Applications Sartobind Membrane Adsorbers

Transcription

1 Platzhalter Bild Applications Sartobind Membrane Adsorbers Dr. Stefan Fischer-Frühholz, Goettingen, July 11, 2007

2 Applications overview Flow through Bind and elute 7/16/2007 page 2

3 Flow through: Contaminant removal in Downstream Processing Purification Cell-Harvest/Clarification Affinity Chrom. Capturing Step Cation Exchange Anion Ex. Sartobind Disposable UF Buffer Exchange 0.2μm Sterile Virus Filter Form and Fill DNA Endotoxins Host cell proteins Leached protein A Viruses 7/16/2007 page 3

4 DNA removal from a therapeutic antibody. Campath 1-H pg DNA pro mg mg Protein / ml Average of three 12,500 liter batches with DNA binding module capacity Af ter pr otein Sartobind A Q Af ter Q-Sepharose Sartobind FF Q J. K. Walter, Bioseparation and Bioprocessing, G. Subramanian (ed.) Wiley VCH, Vol. II p , clears the DNA below detection limit superior pressure/flow relation...the process time is reduced 23-fold, excluding the benefit in handling for set-up....diminish a loss of product...installed as in-line filters and can be disposed after use* Galliher P, Fowler E, Millennium Pharmaceuticals Inc.: Validation of Impurity Removal by the CAMPATH-1H Biomanufacturing Process. IBC s Biopharmaceutical Production Week, Paradise Point Resort San Diego, CA, November 12-15, 2001 FDA approval March /16/2007 page 4

5 Q Membrane in flow-through mode vs. Column Load materials spiked with DNA, HCP and ProA separately cm/hr Conductivity DNA ppb (1000) QSFF ms HCP ppm (1000) ProA ppm (8) 2.1 QSFF ms < Q-membrane Q-membrane Q-membrane ms 7 ms 7 ms <0.86 <0.88 < Zhou, J, Solano, F., Dermawan, S., Hong, T. and Tressel, T., Disposable Anionic Membrane, A Cleaning/Storage-Validation-Free Ultimate Solution to Mab Purification Challenge in Robust Scale Process Chromatography, IBC conference, /16/2007 page 5

6 Dynamic binding capacity for DNA compared to column Dynamic Binding Capacity for DNA: Q Membrane 2.1 ml Q-Sepharose column 10 ml Salmon sperm DNA at 50 μg/ml in sodium acetate buffer Binding capactiy was determinde at the 5% breakthrough point 3.3 g/l 0.12 g/l Rieble, S., Evaluation of a Disposable Q-Membrane Filter in the Purification of a Monoclonal Antibody, European Downstream Techn. Forum, Goettingen, May 9-10, /16/2007 page 6

7 Removal of DNA and HCP at low conductivity Introduction of charged membrane in flowthrough mode was: Beneficial for contaminant removal Successful to reduce process time Successful to minimise complexity Constraints Optimised process for DNA removal Non-binding condition Simple to insert into current process High yield in a short time Solution Results at production scale: Yield 99 ± 3 % 7/16/2007 page 7 DNA Host cell protein 5 ± 8 pg/mg 19x ± 8 <100 ppm AEX in flow-through mode (ph 7, no salt) Acidic condition ph 6.0 Between step 4 and 5 (high conductivity) Charged-membrane technology (Sartobind Q single use) Delvaille, D., Contaminant removal in purification process of recombinant proteins, Serono approach for products in early development, PDA meeting Paris, Dec 6-7, 2006

8 Removal of process impurities Galliher, P., Fowler, E., IBC s Biopharmaceutical Production Week, Paradise Point Resort San Diego, CA, November 12-15, 2001, Validation of Impurity Removal by the CAMPATH- 1H Biomanufacturing Process: Clearance of Sartobind Q reduction value log 10 Endotoxin decreased DNA 3 Host cell protein 1 7/16/2007 page 8

9 Clearance of endotoxin 5 log Clutterbuck, A, Avecia, BioPharm Int. 20 (5), 24 31, 2007 Endotoxins were removed from GST-protease from levels EU/mg to 0.13 EU/mg protein solved in TRIS / NaCl buffer ph 8 with Sartobind Q SingleSep 20 at cgmp runs Run 1 Run 2 Process volume (Start) 50 L 47 L Process volume (End) 60 L 59 L Process time ~10 20 min ~10 20 min Protein concentration (Bradford) 4.10 g/l 4.14 g/l Conductivity ms/cm ms/cm Pre use endotoxin level 26,900 EU/mg 10,100 EU/mg Post use endotoxin level 0.13 EU/mg 0.18 EU/mg Protein recovery over step 81.4 % 84.6 % LRV endotoxins 5 5 7/16/2007 page 9

10 Endotoxin, DNA, HCP removal Clearance of Sartobind Q reduction value log 10 Endotoxin >2.8 DNA > 2 Host cell protein 1.9 Zhang, R. et al, Abgenix Inc (now Amgen), Fremont CA, USA: Viral Clearance Feasability study with Sartobind Q Membrane Adsorber for Human Antibody Production, Poster, IBC BioProduction October 2004, Munich 7/16/2007 page 10

11 Contaminant removal protecting Protein A column 2 x SingleSep 20 inch in series Klaus Kaiser, Bayer Healthcare, Dechema Meeting, Osnabrück May 14-16, /16/2007 page 11

12 Impurity levels after 1 st column and g Mab product 1.5 % aggregate 180 g 1000 ppm CHOP 12 g 200 ppm DNA 2.4 g 30 ppm leached ProA 0.36 g Typical load value: > 2 kg protein per liter Sartobind Q Gerardo Zapata et al., Membrane Chromatography for Purification of Human Antibodies in Commercial Processes, IBC Antibody Meeting 2005

