Intensification of Influenza Virus Purification from Clarified Harvest to Formulated Product in a Single Shift

Intensification of Influenza Virus Purification From Clarified Harvest to Formulated Product in a Single Shift M. Tajnik Sbaizero, M. Wolschek, M. Reiter, T. Muster, P. Gagnon, A. Štrancar nfluenza is a global respiratory disease with an estimated mortality of up to a half million I people per year (1). The majority of traditional influenza vaccines are still produced in eggs. Downstream processing typically consists of Vero cells infected with purified influenza A: Infectious virus particles were stained clarification by centrifugation, with rabbit antiinfluenza A NP-antibody and a goat antirabbit IgG antibody concentration by ultrafiltration, and conjugated with AlexaFluor 488 (green). Cell nuclei are stained blue with DAPI (4‘,6-diamidino-2-phenylindole). purification by ultracentrifugation (2). Recombinant vaccines are most often purified by chromatography. across the field of biopharmaceuticals. help companies focus resources on Chromatographic purification of One of those trends is process essential process functions. viruses already has achieved major intensification, referring to As with many viruses, purification of improvements in recovery and development of processes that influenza virus from clarified cell scalability (3), but it also is important harmonize integration of fewer and culture harvests often begins with because it enables virus purification more capable steps to achieve higher tangential-flow filtration (TFF), which to keep pace with important productivity and reproducibility as simultaneously achieves multiple tasks. regulatory and manufacturing trends well as reduce manufacturing costs. The first is concentration of the virus, Intensification takes many forms. which is retained, while contaminant Continuous processing represents one levels are reduced by passing through PRODUCT FOCUS: VACCINES aspect, and it already has been applied membrane pores. Both capabilities often PROCESS FOCUS: DOWNSTREAM successfully to anion-exchange are referred to as ultrafiltration (UF). PROCESSING purification of influenza A (4). An The third capability is buffer exchange arguably more important aspect by a process called diafiltration (DF). WHO SHOULD READ: PROCESS involves application of tools that The original fluid is replaced gradually DEVELOPMENT AND MANUFACTURING overcome limitations of traditional by buffer with a formulation suitable for options and thereby improve results an initial purification step. In this KEYWORDS: CHROMATOGRAPHY, and/or reduce the number of process discussion, reference to UF–DF is INFLUENZA, PROCESS INTENSIFICATION, steps. Application of single-use simplified to TFF. SINGLE-USE, TANGENTIAL-FLOW FILTRATION, processing materials also contributes to Despite the valuable role of TFF, VERO CELLS intensification. By suspending the its benefits come at a price. Shear LEVEL: INTERMEDIATE validation and manufacturing burdens stress produced during TFF has been of multiple use components, disposables documented to strip outer envelopes from lipid-enveloped viruses and to One key distinction of monolithic influenza B, both lacking TFF steps, fracture brittle capsids of non-lipid− chromatography media compared with and both using a single enveloped species (5, 6). Such liabilities columns packed with particles is that chromatography step with a cation- might be prevented by direct virus monoliths do not have void spaces. exchange monolith on a single-use capture on traditional porous particle Flow through the interconnected basis. The choice of process buffers columns, but this is impractical for channels is laminar, so eddy formation enables final formulation by simple two reasons. An obvious reason is the and shear stress are nil. Another key dilution of the product pool. DNA extended time that would be required distinction is that mass transport in digestion requires two hours. Capture, to load unconcentrated virus. The monoliths is exclusively convective, purification, and formulation are other is that chromatography columns rather than the situation in porous achieved within four hours. Host-cell packed with particles represent an particle columns where flow between DNA and host-cell protein (HCP) are additional source of shear stress. Flow particles is convective, but solutes reduced more than 99%, and final through the irregular spaces between move into and out of the pores virus recovery is 80%. particles causes formation of eddies, exclusively by diffusion. zones of recirculation. When bulk Diffusion is the reason columns MATERIALS AND METHODS flow through the interparticle space packed with particles are slow. Influenza virus was produced in Vero encounters eddy flow in the opposite Diffusion is even slower for large cells. Viruses were constructed as direction, it generates shear stress at products such as viruses because previously described (9) and propagated their interface (Figure 1). Shear stress diffusion constants become slower with in Nunc Cell Factory CF-10 systems