Designing Next Generation Chromatography Media for Modern High-Throughput Plasma Processes. Mats Gruvegård PPB 09 Menorca, Spain

Transcription

1 Designing Next Generation Chromatography Media for Modern High-Throughput Plasma Processes Mats Gruvegård PPB 09 Menorca, Spain

2 utline Introduction Process needs Performance Designing a base matrix Screening and optimization Novel Affinity and Multimodal media Screening strategy Improved productivity in IgG process Conclusions Summary

3 utline Introduction Process needs Performance Designing a base matrix Screening and optimization Novel Affinity and Multimodal media Screening strategy Improved productivity in IgG process Conclusions Summary

4 Process needs Modern Bioprocesses require: Robustness, High Low Cost Capacity, Throughput, Lifetime, Selectivity Low batch to batch variability, Security of supply Bead size, Pore size, Construction material, Ligand, Ligand density, Particle size distribution, Surface modification, Regulatory support, Specification limits, etc

5 Next generation BioProcess media Key driving forces for new media development Higher productivities Improving the overall process economy in down stream purification New selectivities To solve new and old separation challenges that customers are facing now and in the future.

6 What is chromatography media Key components of the construction: Particle size Construction material Surface modification Type of ligand + Ligand Pore size Ligand density Particle and pore size distribution Mode of interaction

7 utline Introduction Process needs Performance Designing a base matrix Screening and optimization Novel Affinity and Multimodal media Screening strategy Improved productivity in IgG process Conclusions Summary

8 Choice of materials for a base matrix Essentially any material that could be formed into porous beads with good mechanical properties Inorganic materials Bio-polymers Synthetic polymers Silica, Glass, Ceramic, Carbon etc Agarose, Cellulose, Dextran, etc Polystyrene, Acrylamide, Methacrylate, etc H Si H Si H H Si Si Si Si Si Si Si Si Si Si H H H H Mechanical properties and chemical stability are key issues to be considered

9 Agarose an ideal material?..for chromatography media Hydrophilic and neutral Protein friendly Chemically and physically stable CIP using NaH Low material content Potentially high capacity pen pore structure Fast mass transport

10 The new High Flow Agarose platform Chemical modification of agarose R H CH 2 H H H Rigidity Pore size High Flow Agarose CH 2 H H R Sepharose FF Sepharose CL Sepharose 4/6B Bead size Capto and MabSelect media for high productivity processes

11 Increasing the window of operation The increased rigidity of the media allows for higher flow rates at large scale and smaller foot print

12 utline Introduction Process needs Performance Designing a base matrix Screening and optimization Novel Affinity and Multimodal media Screening strategy Improved productivity in IgG process Conclusions Summary

13 The ligand The key component for selectivity and capacity in combination with the nature of the base matrix! Affinity ligands Conventional IEX Multimodal S- 3 + H H N N + + H H + N Ligands for plasma processing

14 Established Affinity ligands Affinity chromatography is already a well established technique in plasma processing Heparin Dyes

15 New Affinity Ligands From Single Domain Antibodies A collaboration between BAC and GE Healthcare to develop industrial affinity media -IgGbinder - FVIII binder - AAV binder - kappa fab binder - custom ligands

16 BAC IgG004:10_UV1_280nm BAC IgG004:10_Inject BAC IgG004:10_Logbook IgSelect A new affinity resin for IgG purification mau IgG Subclass Distribution, Load vs. Eluate Serum feed IgSelect eluate MabSelect SuRe eluate ml IgG1 62.6% 62.3% 62.9% Yield: 92% Human serum albumin IgG2 28.4% 28.2% 34.2% IgG3 5.9% 5.9% 0.5% Phosforylas B 97Kd Albumin 66 Kd valbumin 45 Kd Carbanhydrase 30 Kd Trypsin inhibitor 20.1 Kd Heavy chain IgG Light chain IgG IgG4 3.2% 3.7% 2.5% A-Lactalbumin 14.4 Kd Column: Tricorn 5/100, 10 cm bed height LMW Serum Wash Elution diluted1:5 Elution diluted1:10 LMW Commercial IVIG Flow rate: 250 cm/h, corresponding to 2.4 min residence time Loading material: Human serum Equilibration: 20 mm phosphate, 150 mm NaCl, ph 7.4, 10 column volumes Wash: Same as equilibration buffer, 7 column volumes Elution: 0.1 M glycine, ph 3.0, 5 column volumes Yield: %

17 IgSelect - CIP stability Ligand screened for high chemical stability Excellent low ph stability For long lifetime phosphoric acid is suggested Combination with 0.1 M NaH can be considered Leakage assay available from BAC ( Capacity (% of reference) M NaH + 1 M NaCl 0.5 M phosphoric acid Number of CIP cycles

18 Multimodal chromatography HIC/RPC (Hydrophobicity) Ion exchange (Charge) Affinity (Biorecognition)

19 Different types of multimodal media Multimodal chromatography will give multiple type of interactions, provide new selectivities, and are in some cases designed for specific applications. S N N + H Capto MMC (high salt tolerant) Capto adhere (polishing of MAbs, non binding mode) Multiple types of interactions ionic interaction hydrophobic interaction hydrogen bonding thiophilic

20 Multimodal media in plasma process Reference process Plasma Desalting Euglobulin prec. DEAE Sepharose FF Adjustments Q Sepharose FF ph adjustment UF/DF S/D treatment CM Sepharose FF IgG Alternative process Plasma Desalting Euglobulin prec. DEAE Sepharose FF Adjustments Capto adhere ph adjustment UF/DF S/D treatment CM Sepharose FF IgG Replacing Q Sepharose Fast Flow in IgG process. Non-binding mode Initial study: sample loading increased by 100% Doubled capacity for binding impurities Yield: 4.3 g IgG/L starting plasma (vs. 3.4 IgG/L in reference process) Purity of IgG: 97% (gel filtration)

