.0. Static binding capacity was determined. Effect of cleaning agents on chromatography media stability PreDictor NaOH and n-propanol concentrations were varied between 0.01 and 1.09 M and 0% to 10%, respectively. The media were stored in the CIP solution for 18 h, which corresponds to 180 cycles with 30 min CIP/Sanitization-in-place (SIP) every fifth cycle. MODDE™ software (Umetrics, reference 14) was used for setting up the experiments and evaluating the results (Fig 23). MabSelect, 20°C MabSelect SuRe, 20°C 10 10 n-propanol concentration (%) 98 8 8 6 6 90 4 82 74 66 58 50 2 96 94 92 90 88 86 4 2 84 0 0 0.0 0.5 0.0 1.0 MabSelect, 40°C 0.5 1.0 MabSelect SuRe, 40°C 10 10 50 30 8 60 8 20 0 6 40 0 30 20 6 70 4 4 60 80 40 50 10 2 0 0 0.0 0.5 10 2 1.0 90 0.0 70 0.5 1.0 NaOH concentration (M) Fig 23. Contour plots showing remaining IgG binding capacity (%) after exposure to CIP chemicals. PreDictor MabSelect SuRe (alkali-stabilized protein A maintained IgG binding capacity at higher NaOH concentrations and elevated temperatures compared with PreDictor MabSelect (recombinant protein A). Conclusions PreDictor 96-well filter plates filled with chromatography media enable parallel screening of CIP agents for cleaning efficiency and media compatibility studies. This approach allows a rapid design of suitable CIP protocols for different media and feed-stock combinations. NaOH at concentrations from 0.1 to 0.5 M proved effective for cleaning agarose-based protein A media fouled with E. coli lysate. 28-9403-58 AA 27 4.6 Adsorption isotherms with PreDictor plates Adsorption isotherms were constructed for adsorption of human IgG on MabSelect SuRe. A PreDictor MabSelect SuRe adsorption isotherm plate was used and isotherms were constructed from flowthrough and elution data. Experimental: Plate: PreDictor MabSelect SuRe isotherm Equilibration/loading buffer: 20 mM sodium phosphate pH 7.4 Wash buffer: 20 mM sodium phosphate pH 7.4 Elution buffer: 20 mM sodium citrate, pH 3.6, 150 mM NaCl Sample: Polyclonal human IgG, hIgG (Gammanorm™, Octapharma AB) at concentration 4.0 g/l in equilibration/loading buffer Steps: Equilibration 1–3: 200 µl equilibration buffer, 1 min incubation Sample loading: 200 µl sample incubated for 6 h Wash 1–3: 200 µl equilibration buffer, 1 min incubation Elution 1–3: 200 µl elution buffer, 1 min incubation Mixing was done on an orbital shaker at 1100 rpm in all steps. Liquids were removed by vacuum filtration. 1 2 3 4 5 6 7 8 9 10 11 12 A 50 µl 50 µl 20 µl 20 µl 8 µl 8 µl 6 µl 6 µl 4 µl 4 µl 2 µl 2 µl B 50 µl 50 µl 20 µl 20 µl 8 µl 8 µl 6 µl 6 µl 4 µl 4 µl 2 µl 2 µl C 50 µl 50 µl 20 µl 20 µl 8 µl 8 µl 6 µl 6 µl 4 µl 4 µl 2 µl 2 µl D 50 µl 50 µl 20 µl 20 µl 8 µl 8 µl 6 µl 6 µl 4 µl 4 µl 2 µl 2 µl E 50 µl 50 µl 20 µl 20 µl 8 µl 8 µl 6 µl 6 µl 4 µl 4 µl 2 µl 2 µl F 50 µl 50 µl 20 µl 20 µl 8 µl 8 µl 6 µl 6 µl 4 µl 4 µl 2 µl 2 µl G 50 µl 50 µl 20 µl 20 µl 8 µl 8 µl 6 µl 6 µl 4 µl 4 µl 2 µl 2 µl H 50 µl 50 µl 20 µl 20 µl 8 µl 8 µl 6 µl 6 µl 4 µl 4 µl 2 µl 2 µl Fig 24. Plate layout for the adsorption isotherm experiment using PreDictor MabSelect SuRe isotherm plate. Values indicate volumes of MabSelect SuRe in the wells. Calculations: UV absorbance was used to calculate protein concentration. Binding capacities, expressed as mass of protein per unit sedimented medium, were calculated from flowthrough data or elution data. For flowthrough data, c was measured directly from the flowthrough and binding capacity, q calculated from the mass balance equation (Eq. 6). Retained liquid, Vliq (see Section 2.3) was accounted for in the calculations. Elution was performed in three consecutive elutions. Binding capacity, q, was calculated according to Equation 16 summarizing the amount of protein found in each elution (i ). The concentration of the target protein, c, was calculated according to Equation 17. 28 28-9403-58 AA i q= Veluate, i × celuate, i 1 (16) Vmedium c= o –q Vmedium (17) Vliq Results and discussion Binding capacity (solid phase), q (g/l) The obtained isotherms are shown in Fig 25. Values of the parameters in the Langmuir adsorption isotherm (Eq. 13) were estimated from capacities calculated from flowthrough and elution data. The least square estimates for flowthrough data were qmax = 57 g/l and equilibrium dissociation constant, Kd = 0.15 g/l, respectively. For elution data, the least square estimates were qmax = 59 g/l and Kd = 0.25 g/l, respectively. 70 Flowthrough data Elution data 60 50 40 30 20 10 0 0 1 2 3 4 Conc. of target protein (liquid phase), c (g/l) Fig 25. Adsorption isotherms for hIgG on MabSelect SuRe. Capacities were calculated either from flowthrough data (blue) or elution data (red). As mentioned in Section 2.9.6, several different approaches can be used to obtain an isotherm. If single-medium plates are used, the concentration of sample added to the wells can be varied. Isotherm plates containing variable volumes of medium in the wells are an alternative. In this experiment, the latter approach was chosen to avoid sample dilution and volume adjustment. To illustrate the results, capacities were calculated either from flowthrough data or from elution data. In this application, similar results were obtained with both approaches. Note that if elution data are used to create adsorption isotherms, all of the bound material is assumed to be collected in the elution fractions i.e., elution conditions are optimal. Alternatively, flowthrough data may be used to construct the adsorption isotherm. Use flowthrough data when constructing the isotherm. For further reading on adsorption isotherms, see Reference 15. 28-9403-58 AA 29 30 28-9403-58 AA 5. Quality aspects of the PreDictor plate workflow An important aspect of using PreDictor plates is that chromatography media volume reproducibility is good both between wells within a plate and between plates. Studies of reproducibility between wells and plates, replication, and method variability vs media volume variability are presented below (16). 5.1 Functional testing of reproducibility The reproducibility of PreDictor plates was studied by testing the binding capacity of 113 plates from continuous production. Seven media were tested and the volume in each well was measured. Each plate thus gave 96 data points and 10 848 wells were tested in total. High reproducibility within plates was obtained in all cases. Relative standard deviations (RSDs) were in the order of 1% to 5% (Fig 26). PreDictor plate 1 PreDictor plate 2 60 60 Well-binding capacity Well-binding capacity 50 Well-binding capacity (mg IgG/ml medium) Well-binding capacity (mg IgG/ml medium) 50 40 30 20 10 40 30 20 10 0 0 0 20 40 60 80 100 120 0 20 Well number in plate 40 60 80 100 120 Well number in plate Fig 26. Evaluation of the hIgG-binding capacity of two different PreDictor MabSelect SuRe, 6 μl plates. The light blue horizontal bands denote 95% confidence limits for each plate. Each veritical blue bar denotes the binding capacity for a specific well. The Relative Standard Deviation (RSD) was 1% to 1.2% in each plate. 