MabSelect Xtra. GE Healthcare. Recombinant protein A-based, high-capacity affinity medium. Medium characteristics Description

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1 GE Healthcare Data File AB Affinity chromatography MabSelect Xtra Recombinant protein A-based, high-capacity affinity medium Many disorders that are treated with monoclonal antibodies, e.g. cancer and immunological diseases, require prolonged therapy with high doses. Manufacturers of therapeutic antibodies thus need to produce larger quantities of material at the same time as they improve overall process economy. MabSelect Xtra (Fig 1) is a recombinant protein A-based affinity medium engineered to give a dynamic binding capacity higher than other commercially available recombinant protein A-based media. Higher capacity translates directly to lower cost of production. Key performance characteristics of MabSelect Xtra include: Outstanding high dynamic binding capacity. Improved process economy and reduced raw materials costs. High purity capture due to minimal nonspecific binding. Simple scale-up to production-sized columns. Medium characteristics Description MabSelect Xtra is a member of the MabSelect product line of recombinant protein A-based affinity media for capturing monoclonal antibodies. Like its companion product, MabSelect, MabSelect Xtra is based on an innovative, high-flow agarose base matrix, optimized for maximum capacity (Fig ). The recombinant protein A is coupled to the base matrix at the C-terminal cysteine via a stable thio-ether bond. It is the same ligand and coupling chemistry used in MabSelect. This oriented coupling contributes to the increased capacity seen with these media. Fig 1. MabSelect Xtra captures antibodies from high-expression feedstocks with improved efficiency and economy. The MabSelect family of media for process-scale purification of monoclonal antibodies comprises MabSelect, MabSelect Xtra, and MabSelect SuRe TM. MabSelect is designed for highthroughput purification of monoclonal antibodies from large volumes of feed. MabSelect SuRe is composed of the same rigid, high flow agarose matrix, but posesses an alkalistabilized rprotein A-based ligand. The ligand provides greater stability than conventional rprotein A-based media allowing extended use of.1. M snaoh for cleaning-inplace. For more information on MabSelect and MabSelect SuRe, refer to data files and respectively.

2 Table 1. Main characteristics of MabSelect Xtra. Fig. Electron micrographs of a MabSelect Xtra particle. The inset (resolution nm) shows the structure of the high-flow agarose matrix with large pores and high connectivity. MabSelect Xtra is easy to pack and operate in a wide variety of columns from laboratory to production scale. Well-proven packing and cleaning-in-place (CIP) recommendations are available, as are scale-up studies. Table 1 lists the key characteristics of MabSelect Xtra. Dynamic binding capacity Lower cost of production is a key processing goal of monoclonal antibody manufacturers. In upstream operations, cell culture expression levels are steadily increasing and forecasted to reach multi-gram levels per liter medium in the near future. New MabSelect Xtra responds to these improvements in cell culture optimization; the medium is designed for the downstream capture of antibodies from high expression feedstocks. Confirmation of the high dynamic binding capacity (DBC) of MabSelect Xtra compared with competitor high capacity media is seen in Figure 3, where the two media were tested with human IgG. (mg/ml) MabSelect Xtra ProSep -va Ultra Composition Particle size 1 d v 7 µm Ligand Coupling chemistry Dynamic binding capacity Chemical stability 3 Highly cross-linked agarose Recombinant protein A (E. coli) Epoxy Approx. 4 mg human IgG/ml medium (MabSelect approx. 3 mg IgG/ml medium) Stable in all aqueous buffers commonly used in protein A chromatography: 1 mm H 3 PO 4 (ph 1.6), 1 mm HCl (ph ), 1 mm NaOH (ph 1),.1 M % sodium citrate/ HCl (ph 3), 6 M Gua-HCl, 6 M urea, % ethanol, % benzyl alcohol Recommended ph Working range 3 1 Cleaning-in-Place (CIP) 1 Recommended mobile phase 1 3 cm/h (MabSelect velocity 4 1 cm/h) Temperature stability 4 4 C Delivery conditions % ethanol Mammalian product No material of animal origin is contact-free used in the manufacturing process Regulatory support Regulatory Support File 1 d v is the medium particle size of the cumulative volume distribution. Determined at 1% breakthrough by frontal analysis at a nominal mobile phase velocity of cm/h in a column with a bed height of 1 cm (.4 min residence time). 3 No significant change in chromatographic performance after one week storage or 1 cycles normal use at room temperature, which corresponds to a total contact time of 16.7 h (1 mm H 3 PO 4 ; 1 mm NaOH). 4 In a packed bed, the maximum recommended operating velocity for MabSelect Xtra is 3 cm/h at cm bed height, with water at C. Operating pressure is less than bar. Recommended long-term storage conditions: 4 8 C, % ethanol. The outstanding dynamic binding capacity of MabSelect Xtra is also observed when two different monoclonal antibody feedstocks are tested. At a residence time of.4 min the dynamic binding capacity at 1% breakthrough of a humanized IgG 4 (MAb 1) is 4 mg/ml and of a humanized IgG 1 (MAb ) about 3 mg/ml. Increasing the residence time to 4.8 min, increases the dynamic binding capacity by 1% (Fig 4) Residence time (min) Fig 3. At equivalent residence times, MabSelect Xtra has 3% higher dynamic binding capacity than porous glass- based protein A media. Feed concentration was 1.1 mg/ml human polyclonal IgG. Data File AB

