WO2022043926A1

WO2022043926A1 - Process for preparation of recombinant adeno-associated virus particle

Info

Publication number: WO2022043926A1
Application number: PCT/IB2021/057848
Authority: WO
Inventors: Varun LAHOTI; Deepak KANJWANI; Siva BINGI; Priyanka Priyadarsiny; Lakshmikanth Gandikota
Original assignee: Intas Pharmaceuticals Ltd.
Priority date: 2020-08-31
Filing date: 2021-08-27
Publication date: 2022-03-03

Abstract

The present invention relates to the consistent and reproducible method for process scale up and improving yield of AAV production. Further, present invention relates to downstream purification process for purifying AAV8-FIX gene therapy product that include two major steps including affinity followed by density gradient step which can be scalable for purification of different AAV product/serotypes with high purity and more encapsidated virus which is suitable for clinical applications.

Description

PROCESS FOR PREPARATION OF RECOMBINANT ADENO- ASSOCIATED VIRUS PARTICLE

RELATED APPLICATION

This application is related to Indian Provisional Application 202021037364 filed on 31 ^st Aug, 2020 and is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention pertains to process for production and purification of recombinant AAV particle.

BACKGROUND OF THE INVENTION

FIX is a vitamin K-dependent glycoprotein belonging to the serine-protease family, and is synthesized in the liver of man and other animals, including mammals, playing a fundamental role in both intrinsic and extrinsic pathways of the blood coagulation cascade. Human FIX circulates in plasma as a single chain zymogen enzyme composed of 415 amino acids. Human FIX has a molecular weight of 57 kD and a plasma concentration of about 5 pg/ml. The zymogen is activated both by activated factor XI (FXIa), and tissue factor complex (TF) — activated factor VII (FVIIa). The structural organization of FIX is similar to that of other vitamin K-dependent coagulation proteins such as factor VII (FVII), factor X (FX) and protein C (PC). The amino-terminal portion of the molecule comprises the “Gia” domain, a region rich in gamma-carboxy-glutamic residues whose carboxylation is dependent on the presence of vitamin K. The main physiological function of FIX, once activated, is to convert factor X (FX) into activated factor X (FXa) in a process that requires the presence of a phospholipid surface, calcium ions and a protein with co-factor effect, namely activated factor VIII (F Villa). FXa itself is able to convert prothrombin into thrombin which transforms fibrinogen into soluble fibrin which, on polymerization, forms the clot. The action of FXa is enhanced by the presence of activated factor V (FVa).

The human FIX gene is located on chromosome X in position Xq27.1 and contains 8 exons of lengths varying from 25 base pairs (bp) to 2000 bp. Human FIX mRNA is about 3 kb in length and comprises 205 bases which form the 5' UTR region, 1386 bases which encode the FIX polypeptide and 1392 bases of the 3' UTR region. This mRNA encodes the synthesis of 461 amino acids which form the human FIX precursor. This precursor (SEQ ID NO: 1) comprises the following segments and domains: a hydrophobic signal peptide (amino acids 1-28), a propeptide (amino acids 29-46), a Gla-domain (amino acids 47 to 92), an EGF-like 1 domain (amino acids 93 to 129), an EGF-like 2 domain (amino acids 130 to 171), an activation peptide (amino acids 192 to 226) and a serine-protease domain (amino acids 227 to 461). The mature form of human FIX (SEQ ID NO: 2) looses the hydrophobic signal peptide and the propeptide. Consequently, the corresponding amino acid positions of the aforementioned domains become the following: a Gla-domain (amino acids 1 to 46), an EGF-like 1 domain (amino acids 47 to 83), an EGF-like 2 domain (amino acids 84 to 125), an activation peptide (amino acids 146 to 180) and a serine-protease domain (amino acids 181 to 415). SEQ ID NO: 1 (from which SEQ ID NO: 2 is derived) corresponds to the sequence on PubMed (“Protein” category) found by entering accession number AAB59620; this amino acid sequence comprises the signal peptide (46 AA), followed by the amino acid sequence of the mature protein.