5.2 Increasing the robustness of experiments Even though throughput can be maximized by performing singular measurements, replication of experiments is generally recommended. This increases the robustness of the batch experiment by reducing variability and decreasing the risk of repeated runs for specific conditions. The estimated variability within and between plates was used to calculate 95% confidence intervals for triplicate well measurements (Fig 27). 10 Capto Q Capto S Capto DEAE 8 95% of all triplicates within +/- (%) 95% of all triplicates within +/- (%) 10 6 4 2 0 Capto MMC Capto adhere MabSelect MabSelect SuRe 8 6 4 2 0 2 20 50 Chromatography media volume (µl) 6 20 50 Chromatography media volume (µl) Fig 27. A) Reproducibility of PreDictor plates filled with ion exchangers Capto Q, Capto S, and Capto DEAE tested with different proteins. B) Reproducibility of PreDictor Capto MMC, Capto adhere, MabSelect, and MabSelect SuRe tested with polyclonal IgG and BSA. 28-9403-58 AA 31 The uncertainty (+/-) in binding capacity of triplicate samples was in all cases less than 7%. This uncertainty decreases at larger media volumes. However, measuring binding capacity with larger volumes can require unrealistically high initial protein concentrations or excessive sample volumes. 5.3 Method variability vs chromatography media volume variability Blank run variability was used as an estimate of “non-media volume-related” error sources (e.g., factors such as pipetting, plate handling, and detection). UV detection was chosen for this study as it is well-suited for the amounts of protein used. In this case, the contribution from media volume variability was similar to the contribution from plate handling and detection (Fig 28). 6 Overall uncertainty in experiment “Non-media volume-related” error sources Contribution from media volume variability Relative standard deviation, RSD (%) 5 4 3 2 1 0 2 20 Chromatography media volume (µl) Fig 28. Overall RSD and variability contributions from method handling and chromatography media volume. The relative influence of chromatography media volume variability on the total end uncertainty of an experiment may be even smaller when less precise detection methods are used (Fig 29). Relative standard deviation, RSD (%) 12 If detection method has an RSD of 10% Four replicates in detection method, corresponding to a detection method RSD of 5% 16 replicates of detection method, corresponding to a detection method RSD of 2.5% 10 8 6 4 2 0 1 2 3 4 Number of replicates of batch experiment 5 6 Fig 29. The effect of replicating a detection method with an relative standard deviation (RSD) of 10% on the overall uncertainty in the experiment. Different curves show the effect of changing the number of detection replicates. The x-axis shows the effect of replication of the batch experiment. A detection RSD of 2.5% corresponds roughly to UV measurements in PreDictor plates. Calculations are based on a 2 µl chromatography medium volume. Assay replication should be considered if less precise detection methods are used. However, as stated earlier, true experimental replication is still recommended to ensure robust and efficient process development in PreDictor plates. 32 28-9403-58 AA Conclusions The reproducibility of chromatography media volume in PreDictor plates is good (RSD in binding capacity of 1.5% to 5% between wells). Replicate experiments should be performed to maintain the robustness of the high-throughput approach in PreDictor plates. The uncertainty in detection methods is of more concern than the chromatography media volume variability, which is the same level as error sources from UV-detection and liquid handling. 28-9403-58 AA 33 34 28-9403-58 AA 6. Assist software 6.1 Assist software workflow Assist software helps chromatography process developers design and evaluate PreDictor plate experiments by facilitating the high-throughput methodology. The software supports all steps in the workflow by: • Providing guidance to design experiments • Handling experimental data relating to an experiment • Providing tools for data analysis Figure 30 shows the Assist software workflow. Experimental setup Data management Data analysis Type of study Plate data Mass balance Experimental design Replicate plot Adsorption isotherm Plate layout Response curve Experimental protocol Response surface Lab instructions Fig 30. Assist software workflow. 6.1.1 Experimental setup Based on the type of study (i.e., binding, wash, elution, determining adsorption isotherms, or media screening), and on the number of experimental factors (e.g., salt concentration, pH, buffer system, etc.), Assist software suggests different experimental designs that map out the experimental factors in a 96-well filter plate. These designs are suitable for automated workflows using robotic systems or manual workflows using multi-channel pipettes. Importing custom designs into the software using a supplied Microsoft™ Excel™ file is also possible. Since the 96-well plate format allows testing many different experimental conditions simultaneously, the designs include many different factor levels. The physical layout of factors is shown in the plate layout section, either one-by-one as colored gradients in the plate, or all at the same time (see Fig 31). A detailed laboratory protocol based on the experimental steps in the workflow is created in the experimental protocol section. This can be modified and other documentation such as notes, can be added for traceability. The protocol can be printed and used as a laboratory instruction when setting up the experiment, and saved for future documentation. 28-9403-58 AA 35 Buffer pH All factors Salt concentration Fig 31. Factor level layout is either shown one-by-one as colored gradients (e.g., buffer pH) or all at the same time. 6.1.2 Data management The data management section includes two parts: Data loading and raw data management. Data can be loaded from a file or pasted from the clipboard, either as concentration (µg/µl), mass (µg), UV absorbance (AU) or a generic response (any kind of data, e.g., impurity data). Raw data management displays data both in table format and as a replicate plot. The table presents calculated mean values, standard deviations and relative standard deviations (RSD) for the replicates. A statistical way to identify and/or exclude outliers is not available, but a sorting function makes it easy to identify replicates with large variation. Data points can easily be excluded from further analysis and re-included into the analysis again. Excluded data points are indicated in the replicate plot, but not displayed in subsequent plots. 