Using Monolithic Chromatographic Columns to Improve the Efficiency and Robustness of Biopharmaceutical Manufacturing

Transcription

1 Using Monolithic Chromatographic Columns to Improve the Efficiency and Robustness of Biopharmaceutical Manufacturing IFPAC, Arlington, VA, January 25th 28th, 2015 Nika Lendero Krajnc, PhD;

2 Production of biopharmaceuticals

3 Biopharmaceuticals While traditional drugs are usualy small molecules, biopharmaceuticals are large complex bioparticles, with heterogeneus physicochemical properties, sensitive to the environmental conditions (ph, I) and shear forces. antibodies 20 Size (nm) Viruses DNA Adenovirus Retrovirus/Lentivirus Adeno- associated virus Pox/Vaccinia virus 200x450 Proteins Herpes virus Naked DNA Production must be fast and gentle. 100-over 1000

4 USP Production of biopharmaceuticals DSP Alive up-stream process, complex down-stream process. Multistep extensive production, demanding analytical methods. DSP comprises of several chromatographic steps.

5 Biochromatography Diffusion vs. convection

6 Chromatography of large biomolecules Principle of Chromatography: Equilibrium between the molecules in the mobile and stationary phase The movement of the solutes (proteins, DNA, virus particles) between the two phases and through the column - MASS TRANSFER MASS TRANSFER Diffusion: random thermal movement from an area of high concentration to an area of low concentration Convection: movement induced by an external force, such as the flow of buffer, induced by gravity or a pump.

7 Conventional packed media

8 Diffusion limitations: lower flow rates Large molecules have low diffusivity coefficients -they move slowly! Slow flow rates in order for the molecule to reach the binding site. Solute Size K diff (cm 2 /s) Delta BSA Sodium 53 Da 1.4 E-5 > 479x BSA 66 kda 6.7 E-7 = 1x IgG 150 kda 4.9 E-7 < 1.4x IgM 1 MDa 2.6 E-7 < 2.6x CMV 5 MDa 1.2 E-7 < 5.6x TMV 40 MDa 5.0 E-8 < 13.4x DNA 33 kbp 4.0 E-9 < 167x Diffusivities of some of the representative molecules (BSA normalized). BSA = Bovine Serum Albumin, CMV = Cucumber Mosaic Virus, TMV = Tobacco Mosaic Virus. Courtesy of: Pete Gagnon.

9 Diffusion limitations: compromised binding capacity Chromatographic material: Source 30 S Sample: IgG cm/h 200 cm/h 400 cm/h 600 cm/h 0.1 mg/ml 0 C/C elution volume [ml] Higher flow rates lower capacities! Courtesy of Prof. A. Jungbauer, IAM, University Vienna, Austria

10 Detector response Diffusion limitations: compromised resolution Speed limitation: lower resolution in linear gradient elution at higher flow rates f2 > f1 f 1 f 2 Eluted volume [ml] Higher flow rates - wider peaks lower the resolution

11 Limited surface accessibility for large molecules: lower dynamic binding capacity Molecule nm Proteins 1-3 IgM 25 Plasmids Rotavirus 130 Poxvirus 200 x 500 T4 220 x 85 Many plasmids and viruses are larger then pores, which dramatically reduces the binding capacity.

12 Chromatographic monoliths Channels are large (1-2 µm) - optimal for molecules like viruses, virus-like particles and DNA to flow through the channels and bind to the binding sites Binding sites are situated inside the channels no dead end pores no diffusion limitations same performance at lower and at higher flow rates Chromatographic monoliths - enhanced convection mass transport.

13 Flow independent mass transport within the chromatography column unaffected capacity Diffusion Convection Better reproducibility across process scales, as well as faster operation. Courtesy of: Pete Gagnon.

14 Flow independent mass transport within the chromatography column unaffected resolution Diffusion Convection High efficiency and flow-unaffected resolution.

15 Accessibility of the surface - influence on the binding capacity Solute Method Monolith Particle based BSA Ion exchange IgG Affinity IgG Ion exchange IgM Ion exchange DNA Ion exchange Flu virus Ion exchange x 1x Large bioparticles high capacities! Courtesy of: Pete Gagnon.

16 Basic properties of chromatographic monoliths based on the physical structure Low pressure drop Low share forces High surface accessibility High dynamic binding capacities for large molecules Convective transport Flow independent performance; Operating at high flow rates (short method time) Suitable for the separation and purification of large biomolecules; pdna, viruses, proteins.

17 Main Applications molecule type Large proteins Viruses & VLPs CIM Monoliths Plasmid DNA Endotoxins DNA depletion

18 Chromatography as a PAT tool for biopharmaceutical manufacturing processes Process Analytics Technologies

19 Characterization/analytical methods for viruses/vlps Viral potency/efficacy Identity Quantity Residuals (i.e., Triton and deoxiribonuclease) Aggregation Empty capsids Protein content Safety

20 Characterization/analytical methods for viruses/vlps Viral preparation can be charaterised using a variety of different methods based on: determining the viral genetic material (quantitative Polymerase Chain Reaction, qpcr), the ability of viruses to agglutinate red-blood cells (Haemagglutination Assay, HA) protein-antibody interactions (Enzyme-Linked ImmunoSorbent Assay, ELISA and Single Radial Immunodiffusion - SRID) counting viral particles (Transmission Electron Microscopy, TEM and Dynamic Light Scattering, DLS, new technologies such as ViroCyt and Nanosight device) measuring the optical density (OD) of pure virus preparations. Purity - Polyacrylamide Gel Electrophoresis (SDS-PAGE) Western blot - identification of specific viral proteins, DNA, protein specific tests - host cell residuals.

