DOW AMBERCHROM HPS60 Cation Exchange Media. User Guide

Transcription

1 DOW AMBERCHROM HPS60 Cation Exchange Media User Guide

2 Contents Introduction...3 Intended Use...3 Typical Specifications...4 Operator and Equipment Safety...4 First Aid...4 Column Packing...4 Compression Factor...4 Small Scale Column Packing...5 Lab Scale Column Packing Method...5 Large Scale Column Packing cm Diameter QuickScale Column Packing...5 Process Conditions...6 General...6 Recommended Buffers for Cation Exchange...6 Adsorption...6 Adsorption Process Optimization...7 Elution...7 Elution Process Optimization...7 Column Regeneration...9 Cleaning and Sanitization...9 Storage and Handling...9 Ordering Information...9 References...9 Technical Assistance...9 2

3 Introduction DOW AMBERCHROM HPS60 media is a strong cation and exchange absorbent which has been developed to facilitate highly efficient, cost effective purification for proteins including both monoclonal (mab) and polyclonal antibodies (PAb). This media s excellent impurity resolution, capacity and pressure flow properties enable the robust, efficient and cost-effective production of therapeutic and other high value proteins. DOW AMBERCHROM HPS60 media consists of a cross-linked polymethacrylate base matrix modified with a strong cation S ligand. The DOW AMBERCHROM HPS60 base matrix is a semi-rigid, durable, monodisperse particle which exhibits good packed bed permeability and rapid mass transfer. The media has robust chemical and mechanical stability over a range of operating conditions and solutions and does not exhibit significant volumetric changes in common process solutions including ph extremes, varying ionic strength, exposure to detergents and many organic solvents. DOW AMBERCHROM HPS60 media is optimized for efficient mass transfer with a high surface area, open pore structure and monodisperse 58 μm particle size. These properties enable a high dynamic binding capacity with sharp breakthrough curves over a wide range of buffer conditions. DOW AMBERCHROM HPS60 is ideally suited for the efficient, cost-effective purification of monoclonal antibodies and other proteins from laboratory to process and production scales. Intended Use DOW AMBERCHROM HPS60 media provides excellent purification of mono-clonal antibodies, and provides benefits for the purification of many proteins, including fusion proteins, antibody fragments, polyclonal IgG and other recombinant products. DOW AMBERCHROM HPS60 media is designed to provide process flexibility in the large scale purification of proteins. The semi-rigid base matrix allows for good flow at bed heights of 30 cm. The flow properties in combination with the excellent mass transfer enable the use of higher flow rates during loading such as loading a 20 cm bed at 400 cm/hr (3 minute residence time). The mass transfer properties provided by the open pores and monodisperse 58 μm particle size also lead to sharp elution peaks and good resolution of difficult-to-remove impurities. The improved mass transfer makes the binding capacity less dependant on the buffer condition during loading. 3

4 Typical Specifications Support Matrix Particle Size Ligand Cross-linked polymethacrylate 58 μm S type strong cation exchange ph Range 1 to 13 Maximum Flow Rate Recommended Flow Rate Range Lifetime Binding Capacity Shipping Buffer Recommended Storage 600 cm/hr at 20 cm bed height 200 to 500 cm/hr at 20 cm bed height DOW AMBERCHROM HPS60 media is stable over repeated operational cycles when appropriate regeneration and cleaning protocols are used 64 mg/ml (polyclonal IgG) 20% ethanol 20% ethanol, short-term storage in M NaOH Operator and Equipment Safety DOW AMBERCHROM HPS60 media is supplied in buffer containing 20% ethanol. DO NOT mouth pipette. The buffer may be harmful if swallowed. First Aid Eyes Skin Mouth Spills The Material Safety Data Sheet (MSDS) for this product and all reagents used with it should be carefully reviewed prior to beginning work. Work should not commence until the user is thoroughly familiar with the health and safety hazards of all reactants and the hazardous nature of their interactions. Column Packing Rinse thoroughly with water. If discomfort persists obtain medical attention. Wash thoroughly with soap and water. Rinse thoroughly with water. In severe cases, obtain medical attention. Wear appropriate protective clothing. Carefully mop up spill and dispose of in accordance with local regulations. Recommended bed heights are 20 cm to 30 cm, with maximum flow rates of 600 cm/hr and 400 cm/hr respectively. The spherical, monodisperse nature of the media allows for acceptable packed beds with standard flow packing methods. DOW AMBERCHROM HPS60 cation exchange media is supplied in a suspension of 20% ethanol. Prior to column packing, buffer exchange the media by pouring off the storage buffer above the settled media and replace it with the appropriate volume of packing buffer (typically phosphate buffer saline (PBS) or water) to give a 50 to 70% slurry. Gently mix the slurry into a suspension. Note: When slurrying the media, do not use magnetic stirrers, as these can damage the media. Allow the media to settle for approximately 60 minutes, then decant off the supernatant. Add fresh packing buffer and repeat until ethanol is removed. This decanting step can also be used to remove fines (define the media before packing). An assessment of the fines content can be made by measuring the turbidity of the supernatant after allowing the resin to settle for 3 to 4 hours. Keep the turbidity below 100 NTU. If the value is > 100 NTU, repeat the defining steps. Compression factor DOW AMBERCHROM HPS60 media is a semi-rigid, mono-disperse media with a compression factor between 5 and 10%. Using the settled bed volume, the slurry should contain 1.08 times the column s target media volume. 4