21 Virus clearance Capto adhere IgG 1 pool from MabSelect SuRe spiked with stock solutions of: Virus Conductivity (ms/cm) LRF 95% confidence limit MVM (Minute Virus of Mice) Single stranded DNA virus Non-enveloped, nm MuLV (Murine leukemia Virus) Singel stranded RNA Enveloped, nm MVM MVM MuLV ± ± ± 0.4 Applied to Capto adhere in flow through mode, ph 6.75, 10 and 30 ms/cm* MuLV ± 0.4 Very good log10 reduction factors even for conditions where traditional ion exchangers do not work! * Performed by NewLab BioQuality AG

22 utline Introduction Process needs Performance Designing a base matrix Screening and optimization Novel Affinity and Multimodal media Screening strategy Improved productivity in IgG process Conclusions Summary

23 Current Albumin & IgG process at CSL Plasma Plasma Cryosupernatant Supernatant I Delipidated SNI Delipidated & Diafiltered SNI Absorbance 280 nm (mau) IgG in flow through albumin eluted at ph 4.5 Delipidated & Euglobulin depleted SNI Elution volume (ml) Sepharose DEAE-FF chromatography CM Sepharose FF chromatography ph adjusted Crude IgG ph adjusted & concentrated Albumin MacroPrep HQ chromatography Sephacryl S200HR chromatography Concentrated & Diafiltered Pure IgG Albumin Bulk Pasteurisation Low ph caprylate incubation Pasteurised Bulk IgG Formulation (Albumin) Formulation (Immunoglobulin) Albumin IgG

24 bjective of the study Evaluate Capto DEAE as replacement of DEAE Sepharose Fast Flow in IgG / Albumin process Investigate effects of increased protein load flow rates bed height Scale-up studies Life time study Initial discussion with regulatory bodies

25 Effect of increased protein load Flow through % of total loaded Eluate % of total loaded DEAE Sepharose Fast Flow Protein load (g/l) Protein load (g/l) IgG IgA IgM Transferrin Albumin % of total loaded % of total loaded 50 0 Capto DEAE Protein load (g/l) Protein load (g/l) XK16/40 column h=17.5 cm. Flow rate of 100 cm/h., Equilibration ph 5.2 Albumin eluted at ph 4.5, Washed with 1 M NaCl. Protein load could be increased to 100 g/l using Capto DEAE

26 Effect of increased flow rate and bed height 3500 Absorbance 280 nm (mau) , 25 and 30 cm 100, 130 and 150 cm/h Elution volume (ml) Level of IgA depending on residence time could be maintained at same level as today s process Possibility to increase the column size by increasing the bed height Possible to reduce the number of cycles from 10 to 5 or 4 XK16/40 column h=25cm. Flow rates of 100, 130 and 150 cm/h. Equilibration ph 5.2 Albumin eluted at ph 4.5 Washed with 1 M NaCl.

27 Scale-up experiments nm Volume (ml) Flow through Average (3 batches) Eluate Average (3 batches) % recovery % of total % recovery % of total IgG IgG 2 <1 IgA 26 4 IgA 21 1 IgM 60 1 IgM 57 1 Albumin Albumin Transferrin Transferrin 5 <1 α 2 - macroglobulin 22 4 α 2 - macroglobulin 8 <1

28 Life time study Absorbance at 280 nm Cycle 1 Cycle Time (min) No change in performance after >360 cycles including CIP

29 Product equivalence of formulated IgG Test type Limits / Expected values Capto DEAE Laboratory-scale batches (n = 3) Production-scale batches (n = 3) Protein Composition B (SEHPLC) Aggregates 3% Monomer + Dimer 90% Fragments For information Freedom from ACA 10 CH50/mg/hour PKA 28.6 IU/mL < 1 < 1 ABD Titres Anti-A Titre 1: Anti-B Titre 1: Anti-D Titre 1:1 < 1 < 1 Hepatitis B Antibody 0.03 IU/mL Hepatitis A Antibody 3 IU/mL Tetanus Antitoxin 1 IU/mL Appearance Clear or slightly opalescent and colourless or pale yellow Pass Pass Fc function =60% EPBRP

30 utline Introduction Process needs Performance Designing a base matrix Screening and optimization Novel Affinity and Multimodal media Screening strategy Improved productivity in IgG process Conclusions Summary

31 Summary Constant improvements of process chromatography media Give possibility for plasma process improvements Some examples: New base matrix, e.g. High Flow Agarose Established ligands on new base matrix New affinity ligand constructs Multimodal chromatography

32 Acknowledgement GE Healthcare Inger Andersson Anders Ljunglöf Anna Grönberg Lise Lundh Klas Allmér CSL Karl McCann Joe Bertolini

33 Introduction Thank you Performance Screening and optimization Screening strategy Conclusions

34 Capto, MabSelect, MabSelect SuRe, MabSelect Xtra, Sepharose, Sephacryl, SURCE and Tricorn are trademarks of GE Healthcare Ltd, a General Electric Company. GE, Imagination at work and GE Monogram are trademarks of General Electric Company. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. General Electric Company reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation General Electric Company All rights reserved. GE Healthcare Bio-Sciences AB, a General Electric Company. GE Healthcare Bio-Sciences AB, Björkgatan 30, SE Uppsala, Sweden.