6.1.3 Data analysis Data evaluation includes calculating and visualizing mass balance, response curves, and response plots. In this way, the effects of experimental conditions on responses such as binding capacity or yield can easily be identified. The mass balance section shows the mass that has been recovered in each well and for each experimental step where analysis has been performed and data loaded into Assist (see above). This functions as a quality check for the overall recovery and allows detection of outliers. The Response curve (Fig 32) shows a response as a function of one experimental factor, while the Response surface (Fig 33) shows the response as a function of two factors. The first is displayed as a curve and the second as a colored surface based on interpolated data showing the actual experimental results as crosses. Plots can be exported in enhanced metafile format and the tables can be exported as tabseparated text files. They can also be used in other applications such as statistical modeling of the Response surface. 36 28-9403-58 AA Fig 32. Screen dump showing a Response curve, which shows a response as a function of a single experimental factor. Fig 33. Screen dump showing a Response surface, which shows a response as a function of two experimental factors. The colored surface is based on interpolated data showing the actual experimental results as crosses. Adsorption isotherm panes are only available when setting up an adsorption isotherm study. In adsorption isotherm analysis, a Langmuir isotherm is fitted to the data points to determine the equilibrium dissociation constant Kd (µg/µl) and the maximum capacity qmax (µg/µl) from either flowthrough data or from elution data. 28-9403-58 AA 37 38 28-9403-58 AA 7. PreDictor plate selection 7.1 PreDictor Plate selection guide 1. Single-medium plates – For binding, wash, or elution studies – Same medium volume throughout the plate – Plates with three different volumes available. Type of study governs which volume to use (refer to Table 2) 2. Media-screening plates – For binding, wash, and elution studies on multiple chromatography media – Two types of plates available: 1) Anion- and multimodal anion-exchange media plate (Capto Q, Capto DEAE, Q Sepharose Fast Flow, and Capto adhere), 2) cation and multimodal cation exchange media plate (Capto S, SP Sepharose Fast Flow, and Capto MMC). For plate layout, see Section 7.2. – Plates with two different media volumes are available. Type of study governs which volume to use (refer to Table 2). 3. Adsorption isotherm plates – Used for binding studies done under equilibrium conditions to obtain fundamental thermodynamic understanding of the adsorption process. – Single-medium per plate, but with varied medium volume in the wells. This facilities simple and rapid construction of the isotherm as sample manipulation is minimized by avoiding the need to vary the concentration of sample molecule between wells. For plate layout, see Section 7.2. 4. Custom plates - Please contact your GE Healthcare representative to discuss the availability of customized PreDictor plates for your process development needs. When choosing PreDictor plates, consider the amount of material, target protein, and impurities required for final analysis. If a large amount of sample is needed, a larger medium volume and/ or increased number of sample aliquots in the loading step is necessary. Alternatively, several replicates from one plate can be pooled. For binding studies, relatively small volumes of chromatography medium are used. The medium should be overloaded with protein and the amount of unbound protein measured. Alternatively, the amount of bound protein is determined from the elution pool. Generally, single-medium plates with 2 or 6 µl volumes should be used, but in some cases plates with 20 µl or 50 µl can be employed. Check the suitability of a particular plate by performing a rough calculation as described in the example in Section 2.9.1. The properties of the medium dictate the volumes used; for high-capacity ion exchangers, 2 µl is sufficient, while for the other media, 6 µl is required for optimal results. For wash and elution studies, larger medium volumes can be required if sample purity needs to be determined. In such cases, the minimum detectable amount of impurities will govern the choice of PreDictor plate. The first option for wash and elution studies is the 20 µl plate. The screening plates facilitate media screening. Instead of using several single-medium plates to screen different media, one plate contains three (PreDictor CIEX screening plate) or four (PreDictor AIEX screening plate) different media. Adsorption isotherm plates contain wells with media volumes ranging from 2 to 50 µl, which ensures that the amount of target protein bound and final concentration of target protein in the supernatant varies so that an isotherm (capacity vs equilibrium concentration) can be constructed. 28-9403-58 AA 39 Table 2. Predictor plate selection guide by application Single-medium plates – Application: Binding conditions Medium volume in well (µl)1 2 6 20 50 Capto Q Capto S Capto DEAE Capto MMC Capto adhere ++ NA - ++ NA - ++ NA - NA ++ - NA ++ - Q SP MabSelect Sepharose Sepharose family Fast Flow Fast Flow NA NA NA ++ ++ ++ - Single-medium plates – Application: Wash/elution conditions Medium volume in well (µl)1 2 6 20 50 Capto Q Capto S Capto DEAE Capto MMC Capto adhere NA ++2 +3 NA ++2 +3 NA ++2 +3 NA ++2 +3 NA ++2 +3 Q SP MabSelect Sepharose Sepharose family Fast Flow Fast Flow NA NA NA ++2 ++2 ++2 +3 +3 +3 Media-screening plates – Application: Binding conditions Medium volume in well (µl)1 AIEX screening plate4 CIEX screening plate4 2 and 6 ++ ++ 20 - - Media-screening plates – Application: Wash/elution conditions Medium volume in well (µl)1 2 and 6 20 AIEX screening plate4 ++ CIEX screening plate4 ++ Adsorption isotherm plates – Application: Adsorption isotherm studies Variable (2, 4, 6, 8, 20, and 50 µl). Only one type of plate per medium available Medium volume in well (µl)1: ++ + NA 1 2 First choice Possible Not recommended Product not available Corresponds to sedimented volume. The 20 µl plate is the preferred plate for the first set of experiments. 3 The 50 µl plate is used for when protein concentration is high or when large amounts of sample are needed for analysis. 4 AIEX screening plate contains Capto Q, Capto DEAE, Q Sepharose Fast Flow, and the multimodal anion exchanger Capto adhere. CIEX screening plate contains Capto S, SP Sepharose Fast Flow, and the multimodal cation exchanger Capto MMC. 40 28-9403-58 AA 7.2 Available 8.2 Availableplates plates Single-medium plate Medium volume in well (µl) Row 1 2 2* A–H Vmedium Vstorage† 200 Column 2 2 200 3 2 200 4 2 200 5 2 200 6 2 200 7 2 200 8 2 200 9 2 200 10 2 200 11 2 200 12 2 200 6* A–H Vmedium 6 Vstorage† 500 6 500 6 500 6 500 6 500 6 500 6 500 6 500 6 500 6 500 6 500 6 500 20* A–H Vmedium 20 Vstorage† 500 20 500 20 500 20 500 20 500 20 500 20 500 20 500 20 500 20 500 20 500 20 500 50* A–H Vmedium 50 Vstorage† 500 50 500 50 500 50 500 50 500 50 500 50 500 50 500 50 500 50 500 50 500 50 500 * Capto S, Capto DEAE, Capto S available as 2, 20, 50 µl plates. MabSelect, MabSelect SuRe, MabSelect Xtra™, Q Sepharose Fast Flow, SP Sepharose Fast Flow, Capto MMC, and Capto adhere available as 6, 20, or 50 µl plates. † Vstorage: Volume of storage solution in the well. Storage solution is 20% ethanol for