13 Polishing Step: hmab after capture Virus Size (nm) Enveloped LRV Run 1 LRV Run 2 MVM: Minute Virus of Mice No 6.77 ± ± 0.37 Reo-3: Reovirus Type III No 7.28 ± ± 0.29 MuLV: Murine Leukemia Virus Yes 5.57 ± ± 0.32 PRV: Pseudorabies virus Yes 5.67 ± ± 0.23 (log 10 ) removal for DNA : >2 log host cell proteins: 1.9 log endotoxin: >2.8 log Zhang R, Bouamama T, Tabur P, Zapata G, Gottschalk U, Mora J, Reif O. Viral Clearance Feasibility Study With Sartobind Q Membrane Adsorber for Human Antibody Purification. IBC s 3rd European Event BioProduction /16/2007 page 13

14 Polishing Step after Intermediate Purification Impurity Levels after 2nd column: << 100 ppm CHO protein << 20 ppm DNA Typical load value: 10.9 kg antibody/l membrane (180 times more than Q resin) Feng Li et al. J. BioProcessing, 09/10, 23-30, /16/2007 page 14

15 Virus removal study for MAb Virus Enveloped LRV Run 1 LRV Run 2 Virus recovery (%) MVM: Minute Virus of Mice no 6.03 ± ± Reo-3: Reovirus Type III no 7.00 ± ± MuLV*: Murine Leukemia Virus yes 5.35 ± ± 0.27 > 70 PRV: Pseudorabies virus yes 5.58 ± ± ph: 7.2 / conductivity: 4 ms/cm / flow rate: 450 cm/h / 1 % spike protein concentration: 4.3 g/l Mab load: 10.9 kg/l membrane (3 kg/m²) / *LRV = 5.59 at 600 cm/hr Zhou J and Tressel T: Basic Concepts in Q Membrane Chromatography for Large-Scale Antibody Production. Biotechnol Prog., 22 (2) , /16/2007 page 15

16 Virus removal Viruses Antibody A 2100 mg/ml Antibody B 300 mg/ml 1% v/v 5% v/v PPV: Porcine Parvo Virus >6.92 N/A MVM: Pseudorabies virus N/A 3.56 A-MuLV: Murine Leukemia Virus >5.75 >6.30 Membrane Chromatography meets and exceeds viral clearance results compared to Q resin chromatography Diehl, T., Application of Membrane Chromatography in the Purification of Human Monoclonal Antibodies, Downstream Technology Forum, Goettingen, May 9 th, /16/2007 page 16

17 Virus removal at high conductivity Farb, D., Platform for generic validation of virus removal steps, Downstream Techn.Forum, King of Prussia, PA, USA, Sept. 28 th, /16/2007 page 17

18 Bind and elute Purification of Viruses Oligonucleotides Proteins by metal chelate 7/16/2007 page 18

19 Purification of adenoviruses with AdenoPACK G. Lux, Progen, Downstream Techn. Forum, Göttingen, May /16/2007 page 19

20 Foot and Mouth disease Virus (23 nm) Comparison of binding capacity towards inactivated FMD virus in PEG concentrates of Sartobind MA75: S 75 and a conventional 1 ml IEX-chromatography column (S-Type, 90 μm beads) Both devices were treated identical (loaded amount, elution conditions etc.) The FMD virus recovery of Sartobind was 69.5% vs. 12.9% with the conventional column. Although the nominal binding capacity of the conventional column is more than 2x higher Oswald, T. Downstream Processing in Vaccine Manufacturing and Development. Downstream Technology Forum London Marriott 2 nd November /16/2007 page 20

21 Heparin Membrane: Purification of Adeno Associated Viruses Production in SF9 insect cells Cell lysate was centrifuged Dilution 1 : 10 with starting buffer Elution with 0.5 M NaCl Raw lysate Load Elution 1 Elution 2 Silver stain SDS gel analysis displays pure AAV virus particles VP 1 (85 kda) Courtesy: Dr. Jörg Rippmann, VP 2 (73 kda) VP 3 (65 kda) Dr. Sebastian Kreuz Boehringer Ingelheim Pharma GmbH & Co KG, Biberach Germany 7/16/2007 page 21

22 Purification of antisense oligonucleotides Purification of a single stranded phosphothionate of 20 nucleotides. 2 m² Q module (560 ml) is used for purification of crude oligonucleotide samples. Max. flow 6 l/min/bar. Deshmukh, R. R. et al. (ISIS Pharmaceuticals), Large Scale Purification of Antisense Oligonucleotides by high-performance membrane adsorber chromatography. Chromatography A, 890, , /16/2007 page 22

23 Lactoferrin / peptides from whey Breakthrough curve of lactoferrin Weiß et al., Institute for Technical Chemistry, Hanover, Isolation of Lactoferrin with Cation Exchange Membranes in Modern Whey Processing, Poster, /16/2007 page 23

24 Purification of proteins on a metal chelate membrane adsorber The metal chelate membrane: iminodiacetic acid charged with Cu 2+ ions used here are suitable for purification ob both intracellular proteins and of extracellular proteins in the culture supernatant. Isolation of Recombinant Proteins with Membrane Module, Karl Friehs, University Bielefeld, Application note, Sartorius Biotech GmbH 7/16/2007 page 24

25 Enzyme coupling restauration of wall paintings Covalently attached enzymes such as alcalase keep their activity when bound to epoxide activated membrane and can be used for the removal of casein: Andrea Schmid, Diploma thesis 2003/2004 The wall painting Christus and the apostles on western wall of the lower church (Unterkirche), Georg chapel (Georgenkapelle) of St. Peter and Paul in the city of Görlitz, Germany 7/16/2007 page 25

26 More applications on 7/16/2007 page 26