Adenovirus purification method

Abstract

The invention relates to an adenovirus purification method. The invention relates to a virus purification method. The present invention provides downstream methods for purifying an adenovirus from a cell culture harvest. More specifically, it relates to an adenovirus purification method using the steps of virus capture and virus refinement.

Description

This application is a divisional application of the invention patent application with application date of April 24, 2019, application number 201980028038.0, and invention name “Adenovirus purification method”. Field of the Invention

[0001] The present invention relates to a virus purification method. The present invention provides a downstream method for purifying adenovirus from cell culture harvest. More specifically, it relates to an adenovirus purification method using virus capture and virus polishing steps. Background of the Invention Human adenoviruses have been classified into six species (A to F) with 55 known serotypes, causing illnesses ranging from mild respiratory infections in young children (known as the common cold) to life-threatening multi-organ disease in people with weakened immune systems. Adenoviruses are common pathogens that are widely used for gene delivery in oncology, cardiovascular and regenerative medicine, and as vaccine vectors, both experimentally and in completed and ongoing clinical trials.

[0002] Adenoviruses are being evaluated as vaccine delivery systems in many preclinical and clinical studies for various infectious diseases (Kallel, H. and Kamen, AAL. Large-scale adenovirus and poxvirus-vectored vaccine manufacturing to enable clinical trials. Biotechnol J 10, 741–747 (2015). Adenoviruses are also being explored as potential viral vectors for gene therapy and as oncolytic viruses. To enhance productivity and produce more effective and safer vaccines, several generations of recombinant adenovirus vectors have been developed (Appaiahgari, MB and Vrati, S. Adenoviruses as gene / vaccine delivery vectors: promises and pitfalls. Expert Opin Biol Ther 15, 337-351 (2015). The most studied adenovirus vector is the first-generation recombinant adenovirus serotype 5 (AdV5), making it a suitable system for developing adenovirus production methods.

[0003] Chromatographic techniques are used for adenovirus purification, but the results are often inadequate in terms of purity, yield, and capacity.

[0004] Thus, there remains a need to find better and more efficient methods for adenovirus purification, especially in those cases where the virus is to be used as an adenoviral gene delivery vector. SUMMARY OF THE INVENTION The present invention provides an efficient method for adenovirus purification with high capacity and high yield.

[0005] The present invention relates to a method for purifying adenoviruses, comprising the steps of: a) capturing adenoviruses from a cell culture harvest containing adenoviruses on an anion exchange resin; b) eluting the adenoviruses with a shallow conductivity gradient having an increasing salt concentration of 15-25%, preferably 18-20%; c) adding the eluted adenoviruses to a shell bead resin comprising a porous shell and a porous core, wherein the core is provided with a hydrophobic interaction ligand and the shell is not provided with any ligand; and d) eluting the adenoviruses from the shell bead resin into a flow-through, wherein the adenoviruses eluted in step d) contain less than 1 ng / ml of host cell proteins (HCP).

[0006] Preferably, the salt is NaCl and the gradient is an increase of 18-20% and the salt concentration is 0-700 mM, such as 480-570 mM NaCl as in the examples below, or any interval between 0-700 mM corresponding to a shallow gradient of 18-20% increase from the starting salt concentration.

[0007] Preferably, the anion exchange resin is multicomponent Capto Q ImpRes anion exchange resin and the shell bead resin is Capto Core 700 resin.

[0008] The anion exchange resin is packed in a column and the shell bead resin is packed in another column, and the eluted adenovirus is added (loaded) to the shell bead resin in a volume corresponding to 15-30 column volumes (CV), preferably 25-30 CV, of the column containing the shell bead resin. The porosity of the core and shell can be the same or different.

[0009] The adenovirus eluted in step d) is very pure and contains no detectable impurities. The level of host cell proteins (HCP) is undetectable or less than 1 ng / ml.

[0010] As shown in the experimental section, the recovery of adenovirus eluted in step d) was 80-100%.

[0011] A preferred use of adenovirus is as an adenoviral vector for cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 An overview of two two-step chromatographic methods (the method of the invention and the reference method) for adenovirus purification is shown.

[0012] Figure 2Shown are chromatograms from a scaled-up method of the invention using (A) Capto Q ImpRes for the capture step and (B) Capto Core 700 for the polishing step.

