STRATEGIES FOR NANOFILTRATION OF THERAPEUTIC PLASMA PROTEINS

Transcription

1 STRATEGIES FOR NANOFILTRATION OF THERAPEUTIC PLASMA PROTEINS Oral Presentation: Pere Ristol Faro M, Canal JM, Marzo N, Caballero S, Belda F, Gajardo R, Grancha S, Ristol P R&D Area, Instituto Grifols S.A. Barcelona, Spain 1

2 Contents: Historical Summary of the Protein Nanofiltration Rationality for Nanofiltration Grifols Nanofiltration Background Plasma Protein Nanofiltration Development Strategies Case-studies: Fibrinogen: A High Molecular Weight Protein IVIG: A Cost - effectiveness Case Conclusion 2

3 Historical Summary of the Protein Nanofiltration Facts - Transmission of HIV during the 1980s. - Transmission of HCV and other enveloped and non-enveloped viruses during the After the tragedy of HIV transmission by plasma derivates and other viruses (in special HCV), as well as the concerns about emerging & new-born viruses, manufacturers of filters and fractionation industry began to work together for developing a new strategy to reduce the presence of viruses by means of size exclusion. This barrier technology was later called: NANOFILTRATION. The effective nanofiltration was introduced into plasma industry at the end of the 80s and it was growing in the 90s for processing the relative small proteins & enzymes (typically with molecular weight less than ~ 150 kda). Since the end of the 90s new membrane developments made it possible to increase the application of effective nanofiltration to processes up to medium-large proteins (i.e. molecular weight e 150 kda). 3

4 Historical Summary of the Protein Nanofiltration THE MAIN CONSTRAINTS TO CARRY OUT THE NANOFILTRATION ARE: TIME-CONSUMING PROCESS PROTEIN LOSS MEMBRANE AREA NEEDED (= COST) 4

5 First approaches used (in the past & currently) to avoid clogging of the nanometric membrane and to maintain acceptable flow-through Choosing the membrane composition: For minimum protein adsorption (limited fouling) - Regenerated cellulose - Polyvinylidene difluoride (PVDF) Selection of appropriate pore size of membrane according to the protein size by: - Metric dimension of pores (15 nm, 35 nm, 75 nm), or - Molecular weight of proteins (70 kda, 180 kda) Configurations: Historical Summary of the Protein Nanofiltration - Multi-layer membrane (hollow fiber), dead-end filtration: Running by entrapment mecanism - Tangential flow filtration: Minimum and constant protein layer thickness on the membrane during nanofiltration 5

6 Historical Summary of the Protein Nanofiltration The current situation (from the end of the 1990s up today): New developments in membrane composition, more pore sizes and configuration (folder) have improved nanofiltration for general application in almost all plasma proteins. - New Polyethersulphone & new PVDF membranes (+ regenerated cellulose) are available. - More membrane pore sizes: 20 nm and 50 nm (+ 15 nm, 35 nm, 75 nm & 180 kda) can be selected. - Dead-end, single, double or triple layer as well as multi-layer membranes, in cartridge folder or hollow-fiber, are available. 6

7 Historical Summary of the Protein Nanofiltration: Nowadays Application EFFECTIVE NANOFILTRATION OF PLASMA PROTEINS (BY PORE SIZE 20 nm) MARKETED PROTEINS: (11) IN DEVELOPMENT (*): IMMUNOGLOBULINS (IVIG, IMIG) ALBUMIN (JP company) FVIII (pd) (JP & FR company) ANTITHROMBIN-III A1-ANTITRYPSIN FIX PCC C1-ESTERASE INHIBITOR FXI (FR company) THROMBIN PROTEIN C a (JP company) Nowadays application VON WILLEBRAND FACTOR / FVIII FIBRINOGEN IV / FIBRIN-SEALANT PLASMIN IgM / IgA Note (*): Feasible according to published data, and not marketed yet NANOFILTRATION APPLICATION (BY ANY TYPE OF PORE SIZE): ALMOST ALL PLASMA PROTEINS 7

8 Among the Safety Technologies, Nanofiltration is unique: Rationality for Nanofiltration It is a very robust, feasible and flexible clearance technology. It permits efficient removal of enveloped & non-enveloped model viruses, as well as other pathogens. Nanofiltration enables high protein recovery with no undesirable effects on the product. Grifols included this nanotechnology more than 15 years ago. 8