3 Protein A leakage Leakage of protein A from MabSelect Xtra is generally low. However, leakage of protein A is also affected by chromatographic running conditions and the composition of feed stock. A purification study of IgG 4 at pilot scale, which included a blank run and CIP with mm NaOH,. M Na (see Application-Purification of IgG 4 using MabSelect Xtra) showed leakage levels of. 7. ppm (ng/mg), see Figure. In this example, three cycles were performed. Studies with MabSelect (same ligand and coupling chemistry) have shown low stable levels of rprotein A leakage for up to 3 cycles (source: The use of sodium hydroxide for CIP of rprotein A media: a 3 cycle study, Hans J Johansson et al, Amersham Biosciences. Oral presentation at IBC Antibody production and Downstream processing, Sept 4,, London, UK.). To remove loosely bound protein A, a blank run including CIP is recommended before the first run with antibody feed and after storage of the media. Host cell protein study The level of host cell proteins in the eluate from MabSelect Xtra has been assessed following repeated processing of a Chinese hamster ovary (CHO) supernatant spiked with human IgG. Complete cycles (equilibration, loading, elution, washing, and CIP) were run, after which the level of host cell protein in the eluate was measured using a high sensitivity ELISA protocol. A comparative study was made on a protein A medium attached to a porous glass base matrix. Results show that the content of host cell protein in the eluates was relatively constant over cycles (Fig 6). However, normalized levels (in ppm) were approximately 6-fold higher for the glass bead-based medium than for MabSelect Xtra. This result suggests a higher degree of nonspecific adsorption to porous glass beads compared to MabSelect Xtra s hydrophilic agarose base matrix. Operation Method optimization Method optimization should establish conditions that promote binding the highest amount of target antibody in the shortest time with maximum purity and recovery. In general, all antibodies of human origin, except subclass 3, have a high affinity for protein A. To optimize elution, determine the highest ph that allows efficient desorption of antibody. Step-wise elution is preferred at large-scale as the antibody elutes in a smaller pool volume, thus decreasing buffer consumption and shortening cycle times. QB 1% (mg/ml medium) 8 Polyclonal higg 7 Mab-1 6 Mab ppm protein A HCP conc (ppm) normalized Residence time (min) Fig 4. MabSelect Xtra dynamic binding capacity of immunoglobulins at.4 min residence and 4.8 min, respectively. Protein A Run No. 1 Run No. Run No. 3 Fig. The leakage of protein A in the eluted IgG4 material showed protein A levels between. and 7. ppm (ng protein A/mg IgG). Porous glass bead medium MabSelect Xtra Cycle no. Fig 6. Normalized HCP concentration: a comparison between MabSelect Xtra and a porous glass bead, high-capacity medium. Data File AB 3