21 What do we gain by introducing an HPLC method? HPLC analytical assays: RAPID REPRODUCIBLE ACCURATE Excellent resolving power - separation of intact virus particles from other cellular contaminants or virus particle fragments. HPLC methods can therefore be applied for monitoring and quantitation in DSP as well as the USP. In process and final control

22 HPLC analytical assays Fingerprinting methods Quantification methods No info about the exact quantity of your molecule not really needed in many cases Information about the chromatographic profile of your sample (purity/impurity) Decision points/decision criteria: When to stop fermentation When to stop collection you chromatography sample/components cuts Am I doing a right type of gradient?

23 HPLC analytical assays Fingerprinting methods Quantification methods Purified virus preparations - standards with a know concentration: Linear range LOQ and LOD Repeatability, reproducibility

24 Case study: Adenovirus manufacturing processes Process Analytics Technologies

25 Purification scheme for AdV Lysis Nuclease treatment Clarification Ultrafiltration/Diafiltration AEX Chromatography Buffer Exchange End product characterization: Viral potency/efficacy Identity Quantity Residuals (i.e. Triton and deoxiribonuclease) Aggregation Empty capsids Protein content Analytical methods for in-process and final control of AdV: qpcr SDS-PAGE Western blot Bradford PicoGreen. Laborious, time consuming methods lack accuracy and precision!!

26 Fingerprinting method Process Analytics Technologies

27 Optimization of DSP steps: Online nuclease treatment monitoring Lysis Nuclease treatment

28 Optimization of DSP steps: AEX chromatography CIMmultus 1 ml columns QA Conditions: AdV Buffer A: 50 mm Hepes, 2 mm MgCl2, 0.1% Tween-80, ph 7.5 Buffer B: 50 mm Hepes, 2 mm MgCl2, 2M NaCl, 0.1% Tween- 80, ph 7.5 Linear gradients Step gradients

29 Optimization of DSP steps: Testing gradient Loading of 100 ml (approximate conc of AdV in load: /ml) in 5% buffer B, gradient: from 5-50% B in 100 cv, Buffer A: 50 mm Hepes, 2 mm MgCl2, 0.1% Tween-80, ph 7.5, Buffer B: 50 mm Hepes, 2 mm MgCl2, 2M NaCl, 0.1% Tween-80, ph 7.5.

30 Optimization of DSP steps: CIMac Adeno - fraction analysis from AEX run Flow rate: 1 ml/min, Buffer A: 50 mm Tris, ph 8.0, Buffer B: 50 mm Tris + 1M NaCl, ph 8.0, Method: gradient from 0 100% in 70 cvs, Vinj: 1 ml Main elution fraction contains mostly AdV.

31 Optimization of DSP steps: Optimization gradient 1 Loading of 100 ml (approximate conc of AdV: /ml) in 10% buffer B, step 15%, gradient from 15-30% B in 75 CV, step 30%, step 50%, step 100%; Buffer A: 50 mm Hepes, 2 mm MgCl2, 0.1% Tween-80, ph 7.5, Buffer B: 50 mm Hepes, 2 mm MgCl2, 2M NaCl, 0.1% Tween-80, ph 7.5.

32 Optimization of DSP steps: CIMac Adeno - fraction analysis from AEX run 2 lane sample description M Marker 1 22 Load 2 21 FT 3 23 A1-A A5-A A A A A12-B B3-B B B11 cl control lysate c control A5 dia

33 Optimization of DSP steps: Optimization gradient 2 production run Loading of 100 ml (approximate conc of AdV: /ml) in 10% buffer B, step 15%, step 30 %, step 50%, step 100%; Buffer A: 50 mm Hepes, 2 mm MgCl2, 0.1% Tween- 80, ph 7.5, Buffer B: 50 mm Hepes, 2 mm MgCl2, 2M NaCl, 0.1% Tween-80, ph 7.5.

34 Optimization of DSP steps: CIMac Adeno - fraction analysis from first production run Flow rate: 1 ml/min, Buffer A: 50 mm Tris, ph 8.0, Buffer B: 50 mm Tris + 1M NaCl, ph 8.0, Method: gradient from 0 100% in 70 cvs, Vinj: 1 ml

35 In-process control by fingerprinting method Lysis Nuclease treatment Clarification Ultrafiltration/Diafiltration AEX Chromatography Buffer Exchange Loop volume: 1 ml, injected 0.5 ml of 3 times diluted final AdV sample; flow rate: 1mL/min.