A genetic deficiency in FIX can cause a number of coagulation diseases (coagulopathies), for example the haemorrhagic disease known as Haemophilia B in affected males (sex linked genetic disease). Haemophilia B can be classified into three classes, each of which is characterized by the presence of different plasma concentrations of FIX. In severe Haemophilia B the plasma levels of FIX activity are below 1% of normal; in the moderate form, levels are between 1% and 5%; in the mild form, between 5 and 25% of normal levels. There are also healthy carrier individuals who have medium FIX activity levels, between 25% and 50% of normal, but many carriers can have levels even exceeding 50%.

At present, gene transfer mediated by an Adeno-Associated Virus (AAV) vector shows the greatest promise for long-term correction of Haemophilia B in the preclinical setting bleeding disorders. We intend to develop Factor IX (FIX) codon optimized expression vector, which is packaged as a complimentary dimer within a single virion. These self-complementary Adeno-Associated Virus (scAAV) vectors improve transduction efficiency of the liver in mice by at least 20-fold over that achieved with comparable conventional AAV vectors; as they are no longer dependent on the target cell machinery for conversion of the single-stranded AAV genome into transcriptionally active double-stranded forms. The present invention relates to development of an adeno-associated virus based Gene Therapy product for the treatment of Haemophilia B. Our strategy in this direction is to develop AAV8 based gene therapy vectors and targeting the specific gene as a therapeutic option for Haemophilia B. Further, present invention relates consistent, reproducible and robust upstream and downstream process for the production of AAV8-FIX gene therapy product.

OBJECTS OF THE INVENTION

The principal object of the present invention is to provide adeno-associated virus (AAV) vector comprising transgene encoding Factor IX which provides long term sustained expression of FIX protein with increased activity and maintain the FIX expression level. Another object of the present invention is to provide method for the production of AAV vector comprising transgene encoding Factor IX wherein said method comprising use of suspension and serum free cell line.

Another object of the present invention is to provide method for the production of AAV vector comprising transgene encoding Factor IX wherein said method comprising culturing cells in serum-free chemically defined media.

Another object of the present invention is to provide method for the production of AAV vector comprising transgene encoding Factor IX wherein said method comprising steps of: a) Vial thaw at seed density (>0.30) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; b) Seed propagation in a working volume of 80-100 mL at seed density of (0.5-0.7) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; c) Seed propagation in a working volume of 350 mL at seed density of (0.5-0.7) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; d) Production in a wave bioreactor with an inoculation density of (0.4-0.6) x 10⁶ cells/mL, at 37°C temperature, 18-20 Rocks, 8-9° Angle, DO : 50%, pH 7.2 ± 0.1 for 5 to 6 days; e) Production in a stirred tank bioreactor of scale 10L to 200L at high density suspension culture, wherein said suspension culture having a cell density ranging from 1.5 x 10⁶ cells/mL to 2 x 10⁷ cells /mL at 37°C; f) Maintaining dissolved oxygen (DO) in the range of 30-50 % using P/V in the range of 5-30 W/m³ and pH in the range of 6.8 to 7.3; g) Incubating transfected culture with enhancers under or with temperature shift conditions in the range of 30°C to 37°C; wherein post-transfection utilize pH rise as a surrogate indicator of glucose limitation and provide continuous feedback control for glucose delivery; and h) Harvesting whole cell culture containing AAV comprising transgene encoding FIX.

Another object of the present invention is to provide method for purification of adeno- associated virus (AAV) vector comprising a transgene encoding FIX, wherein said method comprising steps of affinity chromatography and density gradient centrifugation.

Another object of the present invention is to provide method for purification of adeno associated virus (AAV) vector comprising a transgene encoding FIX, wherein said method comprising steps of: a) Harvesting whole cell culture along with media comprising recombinant AAV; b) Cell lysis and Clarification; c) Tangential Flow Filtration (IFF); d) Affinity Chromatography; e) Density Gradient Ultracentrifugation; f) Ultra-filtration and Diafiltration (UFDF); and g) 0.2 p Filtration.