4 Process development For initial studies of MabSelect Xtra in small-scale columns, we recommend XK 16 columns with a 1 cm bed height. Choose a residence time that meets the demands on capacity and nominal fluid velocity. Make sure the chosen bed height and velocity do not conflict with the large-scale pressure/flow limitations (see Fig 7). Figure 7 shows the possible operating window with combinations of bed height and operational nominal mobile phase velocity for MabSelect Xtra. The Figure also displays the residence time in minutes for any bed height and velocity. Pressure drop and packing limitations at large scale are also included. The solid line shows the large-scale column pressure restriction (gray background). The dashed line indicates that operating at below 1 cm bed height is not favorable (gray background). The reason for this is that large diameter columns have a very different aspect ratio, and that packing short wide beds is a greater challenge. Use Figure 7 as a guide when determining suitable bed height and operating velocity in terms of residence time and thus capacity and pressure drop. Cleaning and sanitization Media life-length expressed as the number of overall purification cycles is a key parameter when looking to lower cost of production in downstream processing. The lifetime and performance (notably dynamic binding capacity) of MabSelect Xtra were evaluated with CHO supernatant spiked with polyclonal IgG in a 14 cycle study which included complete processing cycles (equilibration, loading, elution, washing and CIP performed after each cycle). The cleaning agent, mm NaOH plus mm Na, gave effective, long-term cleaning. UV curves from chromatograms made during the study were almost identical and no visible differences in purity could be observed (Fig 8). In addition, the backpressure increase was less than.1 MPa for 14 cycles compared to the start pressure. Related protocols that have shown good effect with MabSelect will also serve as the basis for designing a CIP protocol for MabSelect Xtra (see Table ). For example, a CIP protocol comprising mm NaOH with 1 M NaCl has proven to be both effective (> 1 cycles) and economic (sodium hydroxide is a low-cost agent) when tested on MabSelect with real feedstock under real processing conditions Velocity (cm/h) 1 Not recommended area Not recommended area Bed height (cm) Fig 7. Operating window for MabSelect Xtra: Choosing bed height and operating velocity in terms of residence time, pressure restrictions, and large-scale column packing. Recommendations for sanitization of MabSelect Xtra (reducing microbial contamination of the packed bed to a minimum) with a number of well-proven methods are listed in Instruction Absorbance (mau) Fig 8. Selected chromatography runs between cycles 1 and 138 are almost identical. 1 Cycle Cycle Cycle 6 Cycle 1 Cycle Retention (min) 4 Data File AB

5 Table. CIP choices available for MabSelect Xtra. CIP protocol Considerations for use mm NaOH + mm Na Recommended for effective cleaning and long to intermediate life-length. mm NaOH + 1 M NaCl 1 mm NaOH + 1 M NaCl or For better cleaning effect if extended lifetime is not critical. 1 mm NaOH + mm Na 6 M Gua-Hcl Denaturation agent for difficult cases. 6 8 M Urea Poor denaturation agent for many proteins, but may work for some difficult cases. Scale-up After optimizing the antibody purification at laboratory-scale, the process can be scaled up to larger columns. MabSelect Xtra has been successfully packed in large-scale columns in our laboratories (Fig 9). As noted in Table 1, at the 3 cm diameter scale (where wall effects are no longer a consideration), MabSelect Xtra has a mobile phase operating velocity no greater than 3 cm/h at a cm bed height with a low viscosity buffer. Packing methods for large-scale columns Good column packing is essential for any chromatographic process and naturally plays a key role in large-scale commercial manufacture. Proper packing will eliminate concerns such as channeling and compression and ensure a stable bed that performs according to expectations over many processing cycles. Reliable and easy-to-follow pump-based methods have been developed for packing MabSelect Xtra in three types of commonly-used large-scale columns; Chromaflow, BPG, and BioProcess LPLC. Test results show that MabSelect Xtra is both easy to pack and repack reproducibly at large scale. Table 3 lists recommended large-scale column details. Table 3. Large-scale production columns for MabSelect Xtra. Column* Inner diameter Bed volume Bed height (mm) (cm) Lab scale: XK 16/ 16 3 ml 1 1 Production scale * : BPG l 1 3 BioProcess l 1 3 Chromaflow l 1 3 * Packing instructions for large-scale columns are described in Application Note Velocity (cm/h) All large-scale columns can be supplied as variable bed height columns. Note that all bed height and diameter combinations above are possible but not suitable. For example, do not choose large-diameter columns if the bed height is low. Another general recommendation is not to choose a bed height above 3 cm. The methods for large-scale packing of MabSelect Xtra are described in full detail in Application Note Pressure (bar) Fig 9. Typical pressure-flow curve obtained in Chromaflow 6 at cm bed height. Recommended flow velocity for MabSelect Xtra at large scale is 3 cm/h. Data File AB