[0013] Figure 3 Shown are chromatograms for a reference method using A) Sepharose QXL for the capture step and (B) Sepharose 4FastFlow for the polishing step. DETAILED DESCRIPTION OF THE INVENTION The present invention describes the development of a downstream chromatography step for purifying adenovirus from HEK293 cell culture harvest. After screening 10 different anion exchange or multi-resins and 1 anion exchange membrane on a small scale, two resins were selected based on the highest dynamic binding capacity. Process conditions were optimized and a polishing step was included to further reduce impurities to meet regulatory requirements. For the first anion exchange capture step, followed by polishing by size exclusion or using Captocore multi-resin, performance was compared on a small scale. Using 3L cell culture harvest, the reference method and the downstream method developed by the present invention were compared on a large scale. Although the overall method results showed comparable performance between the developed two-step chromatographic methods, the loading capacity was approximately 150 times that for the polishing step of the method of the present invention by using Capto core 700 resin instead of traditional size exclusion.

[0014] Materials and methods Virus propagation and sample preparation HEK293.2sus cells (ATCC) were infected with AdV5 encoding green fluorescent protein (GFP) and the cells were cultured in Xcellerex TM XDR-10 or ReadyToProcess WAVE TM 25 HyClone in a bioreactor system TM CDM4HEK medium. During the 4-hour mixing period, cells were irrigated with 0.5% Tween TM 20 was lysed in the bioreactor. At the same time, 20 U / mL Benzonase TM To digest host cell DNA (hcDNA). Clarification of the harvested material was performed by forward flow filtration (NFF) using ULTA TM A combination of 2μm and 0.6μm GF filters for removal of cell debris and initial impurity reduction. Flux 6 filtration system, using M rHollow fiber filters with a nominal molecular weight cutoff of 300 000 were used for concentration and buffer exchange to 20 mM Tris, pH 8.0. Sodium chloride was used for sample conditioning prior to the capture step.

[0015] Development of the Adenovirus Capture Protocol use pure 25 chromatography system, prepacked HiTrap for 1mL resin TM Column (for SOURCE Q15 TM The dynamic binding capacity (DBC) screening was performed in 1 mL ReadyToProcess Adsorber Q columns and in 1 mL ReadyToProcess Adsorber Q capsules for membranes. DBC was determined at different NaCl concentrations in 20 mM Tris, pH 8.0 (Table 1). Loading of samples subjected to TFF was performed with a residence time of 10 minutes for the column and 0.2 minutes for the capsule until viral penetration.

[0016] Optimization of elution conditions Based on the results from the DBC screening, Capto Q, Capto Q ImpRes, Q Sepharose XL, SOURCE 15Q and Capto adhere ImpRes resins, and ReadyToProcess Adsorber Q membranes were selected for further evaluation. The linear elution gradient was evaluated in a 1 mL pre-packed column for the selected resin, and for the membrane, a 1 mL membrane capsule was used. The column and capsule were balanced with 20 mM Tris, pH 8.0 + 300 mM NaCl (0 mM NaCl for Capto adhere ImpRes). Sample loading was performed at 80% of DBC. Fractions were collected throughout the elution gradient for analysis until 700 mM NaCl.

[0017] Capto Q, Capto Q ImpRes and ReadyToProcess Adsorber Q were selected for further optimization and step elution conditions were evaluated for these adsorbents. After the loading step, a wash step with as high NaCl as possible without virus breakthrough was required, followed by a complete virus elution step with as low conductivity increase as possible to avoid co-elution of DNA with virus. The performance of ReadyToProcess AdsorberQ was evaluated with samples subjected to NFF only or NFF followed by TFF.

[0018] Evaluation of Resins for Adenovirus Polishing Steps For the polishing step, Capto Core 700 (prepacked in 1 mL HiTrap columns) and Sepharose 4 Fast Flow (packed in HiScale TM 16 / 40, 71 mL column volume [CV]) resin. Using Capto Q, Capto Q ImpRes and ReadyToProcess Adsorber Q, the purified material in the capture step evaluated was used as sample. 10% of the CV was loaded on Sepharose Fast Flow, and up to 30 CV was loaded on Capto Core 700. In order to determine the maximum possible sample loading volume for Capto Core 700 at an acceptable virus purity, fractions were collected throughout the sample loading stage and analyzed for impurity content.