SUMMARY OF THE INVENTION

The principal aspect of the present invention is to provide adeno-associated virus (AAV) vector comprising transgene encoding Factor IX which provides long term sustained expression of FIX protein with increased activity and maintain the FIX expression level. Another aspect of the present invention is to provide method for the production of AAV vector comprising transgene encoding Factor IX wherein said method comprising use of suspension and serum-free cell line.

Another aspect of the present invention is to provide method for the production of AAV vector comprising transgene encoding Factor IX wherein said method comprising culturing cells in serum-free chemically defined media.

Another aspect of the present invention is to provide method for the production of AAV vector comprising transgene encoding Factor IX wherein said method comprising steps of: a) Vial thaw at seed density (>0.30) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; b) Seed propagation in a working volume of 80-100 mL at seed density of (0.5-0.7) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; c) Seed propagation in a working volume of 350 mL at seed density of (0.5-0.7) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; d) Production in a wave bioreactor with an inoculation density of (0.4-0.6) x 10⁶ cells/mL, at 37°C temperature, 18-20 Rocks, 8-9° Angle, DO : 50%, pH 7.2 ± 0.1 for 5 to 6 days; e) Production in a stirred tank bioreactor of scale 10L to 200L at high density suspension culture, wherein said suspension culture having a cell density ranging from 1.5 x 10⁶ cells/mL to 2 x 10⁷ cells /mL at 37°C; f) Maintaining dissolved oxygen (DO) in the range of 30-50 % using P/V in the range of 5-30 W/m³ and pH in the range of 6.8 to 7.3; g) Incubating transfected culture with enhancers under or with temperature shift conditions in the range of 30°C to 37°C; wherein post-transfection utilize pH rise as a surrogate indicator of glucose limitation and provide continuous feedback control for glucose delivery; and h) Harvesting whole cell culture containing AAV comprising transgene encoding FIX.

Another aspect of the present invention is to provide method for purification of adeno- associated virus (AAV) vector comprising a transgene encoding FIX, wherein said method comprising steps of: a) Harvesting whole cell culture containing AAV comprising transgene encoding FIX b) Cell Lysis and Clarification; c) Tangential Flow Filtration (IFF); d) Affinity Chromatography; e) Density Gradient Ultracentrifugation; f) Ultra-filtration and Diafiltration (UFDF); and g) 0.2 p Filtration.

BRIEF DESCRIPTION OF DRAWINGS

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein: Figure 1: Schematic Representation scAAV8-hFIXco Construct

Figure 2: Flow Chart for Upstream Process

Figure 3: Flow Chart for Downstream Process

Figure 4: The Viable Cell Density and Cell Viability (%) of Six Batches during Different Time Point

Figure 5: Depicts the CP/mL and VG/mL for the Six Consistency Batches Post Cell Lysis (Upstream Process). The data suggests CP/mL including filled and empty capsids ranged in between 1.41E+11 to 2.23E+11 and VG/mL ranged in between 2.0E+10 to 4.6E+10.

Figure 6: Step Recovery of CP in TFF Step for Six Batches

Figure 7: Step Recovery of CP in Affinity Chromatography Step for Six Batches

Figure 8: VG Enrichment After Ultracentrifuge Density Gradient Step (Downstream Process)

Figure 9: VG Recovery from Six Consistency Batches (Downstream Process)

Figure 10A to 10F: Structural characterization of rAAV8-FIX vector (A) Size distribution by intensity of AAV8-FIX DP batch- 1 showing rAAV8 size of 28.5 nm (B) Size distribution by intensity of AAV8-FIX DP batch-2 showing rAAV8 size of 29.4 nm (C) Size distribution by intensity of AAV8-FIX DP batch-3 showing rAAV8 size of 28.4 nm (D) Size Exclusion Chromatography of AAV8-FIX DP batch- 1 (E) Size Exclusion Chromatography of AAV8-FIX DP batch-2 (F) Size Exclusion Chromatography of AAV8-FIX DP batch-3

Figure 10G: Analytical Ultracentrifugation (AUC) profile of representative AAV8- FIX preparation showing presence of full capsids at sedimentation coefficient of -100S and empty capsids at -60S

Figure 10H: Transmission electron microscopy micrographs of negatively stained rAAV8-FIX representative batch showing full (white) and empty (darker spot) viral particles under 150,000x magnification.