all media except SP Sepharose Fast Flow and Capto S, where the storage solution is 20% ethanol with 0.2 M sodium acetate. AIEX screening plate Column Medium volume in well (µl) Row 2 or 6 A–H Vmedium 20 Capto Q Capto DEAE Q Sepharose Fast Flow Capto adhere 1 2 2 2 3 2 4 2 5 2 6 2 7 6 8 6 9 6 10 6 11 6 12 6 Vstorage 200 200 200 200 200 200 500 500 500 500 500 500 A–H Vmedium 20 20 20 20 20 20 20 20 20 20 20 20 Vstorage 500 500 500 500 500 500 500 500 500 500 500 500 CIEX screening plate Column Medium volume in well (µl) Row 2 or 6 A–H Vmedium Capto S 1 2 Vstorage 200 20 SP Sepharose Fast Flow Capto MMC 2 2 3 2 4 2 5 6 6 6 7 6 8 6 9 6 10 6 11 6 12 6 200 200 200 500 500 500 500 500 500 500 500 20 20 20 20 20 20 20 20 20 20 20 20 Vstorage 500 500 500 500 500 500 500 500 500 500 500 500 A–H Vmedium Adsorption isotherm plate* Medium volume in well (µl) Row 2–50 * Column 1 2 3 4 5 6 7 8 9 10 11 12 50 50 20 20 8 8 6 6 4 4 2 2 Vstorage 500 500 200 200 500 500 375 375 250 250 125 125 A–H Vmedium Same plate design for all media 28-9403-58 AA 41 42 28-9403-58 AA 8. Experimental designs/setups The 96-well plate format allows a large number of experiments to be performed in parallel. Experimental design can vary from single-factor designs to more advanced DoE designs (Fig 34). DoE designs are used to acquire as much information as possible in as few experiments as possible. Because of the large number of experiments that can be performed, information from experimental data can yield information similar to that obtained from statistical modeling of responses. This is shown in Figure 16, which shows surface plots derived from raw data. In general, a larger experimental space can be covered by the plate format because of scale-down effects and parallellization. A) Factor 1 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -0.71 -0.71 -0.71 -0.71 -0.71 -0.71 -0.71 -0.71 -0.71 -0.71 -0.71 -0.71 -0.43 -0.43 -0.43 -0.43 -0.43 -0.43 -0.43 -0.43 -0.43 -0.43 -0.43 -0.43 -0.14 -0.14 -0.14 -0.14 -0.14 -0.14 -0.14 -0.14 -0.14 -0.14 -0.14 -0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.43 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.14 B) Factor 1 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 -1 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 12 0 1 Factor 2 1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 1 1 1 1 1 1 1 1 1 1 1 1 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 0 1 0 1 0 1 0 1 0 1 0 1 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 1 0 1 0 1 0 1 0 1 0 1 0 Factor 3 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 0 -1 0 -1 0 -1 0 -1 0 -1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 Fig 34. Examples of experimental designs/setups. Factor level range from -1 to 1. A) One factor in 8 levels with 12 replicates. B) Central Composite Face design (CCF); eight corner points, six face points, and two center-points. Mapped out in a full PreDictor plate, the design results in six replicates. 28-9403-58 AA 43 44 28-9403-58 AA 9. Steps in process development following screening 9.1 HiScreen and HiTrap columns Fig 35. HiScreen columns are available prepacked with a wide range of chromatography media for screening and optimization studies. Prepacked HiScreen columns (Fig 35) are the natural next step from PreDictor plates for optimization and verification, or further process development studies on binding capacity, robustness, and resolution. Nineteen HiScreen columns are currently available, packed with different media from the MabSelect, Capto, and Sepharose families for affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography. HiScreen columns are prepacked with the same chromatography media that are prefilled in PreDictor plates, which ensures reproducible results when scaling up. HiScreen columns have 10 cm bed height and a small volume (4.7 ml) to keep sample and buffer consumption low. If a larger bed height is required, two columns can be connected in series to achieve a 20 cm bed. In antibody purification, binding capacity is one of the most important parameters. HiScreen columns are a useful tool for verification studies. In Figure 36, dynamic binding capacity was measured at three different residence times for HiScreen MabSelect, HiScreen MabSelect SuRe, and HiScreen MabSelect Xtra. For this monoclonal antibody, HiScreen MabSelect Xtra showed the highest capacity. DBC hIgG (mg/ml medium) 50 HiScreen MabSelect Xtra HiScreen MabSelect SuRe HiScreen MabSelect 40 30 20 0 1 2 500 300 3 4 150 5 Residence time (min) Linear flow rate (cm/h) Fig 36. Comparison of dynamic binding capacity for hIgG on the three columns available for antibody purification. For more information visit www.gelifesciences.com/protein-purification 28-9403-58 AA 45 HiTrap columns in convenient 1 ml and 5 ml sizes complement PreDictor plates and offer an alternative to HiScreen columns if sample volumes are limited. HiTrap columns are available prepacked with the same chromatography media that are available in PreDictor plates and HiScreen columns. For more information, visit www.gelifesciences.com/hitrap 9.2 ÄKTA chromatography systems and UNICORN control software Prepacked HiScreen and HiTrap columns are used with ÄKTA design systems such as ÄKTA avant 25 chromatography system, a liquid chromatography system for chromatography media screening, method scouting, and fast, automated process development. Fig 37. ÄKTA avant 25 is a high-performance system designed for process development, method optimization, and scouting. Increased productivity more efficient method scouting and process development is a key feature of ÄKTA avant 25, which has flow rates and pressure specifications that support use of GE Healthcare’s BioProcess™ media such as MabSelect and Capto. The system, which comprises advanced instrumentation and specially developed UNICORN 6 control software also offers greater security through column recognition and individual column run history data, automatic on-line buffer preparation, and easy protocol transfer during scale-up. An important feature of the UNICORN 6 control software is the Design of Experiments tool, which enables increased efficiency in process development and provides time and cost savings by capturing more precise information in fewer experiments. For more information, visit www.gelifesciences.com/akta 9.3 BioProcess media GE Healthcare’s BioProcess media cover all purification steps from capture to polishing, for all scales of work from development and routine production. BioProcess media are developed for production-scale chromatography to meet the demands of industrial biotechnology. All media are manufactured with validated methods and tested to meet stringent quality requirements. Regulatory Support Files are available to assist process validation and submissions to regulatory authorities. Columns and systems for purification scale-up are also available from GE Healthcare, see www.gelifesciences.com/bioprocess for more information. 46 28-9403-58 AA 10. 