5 Small Scale Column Packing Lab Scale Column Packing Method 1. Buffer exchange the media into the packing buffer, typically phosphate buffer saline (PBS) or water solution by decanting the supernatant 3 5 times. Define the media if needed. Make the final media slurry of 50% (v/v) in the packing buffer. 2. Wet the column outlet frit (i.e. a fixed end) with the packing buffer. Add about 4 cm of buffer into the column and plug the outlet when the liquid drains to 1 to 2 cm. Ensure the column is vertical during the packing. 3. Gently suspend the sample into a homogeneous slurry. Do not use magnetic stirrer as this may damage the media. Shake gently to avoid air entrapment or foaming. 4. Pour the slurry continuously along a glass rod into a 0.66 cm 21 cm column. Let the media settle until at least 1 cm clear supernatant layer is visible. 5. set the pump flow rate to 4 ml/min (or equivalently 700 cm/hr) and purge the header. Remove the column outlet plug and place the top header onto the column. The flow helps speed up media settling. Once the bed height is stable, turn off the pump and remove the column header carefully to avoid disturbance of the settled media. Use a pipette to remove the buffer above the settled media. 6. repeat step 4 until the final bed height reaches approximately 21 cm. Mark the final bed height on the column. 7. Turn the pump off and ensure no air is trapped in the system. Carefully lower the column header to the marked line. The column header will contact the top of packed bed surface. Tighten the column header. 8. set the flow rate at 4 ml/min and flush the column with packing buffer for about 10 minutes. If the bed height changes, mark the new bed height and turn off the pump. Adjust the column header so that the frit is in contact with media surface again. Repeat this step until the packed bed height is stable. 9. Close the column outlet and remove the column. Ensure that no air is introduced into the column during this step. 10. Qualify the column by injecting a pulse of 1M sodium chloride solution, 1% acetone or sodium nitrate and monitor the conductivity or UV changes. Packed Bed Acceptance Criteria Asymmetry (As) = 0.8 to 1.6 HETP 0.06 cm Large Scale Column Packing 45 cm Diameter QuickScale Column Packing 1. Buffer exchange media into water or a packing buffer. Measure out the media volume required to pack a targeted column volume ensuring compression factor has been accounted for (5 10%). Prepare a 50 60% of media slurry in packing buffer by letting the media settle in a container for at least one hour and add or remove buffer to the targeted concentration accordingly. Define the media if needed. Slurry the media to a homogenous suspension before transferring to the column. Do not suspend the resin using a pump in recirculating mode as this may damage the resin. 2. Pour some water into the column and let it flow out from the bottom to purge column downstream. Close the bottom valve. Allow 1 to 2 cm of water to remain in the column. 3. Transfer 50% of the media slurry into the large column. If using a pump, ensure the pumping rate and pressure are not high enough to break the media. Let the media settle by gravity until a clear water bed of 1 to 2 cm is formed at the top. 4. remove the sanitary clamp to disconnect the inlet line from the top header so that the header can be purged. Slowly lower the top header into the column until it touches the top of the water bed. Slightly tighten the header seal so that no water (and possibly media) escapes from the gap between the seal and 5