Figure 11A to 11F: rAAV8-FIX vector characterization (A) RP-HPLC chromatogram of AAV8-FIX DP batch-1 showing viral capsid protein peak separation representative of VP1 and VP2 and VP3 was higher than VP1 and VP2 (B) RP-HPLC chromatogram of AAV8-FIX DP batch-2 showing viral capsid protein peak separation representative of VP1 and VP2 and VP3 was higher than VP1 and VP2 (C) RP-HPLC chromatogram of AAV8-FIX DP batch-3 showing viral capsid protein peak separation representative of VP1 and VP2 and VP3 was higher than VP1 and VP2 (D) rAAV8 capsid thermal stability as relative fluorescence units (RFU) versus temperature (T [°C]) for AAV8-FIX DP batch-1 determined by Thermal shift assay (E) rAAV8 capsid thermal stability as relative fluorescence units (RFU) versus temperature (T [°C]) for AAV8-FIX DP batch-2 determined by Thermal shift assay (F) rAAV8 capsid thermal stability as relative fluorescence units (RFU) versus temperature (T [°C]) for AAV8- FIX DP batch-3 determined by Thermal shift assay

Figure 11G: Silver-stained SDS-PAGE Profile of rAAV8-FIX Preparations (Lane-1: Protein marker; Lane-3: AAV8 assay control; Lane-5, 7 and 9: AAV8-FIX DP Batch 1, 2 and 3)

Figure 11H: Coomassie Brilliant Blue Stained SDS-PAGE Profile of rAAV8-FIX Preparations (Lane-1: Protein marker; Lane-3: AAV8 assay control; Lane-5, 7 and 9: AAV8-FIX DP Batch 1, 2 and 3)

Figure 12A: rAAV8-FIX vector functional activity characterization: Schematic for in-vitro potency determination on HepG2 cell based transduction assay followed by measurement of FIX antigen levels and activity

Figure 12B: rAAV8-FIX vector functional activity characterization: Comparative data of 3 DP batches of rAAV8-FIX for FIX antigen levels measured by FIX antigen ELISA and functional activity as measured by Chromogenic and aPTT based clotting assay DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

It is to be noted that the term "a" or "an" refers to one or more. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of or “consisting essentially of language.

There are several naturally occurring (“wild-type”) serotypes and over 100 known variants of AAV, each of which differs in amino acid sequence, particularly within the hyper variable regions of the capsid proteins, and thus in their gene delivery properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

For the purposes of the disclosure herein, the terminology “AAV” is an abbreviation for Adeno-associated virus, including, without limitation, the virus itself and derivatives thereof. Except where otherwise indicated, the terminology refers to all subtypes or serotypes and both replication-competent and recombinant forms. The term “AAV” includes, without limitation, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3 A (AAV-3 A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV- 5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV- 8 or AAV8), AAV type 9 (AAV-9 or AAV 9), AAV type 10 (AAV- 10 or AAV10 or AAVrhlO), avian AAV, bovine AAV, Porcine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infect primates, "non-primate AAV" refers to AAV that infect non- primate mammals, "bovine AAV" refers to AAV that infect bovine mammals, etc.

The term "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of Adeno- associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

The terminology "helper virus" for AAV refers to a virus that allows AAV (e.g. wildtype AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

The term "gene" refers to a polynucleotide that performs a function of some kind in the cell. For example, a gene can contain an open reading frame that is capable of encoding a gene product. One example of a gene product is a protein, which is transcribed and translated from the gene. Another example of a gene product is an RNA, e.g. a functional RNA product, e.g., an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA for nucleases, etc., which is transcribed but not translated.