[0019] All resins were from GE Healthcare Bio-Sciences AB.

[0020] Scale-up of adenovirus capture and polishing steps Two methods - method 1 (= method of the invention) using samples subjected to TFF and a combination of Capto Q ImpRes anion exchange resin and Capto Core 700 resin; and a reference method using samples subjected to TFF and a combination of Sepharose QXL anion exchange and Sepharose 4 Fast Flow size exclusion chromatography (SEC) resins - were scaled up for processing 3 L of cell culture harvest. For capture, Capto Q ImpRes was packed in a HiScale 26 column (88 mL), while Sepharose QXL was packed in a HiScale 50 (249 mL). For polishing, Capto Core 700 was packed in a HiScale 16 column (10 mL), and Sepharose 4 Fast Flow was packed in a HiScale 50 column (382 mL). The columns were The method was run on a pure 150 system. After the polishing step, a second TFF step was included for concentration, formulation and final sterile filtration. The method was performed in duplicate.

[0021] Analytical methods In StepOnePlus TM Real-Time PCR System, using PureLink TM Viral RNA / DNA MiniKit, TaqMan TMThe total viral titer in triplicate samples was analyzed by qPCR using Universal PCR Master Mix, forward and reverse primers for hexon DNA, and TaqMan MGB 6-FAM probe (all Applied Biosystems). Human AdV5 DNA (3.1 × 10 7 copies / mL) (ViraPur) was used as a standard.

[0022] Infectious virus titers were analyzed in triplicate samples by automated fluorescence microscopy of live cells using the IN Cell Analyzer and expressed as TCID 50 Methodology, analyzing the GFP signal (encoded by the virus) in the images.

[0023] Use 1 mL Tricorn with Q Sepharose XL TM 5 / 50 column, HPLC method was used for analysis of intact virus particles. Elution was performed with a NaCl gradient in 20 mM Tris, pH 7.5 at a flow rate of 1.5 mL / min.

[0024] The BCA assay kit (Thermo Scientific) with albumin standard was used to analyze total protein concentration and the Quant-iT TM PicoGreen TM Total DNA was determined using dsDNA Reagent (Invitrogen). The analysis was performed in duplicate.

[0025] The concentration of host cell protein (HCP) was determined using HEK293 HCP ELISA kit (Cygnus).

[0026] The hcDNA concentration in triplicate samples was determined by qPCR (reagents from Applied Biosystems) using primers for GAPDH (Invitrogen) and purified HEK293 DNA as a standard. TM Residual DNA Sample Preparation Kit, and MagMax TM Express 96 purification instrument (Life Technologies) was used to prepare samples.

[0027] Experimental Section Experiment 1: Adenovirus capture step A comprehensive screening of anion exchange and multivariate chromatographic adsorbents was performed. The screening was performed in 1 mL HiTrap columns, which provided sufficient sample volume for virus detection with available methods. As shown below, membranes and resins based on smaller bead sizes and thus larger virus binding surface areas provided the highest capacity (Table 1).

[0028] Table 1. DBC indicated as viral particles (vp) / mL resin *DBC was determined using samples from different formulations.

[0029] In linear elution gradient experiments, Capto Q ImpRes and Capto Q provided good separation of adenovirus from impurities and were therefore selected for further evaluation (not shown). Although ReadyToProcess Adsorber Q provided insufficient virus purity using a linear elution gradient (not shown), this membrane was also selected for further evaluation due to its high binding capacity.

[0030] Because it was found during process optimization that the conductivity required for complete elution of the virus was significantly higher in the step elution gradient compared to the linear elution gradient for both Capto Q and Capto Q ImpRes (not shown), a linear elution gradient was selected for the resin because the higher the elution conductivity for the virus, the higher the risk of co-elution of the virus and DNA. DNA is negatively charged and strongly binds to anion exchangers, and longer DNA sequences (e.g., DNA that is not completely fragmented after Benzonase treatment) bind more strongly than shorter DNA fragments. Lower elution conductivity results in reduced hcDNA in the virus elution pool.