9 Grifols Nanofiltration Background Grifols experience in plasma proteins nanofiltration 9 Plasma Protein Molecular Weight (kda) Nanofilter Pore size (nm) Thrombin ± 2 α1 Proteinase Inhibitor ± 2 Factor IX ± 2 Antithrombin ± 2 Prothrombin Complex (*) ± 2 Immunoglobulin G ± 2 Fibrinogen ± 2 A width range of molecular weight Plasma Proteins can be nanofiltered through small-virus Retentive Filters membranes ( 20 nm). Note (*): Under development

10 Nanofiltration Development Strategies Methodology Critical parameters that allow optimal nanofiltration performance: - Protein Features - Manufacturing Process - Nanofiltration Performance 10

11 Nanofiltration Development Strategies Protein Features & Manufacturing Process: Selecting the best stage Protein Size: Intrinsic limitation. Isoelectric Point: Effects on protein solubility and aggregation level consider ph & Ω. Impurities and contaminants: Presence of HMW proteins and some reagents (i.e. PEG). Protein Content: Protein concentration Viscosity & protein interaction Amount of protein to be nanofiltered (kg protein/industrial batch) Expected Loading Capacity (L/m 2 ). Stability: Labile biological activity proteins Process time becomes a limiting factor. 11

12 Nanofiltration Development Strategies Nanofiltration Performance itself Nanofilter pore size Foreseen Viral Retention Capacity Membrane composition: protein compatibility, adsorption or fouling Nanofilter structure (asymmetric, multi-layer, ): Impact on membrane Loading Capacity Operative filtration conditions: Tangential flow, dead-end, constant pressure, constant flux, temperature, rinse in pre & post-wash, Cost of goods Impact of Protein Recovery on total Yield Profit product margin 12

13 Nanofiltration Development Strategies Plasma Protein Nanofiltration Development Platform Protein Features Facility fit Recovery & Cost Scalability & Robustness Retention Capacity (LRV) Filterability Performance (Flux, t, Vmax) Manufacturing Process Nanofilter characteristics 13

14 NANOFILTRATION DEVELOPMENT CASE STUDY FIBRINOGEN: A High Molecular Weight Plasma Protein 14

15 Fibrinogen: A High Molecular Weight Case-Study Fibrinogen is a glycoprotein of 340 KDa. Raw material comes from the beginning of plasma fractionation (Cohn s Fraction I) Potential risk of viruses presence. Structurally complex protein, hexamer containing two sets of three different chains (α, β, and γ), linked to each other by disulfide bonds and with tendency to form aggregates. ~ 6.5nm ~ 45nm 15

16 Fibrinogen: A High Molecular Weight Case-Study Grifols aimed to develop a high purity & high safety Fibrinogen incorporating an effective 20 nm nanofiltration technology. Due to Fibrinogen size ( nm), structural complexity and adhesiveness, gentle nanofiltration conditions were tested: Low protein concentration minimizing protein interactions. Addition of stabilizers enhancing monomer content & filamentous tridimensional structure of the protein. High Nanofiltration Temperature (> RT) less viscosity. Location at terminal and higher purity process stages. 16

17 Fibrinogen: A High Molecular Weight Case-Study 1.- Determining maximal Fibrinogen concentration for NF performance: [Fibrinogen] (mg/ml) Filtered protein (at V max ) (g protein/m 2 ) NF Step Recovery (%) /50 61/ Only if Fibrinogen was very diluted, at 1 mg/ml, consistent nanofiltration capacity values (g/m 2 ) were obtained. However, nanofiltration recovery was not acceptable and additional purification was explored. 17

18 Fibrinogen: A High Molecular Weight Case-Study 2.- Effect of an additional intermediate Freezing /Thawing Step before 20 nm nanofiltration: Fibrinogen concent. Volumen filtered Nanofiltration Productivity Filtration Time NF Step Recovery (mg/ml) (g) (g/m 2 /h) (h) (%) Fresh material Filter blocked Not applicable Frozen / thawed material 0.74 >> No flow decay at this point ~90% The intermediate Freezing/Thawing step at defined conditions (thawing temperature, addition of stabilizers ), allowed aggregates and aggregable material to be removed. This additional polishing step enables the Fibrinogen nanofiltration through 20 nm. 18

