A novel AIEX chromatography medium (resin) to remove IgA and IVIG purification process

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1 A novel AIEX chromatography medium (resin) to remove IgA and IVIG purification Guodong Javier Jia 1, Chor Sing Tan 2, Linus Laurin 3, Henrik Ihre 3, Lili Sui 1 1 Fast Trak China, GE Healthcare Life Sciences, No 1800 Cailun Road, Shanghai, China 2 Enterprise Solution, GE Healthcare Life Sciences, 310 Ferntree Gully Road Unit 3, Notting Hill, Victoria, Australia 3 GE Healthcare Life Sciences, R&D, Björkgatan 30, SE Uppsala, Sweden First presented at the 8 th Plasma Product Biotechnology meeting Lanzarote, Spain, May 13-17, 2013

2 Introduction One of the challenges in intravenous IgG (IVIG) chromatography purification is to remove large molecular weight impurities such as IgM and IgA in order to improve the IVIG product safety. Ion Exchange Chromatography is a powerful and well established technique to selectively remove different protein related molecules. (resin), Capto Q XP, was designed by CDM, GE Healthcare Life Sciences, based on a large pore high-flow agarose base matrix modified with conventional strong anion exchange groups to optimize the adsorption of large molecular weight proteins. The dynamic binding capacity and pressure-flow properties were the key attributes considered when screening different prototypes during the development work. The goal of this work was to develop a robust industrial scale IVIG allowing for high purity, yield and productivity. 2

3 Process Design Fraction (I+)II+III from the traditional precipitation was re-suspended and then treated with caprylic acid to remove lipids and other impurities. The resulting supernatant, containing mainly IgG, was purified using two AIEX chromatography steps in flow-through mode. The first chromatography column utilized the standard Capto Q media and was found to be effective in removing all traces of albumin from the Fraction I+II+III supernatant. In addition, approximately 50% of IgA was removed by this first AIEX step. Capto Q XP is used in the second chromatography step to carry out final polishing of the IgG solution obtained from the first AIEX step. Both the IgM and the residual IgA in the IgG solution were effectively removed by the Capto Q XP step. 3

4 IVIG Process design Fraction 00+II+III/ precipitate Low ph solubilization Precipitate impurities Filter and adjust ph ph affects yield and purity Albumin, lipid, fibrinogen Chromatography sample UF/DF by 30K flat membrane Capto Q XP flow through mode Remove IgM, IgA Sample conditition adjust ph and cond. Capto Q flow through mode Remove albumin, IgA Virus filter Formulation Low ph incubation Fig.1. IVIG design from Frac (I)+II+III to final product 4

5 Dynamic binding capacity IgA and IgM dynamic binding capacity Capto Q XP did not show the highest capacity but it is still very promising for industrial scale es due to the excellent pressure-flow properties. MacroCap Q did on the other hand show the highest capacity at lab scale where the column wall support allow for higher flow-rates. However, this medium is less rigid and only possible to operate at flow rates (BPG 300 or larger columns) at around 30cm/h. Hence, not suitable for industrial scale IgM DBC (g/l gel) Fig.2. Dynamic binding capacity of IgA and IgM in Capto Q XP, MacroCap Q and ANX Sepharose Fast Flow Capto Q XP MacroCap Q ANX FF 1.8 5

6 IgG capacity (g/l gel) IgG productivity (L gel x h) IgG productivity for industrial scale For industrial es, not only the capacity but also the operation and flow-rate should be taken into consideration. The productivity describes the production per hour, measured by g protein/(l gel h), which is a key parameter for large industrial producers. As seen in Figure 3, Capto Q XP provide the highest productivity, which is 11.3 g IgG/(L gel h). Therefore, Capto Q XP is the best candidate for industrial purification of IVIG Capto Q XP MacroCap Q ANX FF 0 Capto Q XP MacroCap Q ANX FF Fig. 3A. The IgG capacity, g/l gel Fig. 3B. The IgG productivity, g/(l gel h) 6

7 Results The flow through solution was concentrated, diafiltered and then formulated to a 5% IVIG product. Albumin, IgA and IgM content in the final 5% IVIG products are 1.8mg/L, 11.2mg/ml and 15.4mg/ml respectively. The release test results from 500 L plasma pilot scale run are listed in Table 1. All QC results meet FDA and EMEA requirement. Besides the high quality, the yield is also impressive. In the pre-treatment step the yield is more than 75%, the intermediate Capto Q step show a yield of > 98% and the Capto Q XP step also show a yield > 92%. Finally, the total yield is greater than 65%. Based on 10g IgG/L in starting plasma, the final recovered. IVIG is more than 6.5g/L. The chromatography yield and impurity content are significantly affected by sample ph. Here, the optimized ph is 6.0, which can give both high yield and low impurity content. Table 1. Release test of 5% IVIG Test Electrophoresis cellulose acetate HPLC Monomer + Dimer Target 99.9% 99.8% IgM (mg/l) 15.4 IgA(mg/L) 11.2 Alb(mg/L) 1.8 PKA (U/mL) < 12 ACA(CH50/mg/hr) 2 Anti A < 1:32 Anti B < 1:32 IgG Subclass FC Activity > 80% Appearance Stability No loss from starting material Pass Pass 7

8 Custom Design Media (CDM) Custom Design Media (CDM) has over 20 years experience in the preparation of chromatography media. Development is fast and CDM can manufacture in scale within four months under normal circumstances. New chromatography media will be prepared in close collaboration with the customer to fulfill the requirements of their designated. Several CDM chromatography media have become standard products and are fully supported with regulatory documentation. All projects run according to ISO Media definition Synthesis robustness Full-scale production and validation Ligand and coupling chemistry. Decide type of matrix, define desired product function. Analytical methods. Preliminary specifications, customer evaluation of sample. Pilot scale delivery. Scale up to meet the future delivery plan. Validation of test methods and production. Validation of production. Stability studies, Regulatory Support Files. Full scale delivery. 8

9 Conclusion The results from this study highlighted the application of Capto Q XP in a IVIG purification. The introduction of a large pore sized anion IEX resin (Capto Q XP) increased the binding capacity of large molecular weight proteins, while maintaining outstanding pressure-flow properties. For a typical 5000 L scale plasma fractionation only 150 to 200 L Capto Q and Capto Q XP, respectively, are needed in a single cycle operation with 20 to 30% safety margin built into design, as shown in Table 2. Based on this, the output is more than 13,000 doses of 5% IVIG from 5000 L start plasma. Besides, this resin is also suitable to purify FVIII from cryoprecipitate IgG productivity for industry scale and fibrinogen from fraction. Plasma (L) Capto Q Capto Q XP Media volume (L) Bed height (cm) Column diameter (mm) Media volume (L) Bed height (cm) Column diameter (mm) Table 2. IVIG chromatography scale up to 5000L plasma 9

10 Acknowledgments GE, imagination at work, and GE monogram are trademarks of General Electric Company. Capto, MacroCap, and Sepharose are trademarks of GE Healthcare companies. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information General Electric Company All rights reserved. First published May 2013 GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden 10

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