[0031] Experiment 2: Adenovirus fine processing steps An adenovirus polishing step was included in the purification process to further reduce impurities. For this step, both Capto Core 700 polyplex and Sepharose 4 Fast Flow SEC resins were evaluated. Capto Core 700 consists of an inert shell and a ligand-containing core, providing dual functionality to the resin. The pores in the shell allow small proteins and impurities to enter and be captured in the core, while viral particles pass through in the flow-through.

[0032] Adenovirus recovery and purity were analyzed throughout the process steps. Sepharose 4Fast Flow and CaptoCore 700 resins provided similar performance in terms of adenovirus purity, however, CaptoCore 700 showed a clear advantage over Sepharose 4Fast Flow in terms of sample loading volume. Sample loading for Capto Core 700 reached 30CV, while only 0.1CV was loaded onto the Sepharose 4Fast Flow column. Despite the high variability of the qPCR assay, virus recovery was also determined to be higher for Capto Core 700. The overall downstream process results for a smaller scale are summarized in Table 3.

[0033] Table 3. Results from the adenovirus fine processing step *Total viral titer monitored by qPCR.

[0034] Two numbers indicate that the same sample was analyzed twice.

[0035] LOD = 1 ng / ml.

[0036] Although Capto Q provided higher impurity reduction than Capto Q ImpRes, results from the polishing resin evaluation showed that Capto Core 700 reduced residual impurities in the eluate from both resins to below the limit of detection (LOD). Capto Q ImpRes was selected over Capto Q due to the higher loading capacity it provided, supporting improved process economics.

[0037] Based on these results, two parallel methods were compared: method 1 combining Capto Q ImpRes and Capto Core 700, and the reference ( Figure 1 ).

[0038] Experiment 3: Scale-up of adenovirus purification method Method 1 and the reference method were scaled for purification of adenovirus from 3L cell culture harvest. Method 1 showed clear advantages over the reference method in terms of impurity reduction and yield of the polishing step, but the overall results on the scale-up experiments provided comparable results between the methods. In the scale-up experiments, the hcDNA levels in the final samples were similar.

[0039] However, method 1 reduced HCP levels to below the LOD, while in the reference method, HCP levels in the final sample averaged 22 ng / mL. The amount of total viral particles in the final sample was comparable between the methods (method 1: 4.9x10 13 and 5.3x 10 13 vp; reference method: 3.4x 10 13 and 5.1x 10 13 vp).

[0040] The chromatogram for method 1 is shown in Figure 2 , while the chromatogram for the reference method is shown in Figure 3 The results from the scale-up approach are summarized in Table 4.

[0041] Method 1( Figure 2 A) Samples: Harvests subjected to NFF and TFF in 20 mM Tris, pH 8.0 + 450 mM NaCl Resin: Capto Q ImpRes Column: HiScale 26 Loading: 5.6×10 11 VP / mL resin (Q B10 80% of 10 minutes residence time) Binding buffer: 20 mM Tris, pH 8.0 + 450 mM NaCl, 2 mM MgCl2 Wash: 2CV 20mM Tris, pH 8.0 + 480mM NaCl, 2mM MgCl2 Elution buffer: 2.5 CV of 20 mM Tris, pH 8.0 + 450 to 570 mM NaCl, 2 mM MgCl2 system: pure 150 Method 1( Figure 2 B) Sample: Eluate from capture step Resin: Capto Core 700 Column: HiScale 16 Loading: 9CV Capto Q ImpRes eluate Buffer: 20 mM Tris, pH 8.0 + 500 mM NaCl, 2 mM MgCl2 Wash: 1.5 CV of binding buffer system: pure 150 Reference method ( Figure 3 A) Samples: Harvests subjected to NFF and TFF in 20 mM Tris, pH 8.0 + 350 mM NaCl Resin: Q Sepharose XL Column: HiScale 50 Loading: 8.9×10 11 VP / mL resin (Q B10 80% of 10 minutes residence time) Binding buffer: 20 mM NaP, pH 7.3 + 350 mM NaCl, 2 mM MgCl2, 2% sucrose Wash: 5 CV of 20 mM NaP, pH 7.3, 350 mM NaCl, 2 mM MgCl2, 2% sucrose Elution buffer: 20 mM NaP, pH 7.3 + 500 mM NaCl, 2 mM MgCl2, 2% sucrose system: pure 150 Reference method ( Figure 3 B) Sample: Eluate from capture step Loading: 0.2CV Q Sepharose XL eluate Resin: Sepharose 4Fast Flow Column: HiScale 50 Loading: 0.2CV Buffer: 20 mM sodium phosphate, pH 7.3 + 200 mM NaCl, 2 mM MgCl2, 2% sucrose Wash: 1.5 CV of binding buffer system: pure 150 Table 4. Adenovirus recovery and purity in the method of the present invention (method 1) and the reference method *Analysis is performed only once.