6 Process economy Lower cost of antibody capture An important, if not the most important, consideration in bioprocessing is the economic aspect of the process. This is an especially important issue considering the anticipated increase in fermentation volumes and expression levels. For a primary recovery step, the purification cost can be broken down into two types of contributions, one related to the number of cycles and the other related to the process time. In the first group, the cost of chemicals including chromatography media, as well as quality control are two important factors that need to be considered. In the second group, labour and facility costs may be predominant factors. The increase in dynamic binding capacity affects process economy because a lower number of cycles leads to a less frequent replacement of protein A resin and to a propotionally lower buffer consumption. For example, an increase of 3%, which should be the typical dynamic binding capacity gain of using MabSelect Xtra, results in about 3% lower overall cost of the capture step (Fig 1). Economic gain (%) % DBC increase 3% DBC increase 4% DBC increase Contribution of buffer and affinity medium to the overall process cost (%) Fig 1. Effect of an increase in dynamic binding capacity on process economy. The economic gain is shown for a %, 3%, and 4% increase in DBC. Application purification of IgG 4 The capture of IgG 4 (pi 6. 7.) summarized below was performed on a FineLINE Pilot 3 column packed with MabSelect Xtra using an ÄKTApilot chromatography system. The column was packed with MabSelect Xtra to a bed height of cm and run at a nominal fluid velocity of 3 cm/h, which gave a residence time of 4 min. The purification cycle, which included CIP with mm NaOH, mm Na (see Cleaning and sanitization) was repeated three times. Figure 11 shows the running conditions and chromatogram of the first run. IgG 4 yield and purity were high over all three runs. Average yield was 94% and the monomer form of the material as determined by analytical gel filtration on Superdex HR exceeded 99% (Fig 1). The goal of achieving high recovery and product purity with the capture step run at pilot-scale on the FineLINE Pilot 3 column was clearly reached (the eluted antibody contained more than 99% monomer). In addition, the purification step was very consistent; the same separation and analytical results were found in all three runs. Column: FineLINE Pilot 3 (bed height.1 cm, 1 column volume =193 ml) packed with MabSelect Xtra Sample: IgG 4 clarified NSO supernatant, Sample load:.16 l (. g/l) Buffer A: 1 mm sodium phosphate, 1 mm NaCl, ph 7. Buffer B: 1 mm glycine-hcl, ph 3. Flow rate: 3 cm/h (48 ml/min) Gradient: 1 % B in CV CIP: mm NaOH, mm Na, contact time 1 min (46 ml/min) System: ÄKTApilot TM mau ph 1. 3 Eluted IgG ml Fig 11. Capture of IgG4 on MabSelect Xtra packed in a FineLINE Pilot 3 column. Average yield (94%) and purity (more than 99% monomer) were high over the three runs performed. The NSO cell line feedstock containing the IgG 4 monoclonal antibody was supplied by Lonza Biologics, UK. 6 Data File AB

7 Column: Tricorn 1/3 (bed height 3 cm, 1 column volume = 3.6 ml) Medium: Superdex HR Sample: IgG4 purified on MabSelect Xtra from clarified NSO supernatant, Lonza Biologics Sample volume: µl Buffer A: PBS, ph 7.4 Flow rate: 39 cm/h (. ml/min) System: ÄKTAexplorer 1 1 mau 6 nm ml Fig 1. Dimer/aggregate analyses on Superdex HR of the start material showing clarified IgG4 supernatant (red), flowthrough (green), and eluate after the capture step (blue). Note that no monomer of IgG4 was found in the flowthrough (green line). Ordering information Product Pack size Code No. MabSelect Xtra ml ml l l l l Columns and packing Tricorn 1/1 GL Inquire equipment (prepacked with MabSelect Xtra) Related literature XK 16/ (16 mm i.d.) Packing connector XK XK 16/ tube Data files MabSelect MabSelect SuRe BPG columns BioProcess LPLC columns Chromaflow columns Application notes MabSelect Xtra Column packing MabSelect Xtra IgG purification on ÄKTApilot All bulk media products are supplied in suspension in % ethanol. For additional information, please contact your local GE Healthcare representative. Data File AB 7

8 For contact information for your local office, please visit, GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden imagination at work GE, imagination at work, and GE monogram are trademarks of General Electric Company. ÄKTAexplorer, ÄKTApilot, BioProcess, BPG, Chromaflow, FineLINE, MabSelect, MabSelect SuRe, MabSelect Xtra, Superdex, Tricorn, and Drop design are trademarks of GE Healthcare companies. All third party trademarks are the property of their respective owners. 7 General Electric Company All rights reserved First published Nov. 4 Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US pat,84,933 and US pat,31,663, including corresponding foreign patents (assigne: Hoffman La Roche, Inc). All goods and services are sold subject to the terms and conditions of sale of thecompany within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for themost current information. GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp., 8 Centennial Avenue, P.O. Box 137, Piscataway, NJ , USA GE Healthcare Europe GmbH, Munzinger Strasse, D Freiburg, Germany GE Healthcare Bio-Sciences KK, Sanken Bldg., 3--1, Hyakunincho, Shinjuku-ku, Tokyo, Japan AB 11/7 Elanders Östervåla 7