A "transgene" is used herein to conveniently refer to a polynucleotide or a nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene that encodes a polypeptide or protein.

The main embodiment of the present invention is to provide adeno-associated virus (AAV) vector comprising transgene encoding Factor IX which provides long term sustained expression of FIX protein with increased activity and maintain the FIX expression level.

Another embodiment of the present invention is to provide method for production of AAV vector comprising transgene encoding Factor IX wherein said method comprising use of suspension and serum free cell line.

Another embodiment of the present invention is to provide method for production of AAV vector comprising transgene encoding Factor IX wherein said method comprising culturing cells in serum-free chemically defined media.

Another embodiment of the present invention is to provide method for production of AAV vector comprising transgene encoding Factor IX wherein said method comprising steps of: a) Vial thaw at seed density (>0.30) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; b) Seed propagation in a working volume of 80-100mL at seed density (0.5-0.7) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; c) Seed propagation in a working volume of 350mL at seed density (0.5-0.7) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days; d) Production in a wave bioreactor with an inoculation density (0.4-0.6) x 10⁶ cells/mL, at 37°C temperature, 18-20 Rocks, 8-9° Angle, DO : 50%, pH 7.2 ± 0.1 for 5 to 6 days; e) Production in a stirred tank bioreactor of scale 10L to 200L at high density suspension culture, wherein said suspension culture having a cell density ranging from 1.5 x 10⁶ cells/mL to 2 x 10⁷ cells /mL at 37°C; f) Maintaining dissolved oxygen (DO) in the range of 30-50 % using P/V in the range of 5-20 W/m³ and pH in the range of 6.8 to 7.3; g) Incubating culture with enhancers post-transfection under or with temperature shift conditions in the range of 30°C to 37°C; wherein post-transfection utilize pH rise as a surrogate indicator of glucose limitation and provide continuous feedback control for glucose delivery; and h) Harvesting whole cell culture containing AAV comprising transgene encoding FIX.

Another embodiment of the present invention is to provide method for purification of adeno-associated virus (AAV) vector comprising a transgene encoding FIX, wherein said method comprising steps of affinity chromatography and density gradient centrifugation.

Another embodiment of the present invention is to provide method for purification of adeno-associated virus (AAV) vector comprising a transgene encoding FIX, wherein said method comprising steps of: a) Harvesting whole cell culture containing AAV comprising transgene encoding FIX b) Cell lysis and Clarification; c) Tangential flow filtration (TFF); d) Affinity chromatography; e) Density gradient ultracentrifugation; f) Ultra-filtration and Diafiltration (UFDF); and g) 0.2 p filtration.

The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also form the part of this specification and are intended to be included within the scope of it.

EXAMPLE 1: VECTOR DESIGN

Vector and Transgene:

In-house designed gene constructs were synthesized (outsourced) and sub cloned into suitable plasmid for virus production.

ScAAV8-hFIXco Constructs:

The codon optimized human Factor IX (hFIXco) synthesized gene was cloned under liver specific hAAT promoter and has all the genetic elements as mentioned in Figure 1. The choice of serotype AAV8 would carry self-complimentary adeno-associated virus (scAAV) recombinant genome. scAAV-hFIXco-Padua Construct:

The transgene used in this construct was human FIX with Padua mutation (R338L) that provided an added advantage over the wild type gene. FIX Padua was preferred choice for the development of gene therapy vector for Haemophilia B as this mutation enhanced the specific activity 8-10% of the encoded FIX protein. . The hFIXco-Padua construct had same genetic elements as the wild type construct except it has R384L mutation in FIX preprotein (R338L in secreted FIX). Plasmid constructs (pAAV-FIX^wlld and pAAV-FIX^Padua), Packaging constructs pRep2Cap8 (carrying the Rep2 and Cap8 genes), pAdHelper (carrying adenoviral helper genes (E2, E4 and VA RNA) were synthesized commercially and sub-cloned in pUC19 backbone. These constructs were sub-cloned into desired vector and used for AAV production vector.