36 In-process control by fingerprinting method Lysis Nuclease treatment Clarification Ultrafiltration/Diafiltration AEX Chromatography Buffer Exchange Loop volume: 1 ml, injected 0.5 ml of 3 times diluted final AdV sample; flow rate: 1mL/min.

37 In-process control by fingerprinting method Lysis Nuclease treatment Clarification Ultrafiltration/Diafiltration AEX Chromatography Buffer Exchange Loop volume: 1 ml, injected 0.5 ml of 3 times diluted final AdV sample; flow rate: 1mL/min.

38 In-process control by fingerprinting method Lysis Nuclease treatment Clarification Ultrafiltration/Diafiltration AEX Chromatography Buffer Exchange Loop volume: 1 ml, injected 0.5 ml of 3 times diluted final AdV sample; flow rate: 1mL/min.

39 In-process control by fingerprinting method Lysis Nuclease treatment Clarification Ultrafiltration/Diafiltration AEX Chromatography Buffer Exchange Loop volume: 1 ml, injected 0.5 ml of 3 times diluted final AdV sample; flow rate: 1mL/min.

40 Quantification method Process Analytics Technologies

41 Determination of basic analytical parameters Linearity range: Samples injected in triplicates, Plot of avarage areas as a function of concentration, Linear range: from 4.8E+8 to at least 1.9E+10, LOD: 1.6E+8, LOQ: 4.8E+8.

42 Determination of basic analytical parameters Repetability: 6 consecutive injections of the same sample under the same conditions. VP loaded on the column Area Height [mau] t R [min] 6,82E ,57 6,82E ,56 6,82E ,55 6,82E ,57 6,82E ,59 6,82E ,56 average ,57 st dev ,01 CV [%] 1,4 2,0 0,31

43 Determination of basic analytical parameters using fluorescence detector LOD: 3.5E+07 LOQ: 7.0E+07 Linear range: from 7.00E+07 to at least 1.45E+10 VP 10x lower LOD and LOQ.

44 Quantification HPLC method for influenca viruses Based on native fluorescence: the measured fluorescence is proportional to the particle concentration. Suitable for the analysis of influenza strains B and A. LOD: from 2.07E E+09 Direct quantification of influenza virus particles from cell culture supernatants in-process analysis of influenza virus production in real time.

45 Up-stream process monitoring: Understanding the process Process Analytics Technologies

46 Upstream Processing Downstream Processing

47 Phage titer determination plaque assay Plaque assay Labour very intensive Results after a day or more RSD: 15-30% (at least) Plaque

48 LC/HPLC method for phage determination Phage position Chromatographic analysis of purified phage T4: Conditions: Stationary phase: 0.34mL CIM DEAE disk monolithic column; Mobile phase: Buffer A: 20mM Tris, ph 7.5. Buffer B: 20mM Tris, 1.0M NaCl, ph 7.5; Gradient: linear gradient from 0 to 100% buffer B in 1.5 min; Flow rate: 4 ml/min; Injection volume: 1 ml; Detection: UV at 280 nm. Smrekar et al. J. Sep. Sci., 34 (2011)

49 LC/HPLC method for phage determination Phage position Chromatograms of fermentation broth collected during fermentation: Conditions: Stationary phase: 0.34 ml CIM DEAE disk monolithic column; Mobile phase: Buffer A: 20mM Tris, ph 7.5. Buffer B: 20mM Tris, 1.0M NaCl, ph 7.5; Gradient: linear gradient from 0 to 100% buffer B in 1.5min; Flow rate: 4 ml/min; Injection volume: 1 ml; Detection: UV at 280 nm. Smrekar et al. J. Sep. Sci., 34 (2011)

50 Monitoring of changes in chromatographic profile during fermentation impurities phages DNA Chromatograms of fermentation broth collected during fermentation: Conditions: Stationary phase: 0.34 ml CIM DEAE disk monolithic column; Mobile phase: Buffer A: 20mM Tris, ph 7.5. Buffer B: 20mM Tris, 1.0M NaCl, ph 7.5; Gradient: linear gradient from 0 to 100% buffer B in 1.5min; Flow rate: 4 ml/min; Injection volume: 1 ml; Detection: UV at 280 nm. Smrekar et al. J. Sep. Sci., 34 (2011)

51 Bacteriophage replication lytic bacteriophages From: microbes/viruses-help.php

52 Indirect chromatographic method for phage determination gdna Indirect chromatographic method for phage determination in the fermentation broth. Smrekar et al. J. Sep. Sci., 34 (2011)

53 USP monitoring Conclusions Huge time savings, more samples can be analysed in the same time compared to Plaque assay or TCID50. Very sensitive method. Analysis parallel to the process. Flexible method, can be adjusted to your needs. Fast method enables to take immediate action. Enables quantitative and qualitative analysis of the samples (supported by mathematical data processing).

54 Take-home message CIMac monolithic columns enable: Real time monitoring of USP (5 min analysis). In-process control of DSP steps. Easy optimization of USP and DSP conditions. When validated, a final control method. Improved efficiency and robustness of biopharmaceutical manufacturing.

55 Thank you for your attention!