6 column wall. Keep the seal loose enough to push the header down without much effort. Lower the header into the water bed until all the air trapped in column and header line is purged from the top. 5. start water flow through inlet pipe at a low flow rate (< 50 cm/hr) and connect it to the top header. Immediately open the bottom valve so that flow path is established. 6. increase the flow rate rapidly (600 to 700 cm/hr) and let water flow until the bed compresses to a stable bed height. Mark this height, stop the water flow, close the bottom valve, remove the clamp to disconnect inlet tubing and loosen the header seal to push the header down into the column until it touches the bed surface. 7. After stopping water flow, the media bed will start to rebound and the bed will have regained some of its height by the time top header touches it. Repeat step 6 until the bed does not compress any further and the header is tightly in touch with the bed surface. Observe the appearance of the packed column for any cracks or uneven surface. If column is free of voids or crack, then measure the pressure drop across the column at several different flow rates up to near packing flow rate. 8. equilibrate the column with a low salt buffer (e.g to 0.1M NaCl). Measure the HETP and asymmetry using 1M NaCl solution. Packed Bed Acceptance Criteria Asymmetry (As) = 0.8 to 1.6 HETP 0.06 cm Process Conditions General Ensure all buffers and sample feedstock are filtered prior to use (0.22 or 0.45 μm). Before applying sample to a newly packed column or one that is being reused after storage, run a blank cycle of the equilibration, elution and regeneration buffers. After the blank cycles the column needs to be exchanged back to the equilibration buffer before loading. Recommended Buffers for Cation Exchange Acetate (pka 4.8) Citrate (pka 3.1) Malonate (pka 5.7) 2-(N-morpholino)ethanesulfonic acid or MES (pka 6.15) 3-(N-morpholino)propanesulfonic acid or MOPS (pka 7.2) Phosphate (pka 7.2) Bicine (pka 8.35) Typically 5 CV (column volumes) are sufficient for equilibration, but this should be confirmed by monitoring the column eluate ph. Adsorption Cation exchange chromatography binds positively charged molecules through ionic interactions with negatively charged ligands attached to the media. In general, to bind the molecule of interest to the DOW AMBERCHROM HPS60 media, the buffer ph must be at least one ph unit below the isoelectric point (pi) of the target molecule. In addition, the buffer conductivity should be low (generally below 16 ms). Under these conditions, the target molecule will have a net positive charge and the buffer conductivity will not interfere with binding to the media. The buffer selected should possess a negative charge and be within 0.5 ph units of the buffer s pka. Once an adequate binding buffer has been determined, loading the sample on the column can be optimized (mg of target molecule per ml of media). DOW AMBERCHROM HPS60 media s excellent mass transfer enables loading at higher flow rates (see Figure 1 on page 7). This allows for higher productivity and easier processing of large volumes. 6

7 Qd 5% BT Adsorption Process Optimization Example for Polyclonal IgG The dynamic capacity of large proteins such as monoclonal antibodies (mab) or polyclonal IgG can be dependant on the loading buffer conductivity and ph. [1] It is important to screen conditions of both ph and conductivity to determine the optimal loading buffer. For example, to optimize the loading of DOW AMBERCHROM HPS60 with polyclonal IgG, experiments were conducted using a 0.66 cm x 7 cm column at 3 minute residence time (20 CV/hr). The 50 mm acetate buffer ranged from ph 4.5 to ph 5.5 and conductivities (adjusted with NaCl) from 4 to 16 ms. The dynamic capacity for the mapping experiments at 5% breakthrough are reported in the contour ph Residence Time (min) Figure 1. Binding Capacity vs Residence time for polyclonal IgG. For DOW Amberchrom HPS60 capacities above 60 g/l can be achieved over the broad range of 3 to 6 minute residence time (ms/cm) Figure 2. Dynamic capacity of DOW AMBERCHROM HPS60 For polyclonal IgG from ph 4.5 to 5.5 and conductivities ranging from 4 to 16 ms. DOW Amberchrom HPS60 Commercial Agarose Capacity mg/ml < >50.0 plot in Figure 2 below. From this analysis, it is clear that to achieve a target loading of 50 g/l, the buffer conditions would need to be from ph 4.5 to ph 5 and conductivities from 4 to 7.5 ms; a process window of ± 0.25 ph unit and ± 1.75 ms. Elution To recover and purify the target molecule, a carefully selected increase in buffer conductivity and/or ph is required. The simplest way to determine the conditions for purification and recovery of the molecule of interest is by running a linear gradient, typically increasing the conductivity with a simple salt such as sodium chloride. Begin the gradient with equilibration (loading) buffer with no additional salt. Increase the conductivity by blending in sodium chloride up to a concentration of 0.5M or 1M over the course of 10 to 20 CV. In many cases the proteins will separate during the gradient and elute at different conductivities, resulting in two or more peaks. Often the UV280 can be monitored and the quality of the separation qualitatively judged from the peaks in the chromatogram. In these cases, optimization can be done by monitoring the peak separation as conditions are varied and then running step changes in conductivity or ph similar to the gradient condition where the two proteins show the best separation. Elution Process Optimization Example for Monoclonal Antibody To determine the impurity removal potential for a cation exchange mab polishing step (typically after Protein A affinity chromatography) a gradient elution (10 to 90 CV) can be performed and fractions collected throughout the elution (1 to 2 CV fractions). In this example, a mab Protein A pool was loaded onto DOW AMBERCHROM HPS60 media to 40 g/l with acetate buffer at ph 5.4. A 90 CV gradient from 100% buffer A (equilibration/loading buffer) to 100% buffer B (equilibration buffer plus 0.5M NaCl) was run for elution. Fractions were triggered by monitoring the UV280 and 0.5 CV fractions were collected along the peak width (see Figure 3A on page 8). The fractions were analyzed for host cell proteins, leached Protein A 7