11. References 1. Arve, B. H. and Liapis, A. I. Modelling and analysis of biospecific adsorption in a finite batch. AIChE J. 33(2), 179–193 (1987). 2. Chase, H. A. Prediction of the performance of preparative affinity chromatography. J. Chromatogr. 297, 179–202 (1984). 3. Hunter, A. K. and Carta, G. Protein adsorption on novel acrylamido-based polymeric ion exchanger. J. Chromatogr. 897(1-2), 81–87 (2000). 4. Wesselingh, J. A. and Bosma, J. C. Protein ion-exchange adsorption kinetics. AIChE J. 47(7), 1571–1580 (2001). 5. Lewus, R. K. and Carta, G. Binary Protein adsorption on gel-composite ion-exchange media. AIChE J. 45(3), 512–522 (1999). 6. Bergander, T. et al. High-Throughput Process Development: Biotechnol. Prog. 24(3), 632–639 (2008). 7. Data file: Capto MMC, GE Healthcare 11-0035-45 AA. 8. Data file: Capto adhere, GE Healthcare 28-9078-88 AA. 9. Application note: Screening and optimization of loading conditions on Capto S, GE Healthcare, 28-4078-16, Edition AA (2007). 10. Application note: Screening of loading conditions on Capto S using a new high-throughput format, PreDictor plates, GE Healthcare, 28-9258-40, Edition AA (2007). 11. Harinarayan, et al. An exclusion mechanism in ion exchange chromatography. Biotechnology and Bioengineering 95 (5), 775–787 (2005). 12. Application note: High-throughput screening of elution pH for monoclonal antibodies on MabSelect SuRe using PreDictor plates, GE Healthcare, 28-9277-92, Editon AA (2007). 13. Poster presentation: Rapid development of CIP protocols for affinity media, GE Healthcare, 28-9500-94, Edition AA (2008). Available as PDF only. 14. www.umetrics.com 15. Application note: Adsorption equilibrium isotherm studies using a high throughput method, GE Healthcare, 28-9403-62, Edition AA (2008). 16. Poster presentation: Quality aspects of the microtiter plate workflow in the screening of chromatographic conditions, GE Healthcare, 28-9441-75, Edition AA (2008). Available as PDF only. 17. Poster presentation: Accelerated development of a downstream process purification process for production of monoclonal antibodies: A case study, GE Healthcare, 28-9441-76, Edition AA (2008). Available as PDF only. 28-9403-58 28-9403-58 AA AA 47 43 48 28-9403-58 AA 11. 12. Nomenclature madded Mass of target protein added to the well mbound Mass of target protein bound to the chromatography medium munbound Mass of target protein not bound to the chromatography medium meluate Mass of target protein in eluate fraction(s) mFT Mass of target protein in flowthrough fraction mwash Mass of target protein in the wash fraction(s) c Concentration of target protein in the liquid phase ceq Concentration of target protein in the liquid phase at equilibrium co Initial concentration of target protein in the liquid phase at the start of the experiment, see Section 2.3 csample Concentration of target protein in the sample celuate Concentration of target protein in eluate fraction q Concentration of target protein in the solid phase (chromatography medium) qmax Maximum capacity of the chromatography medium for the target protein (saturation capacity) Vsample Sample volume Vliq Volume of the liquid phase, see Section 2.3 Vmedium Chromatography medium volume Veluate Volume of eluate fraction Vr Volume of retained liquid in a well, see Section 2.3 Phase ratio = Vliq /Vmedium , see Section 2.6 Kd Equilibrium dissociation constant 28-9403-58 AA 49 50 28-9403-58 AA 12. Ordering information PreDictor plates Single-medium plates Quantity PreDictor Capto Q, 2 μl PreDictor Capto Q, 20 μl PreDictor Capto Q, 50 μl PreDictor Capto S, 2 μl PreDictor Capto S, 20 μl PreDictor Capto S, 50 μl PreDictor Capto DEAE, 2 μl PreDictor Capto DEAE, 20 μl PreDictor Capto DEAE, 50 μl PreDictor Capto MMC, 6 μl PreDictor Capto MMC, 20 μl PreDictor Capto MMC, 50 μl PreDictor Capto adhere, 6 μl PreDictor Capto adhere, 20 μl PreDictor Capto adhere, 50 μl PreDictor MabSelect, 6 μl PreDictor MabSelect, 20 μl PreDictor MabSelect, 50 μl PreDictor MabSelect SuRe, 6 μl PreDictor MabSelect SuRe, 20 μl PreDictor MabSelect SuRe, 50 μl PreDictor MabSelect Xtra, 6 μl PreDictor MabSelect Xtra, 20 μl PreDictor MabSelect Xtra, 50 μl PreDictor Q Sepharose Fast Flow, 6 μl PreDictor Q Sepharose Fast Flow, 20 μl PreDictor Q Sepharose Fast Flow, 50 μl PreDictor SP Sepharose Fast Flow, 6 μl PreDictor SP Sepharose Fast Flow, 20 μl PreDictor SP Sepharose Fast Flow, 50 μl 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates Screening plates Quantity PreDictor AIEX screening 2 μl/6 μl PreDictor AIEX screening 20 μl PreDictor CIEX screening 2 μl/6 μl PreDictor CIEX screening 20 μl 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates Adsorption isotherm plates Quantity PreDictor Capto Q isotherm1 PreDictor Capto S isotherm1 PreDictor Capto DEAE isotherm1 PreDictor Capto MMC isotherm1 PreDictor Capto adhere isotherm1 PreDictor MabSelect isotherm1 PreDictor MabSelect SuRe isotherm1 PreDictor MabSelect Xtra isotherm1 PreDictor Q Sepharose Fast Flow isotherm1 PreDictor SP Sepharose Fast Flow isotherm1 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 4 × 96-well filter plates 1 Code no. 28-9257-73 28-9258-06 28-9258-07 28-9258-08 28-9258-09 28-9258-10 28-9258-11 28-9258-12 28-9258-13 28-9258-14 28-9258-15 28-9258-16 28-9258-17 28-9258-18 28-9258-19 28-9258-20 28-9258-21 28-9258-22 28-9258-23 28-9258-24 28-9258-25 28-9432-75 28-9432-76 28-9432-77 28-9432-69 28-9432-70 28-9432-71 28-9432-72 28-9432-73 28-9432-74 Code no. 28-9432-88 28-9432-89 28-9432-90 28-9432-91 Code no. 28-9432-78 28-9432-79 28-9432-80 28-9432-81 28-9432-82 28-9432-83 28-9432-84 28-9432-85 28-9432-86 28-9432-87 Plates are manufactured on request. 28-9403-58 AA 51 Software Assist 1.1 Software package Assist 1-user license 1.1 28-9453-96 28-9453-97 Related products Accessories Quantity Collection plate 96-well 500 µl, V-shaped bottom (not UV-readable) Microplate foil (96-well) 5 × 96 well-plates 28-4039-43 Code no. 100 × self-adhesive, transparent plastic foils BR-1005-78 Prepacked columns* Quantity Code no. HiScreen Capto Q HiScreen Capto S HiScreen Capto DEAE HiScreen Capto MMC HiScreen Capto adhere HiScreen MabSelect HiScreen MabSelect SuRe HiScreen MabSelect Xtra HiScreen IMAC FF HiScreen Q FF HiScreen SP FF HiScreen Q HP HiScreen SP HP HiScreen Phenyl HP HiScreen Butyl FF HiScreen Butyl-S FF HiScreen Octyl FF HiScreen Phenyl FF (high sub) HiScreen Phenyl FF (low sub) 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 1 × 4.7 ml 28-9269-78 28-9269-79 28-9269-82 28-9269-80 28-9269-81 28-9269-73 28-9269-77 28-9269-76 28-9505-17 28-9505-10 28-9505-13 28-9505-11 28-9505-15 28-9505-16 28-9269-84 28-9269-85 28-9269-86 28-9269-88 28-9269-89 * HiTrap columns prepacked with the equivalent chromatography media found in HiScreen columns are available in 5 × 1 ml and 5 × 5 ml pack sizes. Visit www.gelifesciences.com/hitrap for information. 