The present transduced vector containing hFIXco-Padua construct provided long term sustained expression of FIX protein with an increase activity and maintain the FIX expression level to treat mild, moderate and severe Haemophilia B patients.

EXAMPLE 2: PRODUCTION OF rAAV-FIX

Adaptation of Expi293F™ Suspension Cell Line and Seed Preparation:

Expi293 cell line was adapted to grow in BalanCD media. Cells were then expanded and cryopreserved using freezing media. The complete freezing media was formulated using 50% fresh medium, 50% spent medium and 10% DMSO. Expi293 cell line was thawed, cultured and expanded in complete BalanCD medium supplemented with 6mM Glutamine. Shaker Incubators were used to propagate and maintain cells at specific RPM (based on cell culture volumes) at 80% humidity and 5% CO2. The culture was passaged every 3-4 days to ensure the cell count does not exceed 7*10⁶ cells/mL and maintains viability of >95% consistently; the process flow given in Figure 2. The number of cells were counted on the day of passage and seeded in the desired volume to obtain the cell density in the range of 0.5-0.7 10⁶ cells/mL. This cell line was passaged to a maximum of 20 passages post vial thaw. A number of vials were thawed from this cell bank and utilised for process development to develop a scalable manufacturing process using shake flasks and wave bioreactor to generate rAAV8-FIX VLP (Virus-like Particles). Transfection of Suspension Expi293F™ Cells:

On the expected day of transfection, cells were counted using automated cell counter, expected viable density to be in the range of (2.0-3.0) x 10⁶ cells/ mL posttransfection. In our study, three plasmid-based transient transfection approach was performed at 48 hours of seeding the wave bioreactor. Sampling was performed every day for cell count, viability, offline pH, pCO2, and residual glucose estimation.

The transfection cocktail was prepared by mixing the following reagents in this order: BalanCD Medium, Plasmid DNA, and Polyethyleneimine Max (MW 40,000). The transient transfection was performed with plasmid DNA in a ratio of 1 : 1 :2 for R2C8: GOI: Helper (packaging plasmid, GOI and helper plasmid) and 1 pg total plasmid was used per 2 x 10⁶ total cells. PEI Max was used at PEI: DNA ratio of 3: 1. The total volume of the cocktail mix was 10% of the total bioreactor working volume. The cocktail mix was mixed for 5-10 sec prior to being incubated at room temperature for 15-20 minutes. The transfection cocktail mix was then added to the wave bioreactor aseptically. Ig/L of Kolliphor was added to the culture 24 hours post-transfection to act as a shear protectant and possibly minimize clumping.

AAV Production Method of Consistency Batches:

To establish a scalable manufacturing upstream process to produce VLPs utilizing the transient triple transfection method with the plasmids having AAV8-FIX construct, Rep2 Cap8, and pHelper construct in Expi293F™ suspension cell line, six production batches in a wave bioreactor (T20WV0100369 -Batch 1, T20WV0102107 - Batch 2, T20WV0102110 - Batch 3, T20WV0101908 - Batch 4, T20WV0102117 - Batch 5 and T20WV0102120 - Batch 6) were initiated.

The Expi293F™ cells were grown in serum-free suspension media that support growth and transfection efficiency in both shake flasks and bioreactors. A Biostat® B bioreactor equipped with wave-mixed RM rocker was used to produce AAV8-FIX. The flex safe wave bioreactor bags were seeded at a volume that was 85% of the bags’ maximum cell culture volume. Seeding densities for these batches were in the range of (~ 0.4-0.6) x io⁶ cells/ml. The wave bag was rocked at an 8-9° angle and 18-20 RPM for 1-3 hr. The transfection in wave bag was performed as illustrated above. Glucose was supplemented to the culture to make up the final glucose concentration of (3-5) g/L using a 40 % glucose stock solution when residual concentration reached < 3.0 g/L post-transfection. The whole-cell culture was harvested between 65 hours to 96 hours post-transfection with viability of >80% and lysed immediately or stored in an appropriate container at -80°C.

