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Monolithic Columns for Purification and In-Process Control of Viruses and Virus-Like Particles Engineering Conferences International ECI Digital Archives Vaccine Technology IV Proceedings Spring 5-24-2012 Monolithic Columns for Purification and In- process Control of Viruses and Virus-like Particles Lidija Urbas BIA Separations Milos Barut BIA Separations Follow this and additional works at: http://dc.engconfintl.org/vaccine_iv Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Lidija Urbas and Milos Barut, "Monolithic Columns for Purification and In-process Control of Viruses and Virus-like Particles" in "Vaccine Technology IV", B. Buckland, University College London, UK; J. Aunins, Janis Biologics, LLC; P. Alves , ITQB/IBET; K. Jansen, Wyeth Vaccine Research Eds, ECI Symposium Series, (2013). http://dc.engconfintl.org/vaccine_iv/43 This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Vaccine Technology IV by an authorized administrator of ECI Digital Archives. For more information, please contact [email protected]. Monolithic Columns for Purification and In-process Control of Viruses and Virus-like Particles Lidija Urbas, Miloš Barut Faro, May 24th 2012 Outline • Conventional particle based chromatography • Monolith based chromatography • Downstream processing of Ad3 VLPs – Case study • Convective interaction media (CIM) monoliths for in-process control – CIMac monolithic analytical columns as a tool for HPLC based analytical methods BIA Separations • BIA Separations was founded in September 1998. • Headquartes in Austria, R&D and Production in Slovenia. • BIA Separations USA established in September 2007 - sales and tech support office. • BIA Separations China established in January 2011 - sales and tech support office. • Main focus: To develop and sell methacrylate monolithic columns & develop methods and processes for large biomolecules separation and purification . Moved to a new 4.200 m 2 facility in October 2011 Downstream Processing of Viruses and Virus-like Particles • Conventional • Filtration • Ultracentrifugation • UF/TFF • Alternative • Ion exchange chromatography A method of choice • Size exclusion chromatography for • (Pseudo)Affinity chromatography proteins. • Hydrophobic interaction chromatography What about for larger biomolecules? Chromatographic Separations Principle of chromatography: molecule from the mobile phase binds to the stationary phase. Mass transport: •Diffusion – Migration of the solutes from an area of high to an area of low concentration •Convection – Movement induced by external force Conventional Liquid Chromatography Media Mass transport in packed porous particle columns: convection + diffusion Interparticle void volume Intraparticle void volume (preferential flow path) (contains majority of binding sites: > 90 %) Conventional Liquid Chromatography Media Diffusion Limitations Binding capacity at high flow rate: f3 > f2 > f1 > f0 Binding capacity [mg/ml] [mg/ml] BindingBinding capacity capacity f1 f2 f3 Flow rate [ml/min] Porous Particles – Flow Dependent Dynamic Binding Capacity 1.0 Source 30 S 100 cm/h 200 cm/h IgG (c=0.1 mg/mL) 0.8 400 cm/h 600 cm/h C/C 0.6 0 0.4 0.2 0.0 20 40 60 80 100 120 140 elution volume [ml] Courtesy of Prof. A. Jungbauer, IAM, BOKU, Vienna, Austria Diffusion Limitations Resolution in linear gradient elution at high flow rate: f2 > f1 f1 f2 Detector response response Detector Detector Eluted volume [ml] Larger the molecule wider the peak – lower the resolution Difference Between Diffusive and Convective Mass Transport 2 molecule MW De (cm /s) small molecule 58 Da 1.4 x 10 -5 hemoglobin 64kDa 7 x 10 -7 BSA 66 kDa 6.1 x 10 -7 urease 482 kDa 3.5 x 10 -7 Tomatomosaicvirus (ToMV) 40 000kDa 5 x 10 -8 DNA 4.4 kbp 1.9 x 10 -8 DNA 3.7kbp 4,5x 10 -8 The bigger the molecule the lower the rate of diffusion Another Challenge – the Size of the Molecule of Interest Pores become too small for large solutes! (Binding mostly on the outer surface) results in Very low binding capacity for large solutes (behave like nonporous particles) Working with big molecules Molecule size: surface accessibility Molecule nm Proteins 1-5 IgM 25 Plasmids 150-250 Rotavirus 130 Poxvirus 200 x 500 T4 phage 220 x 85 Courtesy P. Gagnon www.validated.com What Are the Alternatives? • Membranes • Monoliths X X X Stacks of thin polymeric layers – supplied in single piece but in fact they are discontinuous unit . CIM Monoliths Monoliths are continuous stationary phases cast as a homogeneous column in a single piece.1 1 G. Iberer, R. Hahn, A. Jungbauer, LC-GC 17 (1999) 998. CIM Monoliths Made of highly cross-linked porous rigid monolithic: O O + O • poly(glycidyl methacrylate-co- O O O O GMA EDMA ethylene dimethacrylate) or