52 28-9403-58 AA Related literature PreDictor literature Data file: PreDictor 96-well filter plates and Assist software Application note: Screening of loading conditions on Capto S using a new high-throughput format, PreDictor plates Poster: High-throughput screening of elution conditions on Capto MMC using PreDictor plates Application note: High-throughput screening of elution pH for monoclonal antibodies on MabSelect SuRe using PreDictor plates Application note: Adsorption equilibrium isotherm studies using a high-throughput method Application note: High-throughput screening and column optimization of a monoclonal antibody capture step Additional literature Data file: Capto S, Capto Q, Capto ViralQ, and Capto DEAE Data file: Capto MMC Data file: Capto adhere Data file: MabSelect Data file: MabSelect SuRe Data file: MabSelect Xtra Data file: Sepharose Fast Flow ion exchangers Application note: Screening and optimization of loading conditions on Capto S Data file: ÄKTA avant 25 Data file: UNICORN 6 control software Application note: Rapid process development for purification of a MAb using ÄKTA avant 25 HiScreen Octyl FF HiScreen Phenyl FF (high sub) HiScreen Phenyl FF (low sub) Code no. 28-9258-39 28-9258-40 28-9277-90 28-9277-92 28-9403-62 28-9403-47 Code no. 11-0025-76 11-0035-45 28-9078-88 18-1149-94 11-0011-65 11-0011-57 18-1020-66 28-4078-16 28-9573-45 28-9573-46 28-9573-47 28-9269-86 28-9269-88 28-9269-89 28-9403-58 AA 53 54 28-9403-58 AA 28-9403-58 AA 55 56 28-9403-58 AA Handbooks from GE Healthcare GST Gene Fusion System Handbook 18-1157-58 Gel Filtration Principles and Methods 18-1022-18 Affinity Chromatography Principles and Methods 18-1022-29 Recombinant Protein Purification Handbook Principles and Methods 18-1142-75 Antibody Purification Handbook 18-1037-46 Protein Purification Handbook 18-1132-29 Ion Exchange Chromatography and Chromatofocusing Principles and Methods 11-0004-21 Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods 11-0012-69 Cell Separation Media Methodology and Applications 18-1115-69 2-D Electrophoresis using immobilized pH gradients Principles and Methods 80-6429-60 Purifying Challenging Proteins Principles and Methods 28-9095-31 Isolation of mononuclear cells Methodology and Applications 18-1152-69 High-throughput Process Development with PreDictor Plates Principles and Methods 28-9403-58 Microcarrier Cell Culture Principles and Methods 18-1140-62 Nucleic Acid Sample Preparation for Downstream Analyses Principles and Methods 28-9624-00 GE Healthcare ÄKTA, ÄKTA avant, AxiChrom, BioProcess, Capto, Deep Purple, Drop design, HiScreen, HiTrap, MabSelect, MabSelect SuRe, MabSelect Xtra, PreDictor, ReadyToProcess, Sepharose, and Tricorn are trademarks of GE Healthcare companies. Capto ViralQ: Separating viral particles with Capto Q products may require a license under United States patent number 6,537,793 B2 and equivalent patents and patent applications in other countries owned by Centelion SAS. Such a license is not included with the purchase of Capto Q but is included with the purchase of Capto ViralQ products. With the purchase of Capto ViralQ the customer is granted a free limited license under US patent 6,537,793 B2 and equivalent patents and patent applications in other countries owned by Centelion SAS to separate viral particles solely through use of the product purchased. Deep Purple Total Protein Stain: Deep Purple Total Protein Stain is exclusively licensed to GE Healthcare from Fluorotechnics Pty Ltd.Deep Purple Total Protein Stain may only be used for applications in life science research. Deep Purple is covered under a granted patent in New Zealand entitled “Fluorescent Compounds”, patent number 522291 and equivalent patents and patent applications in other countries. Tricorn Columns: The Tricorn column and components are protected by US design patents USD500856, USD506261, USD500555, USD495060 and their equivalents in other countries. All third party trademarks are the property of their respective owners. © 2009 General Electric Company – All rights reserved. First published Dec. 2009. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare that supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ 08855-1327, USA GE Healthcare Europe GmbH Munzinger Strasse 5 D-79111 Freiburg, Germany GE Healthcare Japan Corporation Sanken Bldg. 3-25-1 Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan For local office contact information, please visit www.gelifesciences.com/contact High-throughput Process Development with PreDictor Plates – Principles and Methods GE, imagination at work, and GE monogram are trademarks of General Electric Company. High-throughput Process Development with PreDictor™ Plates Principles and Methods www.gelifesciences.com/predictor GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden imagination at work imagination at work 28-9403-58 AA 12/2009
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xmp.iid:EFD76E27C9206811AB08A81F40D5F7C1, xmp.iid:F0D76E27C9206811AB08A81F40D5F7C1, xmp.iid:F1D76E27C9206811AB08A81F40D5F7C1, xmp.iid:F2D76E27C9206811AB08A81F40D5F7C1, xmp.iid:F3D76E27C9206811AB08A81F40D5F7C1, xmp.iid:F4D76E27C9206811AB08A81F40D5F7C1, xmp.iid:F5D76E27C9206811AB08A81F40D5F7C1, xmp.iid:F6D76E27C9206811AB08A81F40D5F7C1, xmp.iid:F7D76E27C9206811AB08A81F40D5F7C1, xmp.iid:F8D76E27C9206811AB08A81F40D5F7C1, xmp.iid:3A729AE1CA206811AB08A81F40D5F7C1, xmp.iid:3B729AE1CA206811AB08A81F40D5F7C1, xmp.iid:3C729AE1CA206811AB08A81F40D5F7C1, xmp.iid:3D729AE1CA206811AB08A81F40D5F7C1, xmp.iid:3E729AE1CA206811AB08A81F40D5F7C1, xmp.iid:3F729AE1CA206811AB08A81F40D5F7C1, xmp.iid:40729AE1CA206811AB08A81F40D5F7C1, xmp.iid:41729AE1CA206811AB08A81F40D5F7C1, xmp.iid:42729AE1CA206811AB08A81F40D5F7C1, xmp.iid:43729AE1CA206811AB08A81F40D5F7C1, xmp.iid:44729AE1CA206811AB08A81F40D5F7C1, xmp.iid:26729DCECB206811AB08A81F40D5F7C1, xmp.iid:27729DCECB206811AB08A81F40D5F7C1, xmp.iid:28729DCECB206811AB08A81F40D5F7C1, xmp.iid:29729DCECB206811AB08A81F40D5F7C1, xmp.iid:2A729DCECB206811AB08A81F40D5F7C1, xmp.iid:2B729DCECB206811AB08A81F40D5F7C1, xmp.iid:2C729DCECB206811AB08A81F40D5F7C1, xmp.iid:2D729DCECB206811AB08A81F40D5F7C1, xmp.iid:2E729DCECB206811AB08A81F40D5F7C1, xmp.iid:2F729DCECB206811AB08A81F40D5F7C1, xmp.iid:30729DCECB206811AB08A81F40D5F7C1, xmp.iid:40FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:41FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:42FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:43FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:44FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:45FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:46FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:47FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:48FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:49FB5B20CD206811AB08A81F40D5F7C1, xmp.iid:4AFB5B20CD206811AB08A81F40D5F7C1, xmp.iid:1058D271D3206811AB08A81F40D5F7C1, xmp.iid:1158D271D3206811AB08A81F40D5F7C1, xmp.iid:1258D271D3206811AB08A81F40D5F7C1, xmp.iid:1358D271D3206811AB08A81F40D5F7C1, xmp.iid:1458D271D3206811AB08A81F40D5F7C1, xmp.iid:6EDDB9542A20681192B0CD41668CCA24, xmp.iid:6FDDB9542A20681192B0CD41668CCA24, xmp.iid:70DDB9542A20681192B0CD41668CCA24, xmp.iid:71DDB9542A20681192B0CD41668CCA24, xmp.iid:72DDB9542A20681192B0CD41668CCA24, xmp.iid:73DDB9542A20681192B0CD41668CCA24, xmp.iid:74DDB9542A20681192B0CD41668CCA24, xmp.iid:2C8B956C4420681192B0CD41668CCA24, xmp.iid:2D8B956C4420681192B0CD41668CCA24, xmp.iid:2E8B956C4420681192B0CD41668CCA24, xmp.iid:2F8B956C4420681192B0CD41668CCA24, xmp.iid:308B956C4420681192B0CD41668CCA24, xmp.iid:318B956C4420681192B0CD41668CCA24, xmp.iid:328B956C4420681192B0CD41668CCA24, xmp.iid:338B956C4420681192B0CD41668CCA24, xmp.iid:348B956C4420681192B0CD41668CCA24, xmp.iid:358B956C4420681192B0CD41668CCA24, xmp.iid:368B956C4420681192B0CD41668CCA24, xmp.iid:0C9B302B4620681192B0CD41668CCA24, xmp.iid:0D9B302B4620681192B0CD41668CCA24, xmp.iid:0E9B302B4620681192B0CD41668CCA24, xmp.iid:0F9B302B4620681192B0CD41668CCA24, xmp.iid:109B302B4620681192B0CD41668CCA24, xmp.iid:119B302B4620681192B0CD41668CCA24, xmp.iid:129B302B4620681192B0CD41668CCA24, xmp.iid:139B302B4620681192B0CD41668CCA24, xmp.iid:149B302B4620681192B0CD41668CCA24, xmp.iid:159B302B4620681192B0CD41668CCA24, xmp.iid:169B302B4620681192B0CD41668CCA24, xmp.iid:FF7F117407206811B699D08D9460CAB1, xmp.iid:0080117407206811B699D08D9460CAB1, xmp.iid:8C1836F519206811B699D08D9460CAB1, xmp.iid:8D1836F519206811B699D08D9460CAB1, xmp.iid:8E1836F519206811B699D08D9460CAB1, xmp.iid:8F1836F519206811B699D08D9460CAB1, xmp.iid:901836F519206811B699D08D9460CAB1, xmp.iid:921836F519206811B699D08D9460CAB1, xmp.iid:931836F519206811B699D08D9460CAB1, xmp.iid:941836F519206811B699D08D9460CAB1 History When : 2009:11:18 10:11:47+01:00, 2009:11:18 10:11:48+01:00, 2009:11:18 10:12:31+01:00, 2009:11:18 10:21:26+01:00, 2009:11:18 10:23:11+01:00, 2009:11:18 10:30:20+01:00, 2009:11:18 10:30:54+01:00, 2009:11:18 10:33:37+01:00, 2009:11:18 10:42:09+01:00, 2009:11:18 10:47:42+01:00, 2009:11:18 10:47:48+01:00, 2009:11:18 10:48:27+01:00, 2009:11:18 10:48:39+01:00, 2009:11:18 10:49:16+01:00, 2009:11:18 10:55:46+01:00, 2009:11:18 10:56:52+01:00, 2009:11:18 10:57:12+01:00, 2009:11:18 10:57:45+01:00, 2009:11:18 10:59:31+01:00, 2009:11:18 11:00:19+01:00, 2009:11:18 11:02:36+01:00, 2009:11:18 11:02:58+01:00, 2009:11:18 11:03:36+01:00, 2009:11:18 11:05:47+01:00, 2009:11:18 11:06:28+01:00, 2009:11:18 11:08:44+01:00, 2009:11:18 11:08:48+01:00, 2009:11:18 11:12:03+01:00, 2009:11:18 11:12:56+01:00, 2009:11:18 11:25:37+01:00, 2009:11:18 11:26:41+01:00, 2009:11:18 11:26:50+01:00, 2009:11:18 11:28:29+01:00, 2009:11:18 11:29:02+01:00, 2009:11:18 11:31:23+01:00, 2009:11:18 11:32:03+01:00, 2009:11:18 11:33:03+01:00, 2009:11:18 11:35:25+01:00, 2009:11:18 11:36:45+01:00, 2009:11:18 11:36:54+01:00, 2009:11:18 11:37:05+01:00, 2009:11:18 11:37:10+01:00, 2009:11:18 11:38:31+01:00, 2009:11:18 11:38:38+01:00, 2009:11:18 11:51:14+01:00, 2009:11:18 11:52:43+01:00, 2009:11:18 11:54:37+01:00, 2009:11:18 11:55:03+01:00, 2009:11:18 11:59:47+01:00, 2009:11:18 12:01:24+01:00, 2009:11:18 12:04:48+01:00, 2009:11:18 12:05:41+01:00, 2009:11:18 12:06:02+01:00, 2009:11:18 12:09:16+01:00, 2009:11:18 12:15:38+01:00, 2009:11:18 12:15:43+01:00, 2009:11:18 12:15:53+01:00, 2009:11:18 12:16:01+01:00, 2009:11:18 12:17:23+01:00, 2009:11:18 12:17:33+01:00, 2009:11:18 12:17:39+01:00, 2009:11:18 12:17:43+01:00, 2009:11:18 12:17:49+01:00, 2009:11:18 12:17:54+01:00, 2009:11:18 12:19:25+01:00, 2009:11:18 12:19:36+01:00, 2009:11:18 14:07:36+01:00, 2009:11:18 14:10:41+01:00, 2009:11:18 14:12:10+01:00, 2009:11:18 14:12:25+01:00, 2009:11:18 14:12:56+01:00, 2009:11:18 14:13:08+01:00, 2009:11:18 14:14:03+01:00, 2009:11:18 14:14:53+01:00, 2009:11:18 14:14:59+01:00, 2009:11:18 14:17:01+01:00, 2009:11:18 14:18:19+01:00, 2009:11:18 14:18:33+01:00, 2009:11:18 14:19:57+01:00, 2009:11:18 14:20:29+01:00, 2009:11:18 14:21:18+01:00, 2009:11:18 14:23:39+01:00, 2009:11:18 14:23:45+01:00, 2009:11:18 14:23:53+01:00, 2009:11:18 14:24:52+01:00, 2009:11:18 14:25:02+01:00, 2009:11:18 14:30:45+01:00, 2009:11:18 14:31:13+01:00, 2009:11:18 14:32:40+01:00, 2009:11:18 14:36:19+01:00, 2009:11:18 14:36:30+01:00, 2009:11:18 14:36:35+01:00, 2009:11:18 14:36:53+01:00, 2009:11:18 14:37:18+01:00, 2009:11:18 14:39:24+01:00, 2009:11:18 14:39:35+01:00, 2009:11:18 14:39:54+01:00, 2009:11:18 14:42:01+01:00, 2009:11:18 14:42:11+01:00, 2009:11:18 14:43:40+01:00, 2009:11:18 14:43:58+01:00, 2009:11:18 14:44:05+01:00, 2009:11:18 14:44:32+01:00, 2009:11:18 14:44:48+01:00, 2009:11:18 14:45:23+01:00, 2009:11:18 14:45:45+01:00, 2009:11:18 14:46:38+01:00, 2009:11:18 14:46:44+01:00, 2009:11:18 14:46:57+01:00, 2009:11:18 15:38:50+01:00, 2009:11:18 15:39:39+01:00, 2009:11:18 15:40:40+01:00, 2009:11:18 15:40:51+01:00, 2009:11:18 15:41:23+01:00, 2009:11:18 15:41:34+01:00, 2009:11:18 15:41:45+01:00, 2009:11:18 15:42:11+01:00, 2009:11:18 15:42:21+01:00, 2009:11:18 16:12:30+01:00, 2009:11:18 16:13:16+01:00, 2009:11:18 16:15:10+01:00, 2009:11:18 16:19:02+01:00, 2009:11:18 16:19:30+01:00, 2009:11:18 16:20:11+01:00, 2009:11:18 16:21:40+01:00, 2009:11:18 16:21:54+01:00, 2009:11:18 16:22:31+01:00, 2009:11:18 16:22:41+01:00, 2009:11:18 16:24:09+01:00, 2009:11:18 16:24:21+01:00, 2009:11:18 16:25:37+01:00, 2009:11:18 16:27:20+01:00, 2009:11:18 16:27:40+01:00, 2009:11:18 16:27:59+01:00, 2009:11:18 16:28:13+01:00, 2009:11:18 16:28:35+01:00, 2009:11:18 16:28:42+01:00, 2009:11:18 16:28:52+01:00, 2009:11:18 16:29:03+01:00, 2009:11:18 16:29:24+01:00, 2009:11:18 16:31:07+01:00, 2009:11:18 16:31:20+01:00, 2009:11:18 16:31:33+01:00, 2009:11:18 16:31:47+01:00, 2009:11:18 16:32:03+01:00, 2009:11:18 16:32:53+01:00, 2009:11:18 16:33:03+01:00, 2009:11:18 16:33:22+01:00, 2009:11:18 16:33:28+01:00, 2009:11:18 16:34:35+01:00, 2009:11:18 16:36:29+01:00, 2009:11:18 16:36:59+01:00, 2009:11:18 16:37:03+01:00, 2009:11:18 16:37:11+01:00, 2009:11:18 16:37:17+01:00, 2009:11:18 16:37:44+01:00, 2009:11:18 16:37:48+01:00, 2009:11:18 16:37:53+01:00, 2009:11:18 16:38:35+01:00, 2009:11:18 16:38:48+01:00, 2009:11:18 16:43:34+01:00, 2009:11:18 16:44:15+01:00, 2009:11:18 16:44:24+01:00, 2009:11:18 16:45:51+01:00, 2009:11:18 16:46:12+01:00, 2009:11:18 16:46:20+01:00, 2009:11:18 16:46:38+01:00, 2009:11:18 16:48:26+01:00, 2009:11:18 16:48:39+01:00, 2009:11:18 16:49:10+01:00, 2009:11:18 16:49:19+01:00, 2009:11:18 16:49:25+01:00, 2009:11:18 16:49:39+01:00, 2009:11:18 16:49:49+01:00, 2009:11:18 16:50:43+01:00, 2009:11:18 16:52:06+01:00, 2009:11:18 16:53:39+01:00, 2009:11:18 16:53:53+01:00, 2009:11:18 16:53:59+01:00, 2009:11:18 16:54:08+01:00, 2009:11:18 16:55:11+01:00, 2009:11:18 16:55:58+01:00, 2009:11:18 16:56:02+01:00, 2009:11:18 16:57:30+01:00, 2009:11:18 16:57:59+01:00, 2009:11:18 16:59:13+01:00, 2009:11:18 16:59:19+01:00, 2009:11:18 17:00:01+01:00, 2009:11:18 17:00:28+01:00, 2009:11:18 17:00:32+01:00, 2009:11:18 17:01:24+01:00, 2009:11:18 17:01:28+01:00, 2009:11:18 17:03:41+01:00, 2009:11:18 17:04:34+01:00, 2009:11:18 17:05:05+01:00, 2009:11:18 17:05:13+01:00, 2009:11:18 17:05:25+01:00, 2009:11:18 17:06:42+01:00, 2009:11:18 17:07:18+01:00, 2009:11:18 17:07:41+01:00, 2009:11:18 17:09:29+01:00, 2009:11:18 17:09:44+01:00, 2009:11:18 17:09:55+01:00, 2009:11:18 17:10:14+01:00, 2009:11:18 17:11:59+01:00, 2009:11:18 17:13:45+01:00, 2009:11:18 17:13:50+01:00, 2009:11:18 17:13:56+01:00, 2009:11:18 17:14:21+01:00, 