19 Fibrinogen: A High Molecular Weight Case-Study A deep knowledge of the protein features and manufacturing process allowed the development of a 20 nm nanofiltration step for a HMW protein. Amount Protein Filtered (g/m 2 ) Productivity Consistency (n=5) Time (min) Flow decay (%) Flow decay Consistency (n=5) V Amount Filtered Solution (L/m 2 ) Due to the Flux decay profile reported Nanofilter Capacity becomes a limiting factor and Gradual Pore-Plugging Model could be applicable to predict the maximal & optimal throughput of the nanofilter: Linear equation: 1/Q = A x t + B ; A (slope) = 1 / Vmax Voptimum = 0.5 x V max = V Q75 = V (Throughput at 75% of Flux decay) 56 L/m 2 19

20 Fibrinogen: A High Molecular Weight Case-Study Conclusion The optimized process developed was very consistent at manufacturing scale, resulting in a less than 6 hours process time, high capacity (>56 L/m 2 ) and therefore costeffective nanofilter surface area. The designed NF process allows obtaining a highly safe Fibrinogen product: Reduction factors (log10/ml) 4 for smaller non-enveloped viruses (PPV, B19 Virus model, nm). 20

21 NANOFILTRATION DEVELOPMENT CASE STUDY IVIG: Increasing biological safety while maintaining a high product yield 21

22 Nanofiltration Development Strategies IVIG: A cost-effective Case-Study Grifols aimed to incorporate a 20nm filtration step in the most costeffective way: Main NF Targets: Maximal protein recovery & Lowest cost of goods Effective retention of smallest viruses ( 4 LVR) Secondary NF Requirements: Process time As terminal location as possible (facility fit). 22

23 IVIG: A cost-effective Case-Study Determining optimal IVIG concentration on NF by different nanofilter pore-sizes: 8 1,8 Capacity (kg protein/m2) nm (P35N) 20 nm (PVDF) 15 nm (P15N) Protein Concentration (%) Capacity (kg protein/m2) 1,6 1,4 1,2 1 0,8 0,6 0,4 0, (20 nm (Last Previous generation PVDF) Protein Concentration (%) Optimal [IVIG] conc. depends on filter pore-size. P35N shows a capacity increase vs. [protein] Low protein recovery and low capacity make the filtration of IVIG through 15 nm unfeasible. Optimal [IVIG] for nanofiltration by 20nm at ~20 h process time is ~3%, but capacity was not enough 23

24 IVIG: A Cost-effective Case-Study Once the 20-nm hollow-fiber filter was available a better performance was observed, display a Stable Flux Profile leading to high NF Capacity ( V max ~800 L/m 2 ; V 75 = 400 L/m 2 ; then, Process time = 45 h & total Area = 4 m 2 /batch). In this case, factors other than Capacity become limiting a compromise between Process Time and Surface Area was needed: How to determine it? OPTIMIZING NANOFILTER SURFACE AREA vs PROCESS TIME: Area x Industrial batch (m 2 ) Nanofilter Sizing (m 2 ) vs NF Time ~15 h Time (hours) 20 nm Multi-layer Hollow Fiber Biphasic kinetics: 1 st Phase: Drastic Reduction of nanofilter area with time. 2 nd Phase: The increase in process time impacts on a minimal reduction area. TOTAL AREA NEEDED = 8 m2 / BATCH PROCESS TIME = 15 h 24

25 IVIG: A Cost-effective Case-Study Kinetic profile of new generation 20 nm filters of lower cost are promoting new evaluations on licensed nanofiltered products: 200 Loading Ratio (L/m 2 ) Loading Ratio (L/m2) Time (h) Pre-established IVIG Capacity Target 20 nm NANO A (Reg.cellulose) 20 nm NANO B (PVDF) 20 nm NANO C (PES) 20 nm NANO D (PES) Very different performance is observed with different nanofilters under identical process & protein conditions. The operative optimal area is adjusted to Plugging Model or Area-Time graphic according to kinetics. 25

26 Nanofiltration: Future TREND OF NANOFILTRATION IN THE PLASMA PROTEINS INDUSTRY To explore the application of effective nanofiltration for all proteins as much as possible (preferentially to 20 nm size). To search better cost-effective nanofilters. Interchangeability in the use of commercial nanofilters with equivalent performance for licensed proteins. Simplicity for handling and assembling in process as well as in testing the post-use integrity (automatic control). 26

27 Nanofiltration Development Strategies Conclusion Grifols experience shows that dedicated work is needed to fit a nanofiltration step in every plasma protein manufacturing process, taking into account: - Critical aspects that allow an optimal NF performance are specific of every protein & every manufacturing process & every nanofilter However, once successfully achieved, robust and consistent virus retention capacity has been found in Grifols validation studies, under a wide range of process conditions. 27

28 28 Thanks for your attention!