Glycidyl methacrylate Ethylene glycol dimethacrylate • poly(styrene-divinylbenzene) O O O O polymers O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Poly(glycidyl methacrylate-co -ethylene dimethacrylate) What distinguishes monoliths from conventional supports? Structure of the monoliths • High porosity (over 60 %): • Flow-through channels (“pores”) having large diameter (1.5 µm), • Uniform channel connectivity in 3D (homogeneous structure ). CONSEQUENCE • Low pressure drop • High surface accessibility – no dead end pores • High dynamic binding capacities for large molecules • Mass transfer predominantly based on CONVECTION What distinguishes monoliths from conventional supports? Convective transport – flow independent properties 60 1 3 300 2 100 50 250 Flow rate 40 ml/min 80 35.9 ml/min 40 200 80 ml/min 76.2 ml/min 120 ml/min 152.7 ml/min 60 160 ml/min 224.6 ml/min 30 150 200 ml/min 681 ml/min 240 ml/min 40 BufferBuffer[%] [%] AA 20 100 Absorbance Absorbance Absorbance 280 280 at(mAU) at(mAU) nm nm 10 20 50 AbsorbanceAbsorbance[mAU] [mAU] atat280280 nm nm 0 0 0 0 50 100 150 200 250 300 0 100 200 300 400 500 600 700 800 900 1000 Volume [ml] Elution volume (ml) Podgornik et al., Anal. Chem. 72 (2000) 5693 Avaliable CIM Monolithic Columns 0.1 0.34 1 8 80 800800 8000 ml CIMmultus (multi-use disposable columns) CIM Monolith Column Capacity Flu vaccine 24.000 doses/run pDNA .48 g/run 240.000 doses/run 4.8 g/run 2,400.000 doses/run 48 g/run Main CIM Monoliths Applications Area Endotoxin DNA depletion Large proteins CIM® Monoliths for Production of Complex Biomolecules • Drug Master Files (DMF) for CIM ® DEAE, QA and SO3 columns in place, HIC in preparation. • First drug purified using CIM ® monoliths passed CPIII trial (pDNA for gene therapy). • More than 25 projects in CPI – CPIII trials (various Influenza, various Adenovirus, bacteriophages, various IgMs, Inter -alpha - inhibitors). • More than 200 projects in pre-clinical trials (Influenza A and B virus (eggs, Vero and MDCK cells), Rabies virus, Rotavirus, AAV, various Adenovirus subtypes, Hepatitis A, Mulv, MVM, Feline calicivirus, Japanese encephalitis, Crimean-Congo hemorrhagic fever, Hantaan virus, VLP (Hepatitis B, HPV, Influenza, Adenovirus), bacteriophages (Lambda, T4, VDX10, Pseudomonas phage), Tomato and Pepino Mosaic virus, pDNA, IgM, various proteins). Downstream Processing of Virus-like Particles (VLPs) Case study Purification of adenovirus type 3 dodecahedric VLPs for biomedical applications Work done with the Institute of Biochemistry and Biophysics, Polish Academy of Science (Ewa Szolajska and Jadwiga Chroboczek). Dodecahedric VLPs of Adenovirus Serotype 3 (Ad3 VLPs) Ad3 capsid Ad3 VLPs penton fiber hexon penton base MW (Ad3 VLPs): 3.6 MDa MW (base): 63 kDa Production of Ad3 VLPs Expression in baculovirus/insect cells system Cell lysis 5 days Ultracentrifugation in a sucrose density gradient Chromatography on an agarose based matrix Ad3 VLPs A delivery vector for bleomycin (BLM) Goal: • Evaluation of CIM monoliths – Chromatographic step • Direct purification of crude lysate using CIM monoliths Zochowska et al, Plos One, 2009 Method Development • HPLC system – detection at 280 nm • CIMac analytical column (5 x 5.2 mm ID, V = 0.1 mL) • Ad3 VLPs samples: – UC sample (sample that was prepurified with ultracentrifugation) – Cell lysate sample • Methods: – Ad3 VLPs content - SDS PAGE – Protein content - BCA, Bradford assay – DNA content - PicoGreen Analysis, agarose electrophoresis – Vector internalisation - Confocal Microscopy Screening of Stationary and Mobile Phases • Initial screenings performed using the UC (purified) sample, • Various cation- and anion-exchange monolithic columns, • Various buffering systems - composition and pH. Cation Exchangers 1200 100 1000 Ad3 VLPs 80 800 60 600 A [mAU] [mAU] AA 40 400 Gradient B [%] [%] B B Gradient Gradient 200 20 0 0 0 0,5 1 1,5 2 2,5 3 t [min] Conditions: buffer A: 20 mM Tris, pH 7.5 + 2 mM EDTA + 5% glycerol, buffer B: buffer A + 1 M NaCl, pH 7.5; V: 150 mL 2 ×diluted UC sample; detection at 280 nm. Anion Exchangers 200 100 180 Ad3 VLPs DNA 160 80 140 120 60 100 A[mAU] A[mAU] A[mAU] 80 40 GradientGradient B B [%] [%] 60 GradientGradientGradientGradient BBBB [%] [%] [%] [%] 40 20 20 0 0 0 2 4 6 8 t[min] DEAEDEAEDEAEQA QA EDA Conditions: buffer A: 20 mM Tris, pH 7.5 + 2 mM EDTA + 5% glycerol, buffer B: buffer A + 1 M NaCl, pH 7.5; V: 60 mL 2 ×diluted UC sample; detection at 280 nm. Agarose based matrix and CIMac QA 90 FT E1 100 80 70 80 Ad3 VLPs 60 DNA 60 50 E2 E4 40 E3 A[mAU] 40 30 Gradient B [%] Gradient 20 20 TEM analysis of E2. 10 0 0 0 5 10 15 20 25 SDS PAGE; M: protein t [min] marker, L: loading sample, FT-E: collected fractions.
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