in column void spaces is directly increasing particle size. By contrast, (Thermo Fisher Scientific) using proportional to flow rate (7). convective flow can be thought of as a serum-free medium Opti Pro SFM river. Objects flow at the same rate as (Invitrogen, Thermo Fisher Scientific) Figure 1: Shear stress in the void volume of the current no matter what size they supplemented with 4 mM GlutaMax I packed particle columns; solid areas represent are. The result is that binding (Invitrogen, Thermo Fisher Scientific). particle surfaces. Interparticle void space is indicated in white. Black arrows indicate flow, efficiency, dynamic binding capacity, Upon infection, cells were grown using and line thickness indicates flow velocity. and elution efficiency in monoliths are recombinant trypsin (Thermo Fisher Circular patterns indicate eddies. Areas marked all independent of product size and Scientific) and incubated at 5.0% CO2 in red highlight zones of countercurrent flow flow rate. Flow rates can be 20–50× and 95% relative humidity at 37 °C that generate shear stress. Axial shear stress at higher than in packed-particle columns (influenza A) or 33 °C (influenza B) particle surfaces is negligible with flow rate approaching zero there because of friction. without compromising performance (8). for three days. Harvests were clarified Altogether, the combination of by low-speed centrifugation. Clarified convective efficiency and laminar flow harvest was treated with 20 U/mL enables rapid virus concentration on Benzonase endonuclease (Merck, monoliths without the flow-rate Germany) in the presence of 2 mM restrictions of traditional columns and magnesium for two hours at room without the shear forces created by temperature. either TFF or particle-packed Virus purification was conducted columns. In short, they enable the with 1, 8, or 80 mL CIMmultus™ SO3 sequential functions provided by TFF cation-exchange monoliths. They were and traditional column capture to be sanitized in advance with 1 M NaOH, combined into a single faster process then washed with water and equilibrated step with less risk of damaging a to 50 mM HEPES, 200 mM sucrose, product. pH 7.0. Influenza A virus was diluted In this report we describe processes 2:1 (sample:buffer) and applied to the for purification of influenza A and monoliths without further preparation. Influenza B virus was diluted 1:1 with Figure 2: Elution of Influenza B from a 1-mL CIMmultus SO3 monolith — the inset represents a zoomed image of the elution step; blue = UV absorbance at 280 nm; red = UV absorbance at equilibration buffer, then loaded at the 260 nm; black = conductivity same flow rate. The monolith was washed with 50 mM HEPES, 200 mM ) 2.5 300 Conductivity (mS/cm) Inuenza B 2.5 50 Elution sucrose, pH 7.5 containing 100 mM claried harvest 2.0 2.0 40 250 NaCl (influenza A) or 50 mM NaCl CIMmultus SO3 1.5 30 200 280 nm 1.5 (influenza B) and eluted with a linear 1.0 20 150 gradient. The gradient endpoint buffer 1.0 0.5 10 100 in both cases was 50 mM HEPES, 200 0.0 0 260 nm, 0.5 Load 630 635 640 50 mM sucrose, 2.0 M NaCl, pH 7.5. CIP Specific gradient configurations for AU ( 0.0 0 0 100 200 300 400 500 600 700 influenza A and B are discussed in the Elution Volume (CV) next section. Columns were cleaned and Figure 3: Elution of Influenza A from a 1 mL CIMmultus SO3 monolith — right image represents a zoomed view of the elution step; blue = UV absorbance at 280 nm; red = UV absorbance at 260 nm; brief wash step with pH 7.5 buffer black = conductivity containing 50 mM NaCl, the virus was ) 400 300 Conductivity (mS/cm) eluted with a 50-CV linear gradient to 250 100% buffer B. The six-hour total 300 Load 100 chromatography time included 80 200 280 nm sanitization, equilibration, load, wash 200 60 150 Inuenza A 40 elution, and cleaning in place (CIP). 20 Elution CIP 100 100 claried harvest The infectious virus was recovered in a 260 nm, 0 50 CIMmultus SO3 525 535 545 555 4.1-mL fraction (indicated in the AU ( 0 0 colored background area) representing 0 100 400 500 600 Elution Volume (CV) an overall concentration factor of 73-fold from the undiluted cell culture. Virus concentration was 2.43 × 1010 Figure 4: Elution of Influenza B from an 80 mL CIMmultus SO3 monolith with 2 µm channels; FFU/ mL, and recovery was 74%. The blue = UV absorbance at 280 nm; red = UV absorbance at 260 nm; black = conductivity; virus fraction was formulated with CV = column volume about a fourfold dilution to lower buffer ) capacity, adjust salt concentration, and 2.5 Inuenza B 300 Conductivity (mS/cm) introduce a nonanimal−derived claried harvest 250 2.0 stabilizer before final sterile filtration. CIMmultus SO3 200 280 nm 1.5 Elution Figure 3 shows the elution profile of 150 influenza A from a 1-mL CIMmultus 1.0 2.5 h 100 SO3 monolith. Initial material was 260 nm, 0.5 Load CIP 50 diluted 1:2 (buffer:sample) with AU ( 0.0 0 equilibration buffer at pH 7.0. Similar to 0 20 40 60 340 360 380 400 420 the influenza B process, loading volume Elution Volume (CV) was >500 CV.

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