2009:11:18 17:14:43+01:00, 2009:11:18 17:15:31+01:00, 2009:11:18 17:15:51+01:00, 2009:11:18 17:15:55+01:00, 2009:11:18 17:16:10+01:00, 2009:11:18 17:17:40+01:00, 2009:11:18 17:18:42+01:00, 2009:11:18 17:18:50+01:00, 2009:11:18 17:20:38+01:00, 2009:11:18 17:21:36+01:00, 2009:11:18 17:21:47+01:00, 2009:11:18 17:21:52+01:00, 2009:11:18 17:23:12+01:00, 2009:11:18 17:23:40+01:00, 2009:11:18 17:24:04+01:00, 2009:11:18 17:24:13+01:00, 2009:11:18 17:25:08+01:00, 2009:11:18 17:25:18+01:00, 2009:11:18 17:25:25+01:00, 2009:11:18 17:26:07+01:00, 2009:11:18 17:26:14+01:00, 2009:11:18 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/, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, /, / Manifest Link Form : ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream, ReferenceStream Manifest Placed X Resolution : 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 300.00, 72.00, 300.00, 72.00, 72.00 Manifest Placed Y Resolution : 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 72.00, 300.00, 72.00, 300.00, 72.00, 72.00 Manifest Placed Resolution Unit : Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches, Inches Manifest Reference Instance ID : xmp.iid:F77F11740720681181869B96A5B54FDE, xmp.iid:068011740720681195FEAFF493D187D1, xmp.iid:078011740720681195FEAFF493D187D1, xmp.iid:088011740720681195FEAFF493D187D1, xmp.iid:098011740720681195FEAFF493D187D1, xmp.iid:0A8011740720681195FEAFF493D187D1, xmp.iid:0ADA1DFF1020681195FEAFF493D187D1, xmp.iid:0BDA1DFF1020681195FEAFF493D187D1, xmp.iid:0CDA1DFF1020681195FEAFF493D187D1, xmp.iid:0DDA1DFF1020681195FEAFF493D187D1, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, xmp.iid:0EDA1DFF1020681195FEAFF493D187D1, xmp.iid:0FDA1DFF1020681195FEAFF493D187D1, uuid:a6951510-93d3-134f-8fd8-d55f6b8a42f3, xmp.iid:11DA1DFF1020681195FEAFF493D187D1, uuid:c16b2e92-f065-0c4e-aa85-780c26d3caa9, xmp.iid:14DA1DFF1020681195FEAFF493D187D1, xmp.iid:489CD1031220681195FEAFF493D187D1, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, xmp.iid:499CD1031220681195FEAFF493D187D1, xmp.iid:4A9CD1031220681195FEAFF493D187D1, uuid:47ebdcee-60e3-2340-a0dc-734edaeb43bb, xmp.iid:4C9CD1031220681195FEAFF493D187D1, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, xmp.iid:4D9CD1031220681195FEAFF493D187D1, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, xmp.iid:4E9CD1031220681195FEAFF493D187D1, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, xmp.iid:4F9CD1031220681195FEAFF493D187D1, xmp.iid:509CD1031220681195FEAFF493D187D1, xmp.iid:519CD1031220681195FEAFF493D187D1, xmp.iid:529CD1031220681195FEAFF493D187D1, xmp.iid:C28395D31220681195FEAFF493D187D1, xmp.iid:C38395D31220681195FEAFF493D187D1, xmp.iid:C48395D31220681195FEAFF493D187D1, xmp.iid:C58395D31220681195FEAFF493D187D1, xmp.iid:C68395D31220681195FEAFF493D187D1, xmp.iid:C88395D31220681195FEAFF493D187D1, xmp.iid:C78395D31220681195FEAFF493D187D1, xmp.iid:C98395D31220681195FEAFF493D187D1, xmp.iid:CA8395D31220681195FEAFF493D187D1, xmp.iid:CB8395D31220681195FEAFF493D187D1, xmp.iid:CC8395D31220681195FEAFF493D187D1, xmp.iid:709C0AC41320681195FEAFF493D187D1, xmp.iid:719C0AC41320681195FEAFF493D187D1, xmp.iid:729C0AC41320681195FEAFF493D187D1, xmp.iid:739C0AC41320681195FEAFF493D187D1, xmp.iid:749C0AC41320681195FEAFF493D187D1, uuid:f0419e51-0f69-43cf-974e-f42de348b05e, xmp.iid:759C0AC41320681195FEAFF493D187D1, xmp.iid:769C0AC41320681195FEAFF493D187D1, xmp.iid:779C0AC41320681195FEAFF493D187D1, xmp.iid:789C0AC41320681195FEAFF493D187D1, xmp.iid:799C0AC41320681195FEAFF493D187D1, xmp.iid:7A9C0AC41320681195FEAFF493D187D1, xmp.iid:6C4D52A11420681195FEAFF493D187D1, xmp.iid:6D4D52A11420681195FEAFF493D187D1, xmp.iid:FD7F117407206811B8409F1765462998, xmp.iid:FE7F117407206811B8409F1765462998, xmp.iid:6E4D52A11420681195FEAFF493D187D1, xmp.iid:F07F117407206811B5D18C0E55AF7BC7, xmp.iid:6F4D52A11420681195FEAFF493D187D1, xmp.iid:F27F117407206811B5D18C0E55AF7BC7, uuid:5504fa7a-0d45-c449-9586-a9181dedf52b, xmp.iid:FF7F117407206811B8409F1765462998 Manifest Reference Document ID : xmp.did:F77F11740720681181869B96A5B54FDE, xmp.did:068011740720681195FEAFF493D187D1, xmp.did:078011740720681195FEAFF493D187D1, xmp.did:088011740720681195FEAFF493D187D1, xmp.did:098011740720681195FEAFF493D187D1, xmp.did:0A8011740720681195FEAFF493D187D1, xmp.did:0ADA1DFF1020681195FEAFF493D187D1, xmp.did:0BDA1DFF1020681195FEAFF493D187D1, xmp.did:0CDA1DFF1020681195FEAFF493D187D1, xmp.did:0DDA1DFF1020681195FEAFF493D187D1, uuid:C6836B657BF8DD11A85DA8534E6E670D, xmp.did:0EDA1DFF1020681195FEAFF493D187D1, xmp.did:0FDA1DFF1020681195FEAFF493D187D1, xmp.did:10DA1DFF1020681195FEAFF493D187D1, xmp.did:11DA1DFF1020681195FEAFF493D187D1, xmp.did:12DA1DFF1020681195FEAFF493D187D1, xmp.did:14DA1DFF1020681195FEAFF493D187D1, xmp.did:489CD1031220681195FEAFF493D187D1, uuid:C6836B657BF8DD11A85DA8534E6E670D, uuid:C6836B657BF8DD11A85DA8534E6E670D, xmp.did:499CD1031220681195FEAFF493D187D1, xmp.did:4A9CD1031220681195FEAFF493D187D1, xmp.did:4B9CD1031220681195FEAFF493D187D1, xmp.did:4C9CD1031220681195FEAFF493D187D1, uuid:C6836B657BF8DD11A85DA8534E6E670D, uuid:C6836B657BF8DD11A85DA8534E6E670D, uuid:C6836B657BF8DD11A85DA8534E6E670D, uuid:C6836B657BF8DD11A85DA8534E6E670D, xmp.did:4D9CD1031220681195FEAFF493D187D1, uuid:C6836B657BF8DD11A85DA8534E6E670D, xmp.did:4E9CD1031220681195FEAFF493D187D1, uuid:C6836B657BF8DD11A85DA8534E6E670D, xmp.did:4F9CD1031220681195FEAFF493D187D1, xmp.did:509CD1031220681195FEAFF493D187D1, xmp.did:519CD1031220681195FEAFF493D187D1, xmp.did:529CD1031220681195FEAFF493D187D1, xmp.did:C28395D31220681195FEAFF493D187D1, xmp.did:C38395D31220681195FEAFF493D187D1, xmp.did:C48395D31220681195FEAFF493D187D1, xmp.did:C58395D31220681195FEAFF493D187D1, xmp.did:C68395D31220681195FEAFF493D187D1, xmp.did:C88395D31220681195FEAFF493D187D1, xmp.did:C78395D31220681195FEAFF493D187D1, xmp.did:C98395D31220681195FEAFF493D187D1, xmp.did:CA8395D31220681195FEAFF493D187D1, xmp.did:CB8395D31220681195FEAFF493D187D1, xmp.did:CC8395D31220681195FEAFF493D187D1, xmp.did:709C0AC41320681195FEAFF493D187D1, xmp.did:719C0AC41320681195FEAFF493D187D1, xmp.did:729C0AC41320681195FEAFF493D187D1, xmp.did:739C0AC41320681195FEAFF493D187D1, xmp.did:749C0AC41320681195FEAFF493D187D1, uuid:C6836B657BF8DD11A85DA8534E6E670D, xmp.did:759C0AC41320681195FEAFF493D187D1, xmp.did:769C0AC41320681195FEAFF493D187D1, xmp.did:779C0AC41320681195FEAFF493D187D1, xmp.did:789C0AC41320681195FEAFF493D187D1, xmp.did:799C0AC41320681195FEAFF493D187D1, xmp.did:7A9C0AC41320681195FEAFF493D187D1, xmp.did:6C4D52A11420681195FEAFF493D187D1, xmp.did:6D4D52A11420681195FEAFF493D187D1, xmp.did:FD7F117407206811B8409F1765462998, xmp.did:FE7F117407206811B8409F1765462998, xmp.did:6E4D52A11420681195FEAFF493D187D1, uuid:5224134117DADC1181AD80DEF2582423, xmp.did:6F4D52A11420681195FEAFF493D187D1, xmp.did:49442AC11C2068119160A8C421B0E112, adobe:docid:indd:e690dc80-9176-11dd-94ac-f378f3af1fc7, xmp.did:FF7F117407206811B8409F1765462998 Doc Change Count : 3495 Format : application/pdf Producer : Adobe PDF Library 9.0 Trapped : False Page Count : 60 Page Layout : SinglePage Creator : Adobe InDesign CS4 (6.0.4)EXIF Metadata provided by EXIF.tools