[0042] Two numbers indicate that the same sample was analyzed twice.

[0043] LOD = 1 ng / ml.

[0044] A shallow salt gradient of Capto Q Impres from 480 mM to 570 mM NaCl is crucial for the isolation of DNA fragments from viral particles. This corresponds to a 19% increase in salt concentration across the gradient, or a 7.5% increase in salt concentration per CV in the gradient (gradient calculation: 90 mM change over 2.5 CV, 36 mM / CV). Compared to the reference method (Sepharose QXL step elution followed by size exclusion, Figure 3 A and Figure 3 B, Table 4), polishing with Capto core 700 as the second step resulted in a final bulk with significantly reduced debris or impurities as imaged by transmission electron microscopy (TEM). This procedure is expected to perform equally well with gradients of KCl or LiCl, or any combination of NaCl, KCl, and LiCl.

[0045] From the above, it is clear that Method 1 using a combination of Capto Q ImpRes anion exchange resin and Capto Core 700 resin has several advantages over the reference method.

[0046] On a smaller scale, Method 1 showed a clear advantage in impurity reduction. In scaled-up experiments, Method 1 showed better HCP reduction (<LOD vs 22 ng / ml). This feature and other features make the method of the present invention a suitable method for purifying adenovirus vectors for cell therapy.

[0047] Another major benefit of the present invention is that the Capto Core 700 column in Method 1 can be loaded up to 30 column volumes (CV), while in the reference method it can only be loaded with 0.2 CV (150-fold loading capacity). In addition, compared to size exclusion chromatography SEC, the yield is significantly better for the polishing step using the shell beads in Method 1 (Table 3).

Claims

1. A method for purifying adenovirus comprising the steps of: a) capturing adenovirus from a cell culture harvest containing adenovirus on an anion exchange resin; b) eluting the adenovirus using a shallow conductivity gradient having an increasing salt concentration of 15-25%, preferably 18-20%, over the entire gradient; c) applying the eluted adenovirus to a shell bead resin comprising a porous shell and a porous core, wherein the core is provided with a hydrophobic interaction ligand and the shell is not provided with any ligand; and d) eluting the adenovirus from the shell bead resin into a flow-through, wherein the adenovirus eluted in step d) contains less than 1 ng / ml of host cell protein (HCP).

2. The method according to claim 1, wherein the salt is selected from the group consisting of NaCl, KCl and LiCl, or any combination thereof.

3. The method according to claim 1 or 2, wherein the salt is NaCl and the gradient is an increasing 18-20% and the salt concentration is 0-700 mM.

4. The method according to one or more of the preceding claims, wherein the anion exchange resin is packed in a column and the shell bead resin is packed in another column and wherein the adenovirus eluted from the anion exchange resin is added to the shell bead resin in a volume corresponding to 15-30 column volumes (CV) of the column comprising the shell bead resin.

5. The method according to claim 4, wherein the adenovirus eluted from the anion exchange resin is added to the shell bead resin in a volume corresponding to 25-30 column volumes (CV) of a column comprising the shell bead resin.

6. The method according to one or more of the preceding claims, wherein the porosity of the core and shell is the same as the shell bead resin.

7. The method according to one or more of the preceding claims 1 to 5, wherein the porosity of the core and shell is different from that of the shell bead resin.

8. A composition comprising an adenovirus purified according to one or more of claims 1 to 7, wherein the host cell protein (HCP) is less than 1 ng / ml.

9. The composition according to claim 8, wherein the adenovirus is an adenoviral vector suitable for cell therapy.

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