8 and mab aggregates. For this polishing step, removal of aggregates was the key separation. Analysis of fractions from the gradient experiment showed the aggregates eluted very late with the elution peak, suggesting a step elution might work to remove the higher molecular weight species. A 280 Off Scale 100% B, EQ Buffer with 0.5M NaCl Step elution to 100% buffer B Elution Pool Volume 2 CV Aggregate > 2% Off Scale 100% B, EQ Buffer with 0.5M NaCl 100% A EQ Buffer Regen Peak 1M NaCl Collect 2 CV Pool A 280 Gradient Elution Column Volumes (CV) Figure 3B. Chromatogram for high salt step elution 100% A EQ Buffer Regen Peak 1M NaCl Off Scale Step elution to 18% buffer B Collect Fractions every 0.5 CV Figure 3A. Chromatogram for gradient elution screening A 280 Elution Pool Volume 3 CV Aggregate < 1% Regen Peak 1M NaCl Conversion from Gradient to Step Elution Typically conversion from a gradient elution to a step elution requires some trial and error. If the salt step elution is too high in conductivity (as seen in Figure 3B), the elution pool volume will be reduced, but at the expense of purification. The elution pool in 3B contained a significant amount of aggregates, thus rendering the purification ineffective. If the salt step is too low in conductivity, the elution will be incomplete. This is easiest to observe by a large increase in the regeneration peak as observed in Figure 3C. The aggregate removal for the elution pool shown in 3C is very good, but along with the aggregate in the regeneration peak, a significant amount of mab monomer was observed. When the step elution uses the correct conductivity, the elution pool is slightly larger than the high salt case, but the purity (< 1% aggregate) and mab monomer yield (89%) are very good. The regeneration peak contains primarily aggregate, a good indication the process optimization is nearly complete. Figure 3C. Chromatogram for low salt step elution A % A EQ Buffer 100% A EQ Buffer Off Scale Collect 3 CV Pool Column Volumes (CV) Step elution to 24% buffer B Elution Pool Volume 3 CV Aggregate < 1% Collect 3 CV Pool Column Volumes (CV) 18% B, EQ Buffer with 0.5M NaCl 24% B, EQ Buffer with 0.5M NaCl Regen Peak 1M NaCl Figure 3D. Chromatogram for intermediate salt step elution 8

9 Column Regeneration After elution, column regeneration can be done by flowing 3 to 10 CV of high conductivity buffer (typically 1 to 2 M NaCl). This step can be optimized by monitoring the UV280 and stopping the regeneration once the UV signal has reached the original baseline. Cleaning and Sanitization Cation exchange columns require periodic cleaning and sanitization as part of normal operation. Clean in place (CIP) can typically be accomplished with 0.5 M NaOH for 2 3 CV or 15 minute exposure. Other cleaning solutions can be used with DOW AMBERCHROM HPS60 media including 70% ethanol, 6M guanidine hydrochloride and 0.1M Phosphoric Acid. Storage and Handling DOW AMBERCHROM HPS60 media should be stored in 20% ethanol for long term storage. DOW AMBERCHROM HPS60 media can be stored in 0.05M to 0.1M NaOH for up to 24 months depending on the storage conditions. Store between 4 and 30ºC. DO NOT FREEZE. If used under the recommended conditions, the product will be reusable over many cycles without significant loss of performance. Ordering Information Description Primary Use Qty Catalogue No. 100 ml Resin Bench top Packing and Process Development 1 x 100 ml ml Resin Packing and Scaling Trials 1 x 1000 ml ml Resin Small Scale Manufacturing 1 x 5000 ml L Resin Bulk Drum Large Scale Manufacturing 1 x 50 L References 1. C. Harinarayan, et.al.; Biotechnology and Bioengineering, Vol. 95, No. 5, December 5, Technical Assistance For more information contact or dowcig@dow.com 9

10 NOTICE: No freedom from infringement of any patent owned by Dow or others is to be inferred. Because use conditions and applicable laws may differ from one location to another and may change with time, Customer is responsible for determining whether products and the information in this document are appropriate for Customer s use and for ensuring that Customer s workplace and disposal practices are in compliance with applicable laws and other government enactments. The product shown in this literature may not be available for sale and/or available in all geographies where Dow is represented. The claims made may not have been approved for use in all countries. Dow assumes no obligation or liability for the information in this document. References to Dow or the Company mean the Dow legal entity selling the products to Customer unless otherwise expressly noted. NO WARRANTIES ARE GIVEN; ALL IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE ARE EXPRESSLY EXCLUDED. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow Form No