Six production batches at the 20L scale (wave bag bioreactor , working volume 10L - 12L) were evaluated for process consistency and performance to produce rAAV8- FIX VLPs. Cell growth profile, capsid productivity, and encapsidation of vector genome for the batches of rAAV8-FIX were assessed.

The viable cell density in all the six batches consistently ranged between (5.7-6.7) x 10⁶ cells/ml. Viability at harvest in the six batches was >80.0% as shown in Figure 4. The harvest viable cell density was consistent across the batches. The culture and process parameters, i.e., temperature, pH, and DO profile generated using in-line bioreactor sensors, were similar for the six batches during rAAV production.

Harvest titers for all the six batches have been reported as CP/mL for capsid produced in the batches, estimated using AAV8 serotype capsid ELISA and volumetric yield (VG/mL) was estimated by qPCR. Figure 5 depicts the CP/mL and VG/mL for the six consistency batches. The data suggest that CP/mL, produced from a 10L culture, ranged between 1.41E+11 to 2.23E+11 and VG/mL ranged between 2.0E+10 to 4.6E+10 at the harvest stage. The findings suggest that a total of 1E+15 capsid and 1E+14 vector genome can be produced consistently at 10L scale. The encapsidation ranged between 10 to 25% (as shown in Figure 8), establishing that the transient transfection process with suspension cell line was scalable.

The data suggested upstream process for AAV8-FIX was consistent across the six batches in terms of the cell culture growth profiles. Capsid titer and vector genome titer were well within the ±2 SD range. With reference to the above results and trends it was concluded that the upstream process performance of the six batches of AAV8- FIX executed at 20L wave bioreactor were consistent and demonstrated reproducibility and robustness. The present invention provided the consistent and reproducible method for upstream process and improving yield of AAV production.

EXAMPLE 3: PURIFICATION AND CONCENTRATION OF rAAV-FIX

Downstream Process for Purification:

The purification process of AAV8-FIX starts with whole cell culture harvest containing the VLPs of AAV8-FIX (product of interest); the process flow given below in Figure 3.

Lysis and Clarification of Whole Cell Culture Harvest:

The whole cell culture (cells along with culture medium) was lysed to release the intracellular product produced in cells. For this, the 10 L of harvest was conditioned by adding 20 mM Tris, 150 mM NaCl and 10 mM MgCb. The cell lysis was induced by addition of Triton XI 00 to final concentration of 0.5% in the harvest culture and maintaining the pH around 7.5. Following the addition of benzonase the lysate was incubated at temperature of 37°C for 1 hour. After complete cell lysis, the lysate was clarified for removal of suspended particles, cell debris etc. by centrifugation at 15,900 g at 10°C for 30 minutes or depth filtration. The supernatant was then passed through 0.22 p filtration before ultrafiltration step. Tangential Flow Filtration of Clarified Lysate:

For concentration and diafiltration operation, membranes with molecular weight cutoff of 300 kDa were used. Briefly, the filtrate was concentrated 4 to 8 fold using 0.11m² membrane area, 300 kDa polyethersulfone membrane at a TMP of 0.3 bar, followed by filtration using 0.22 p filter.

Chromatographic Purification of Adeno-Associated Virus:

The affinity chromatography using POROS™ Captures elect™ AAVX was employed as the capture step in the AAV8-FIX purification. The AAV particles bind to POROS™ Captures elect™ AAVX ligand through affinity interactions. This step removed majority of process related impurities (media components and coloring matter). The resin was equilibrated with 20 mM Tris buffer containing 150 mM NaCl at pH 7.5, following loading, washing with equilibration buffer followed by high salt wash buffer (20 mM Tris, 500 mM NaCl, pH 7.0) and low pH wash buffer (100 mM Sodium acetate pH 5.0) were given before elution of bound AAV. The bound AAV was eluted with low pH buffer (100 mM Glycine, pH 2.7).

Density Gradient Ultracentrifugation:

The removal of empty virions from the mixture of empty and filled virions was achieved by iodixanol based density gradient separation using ultracentrifugation. 19 ml of viral suspension was deposited through a glass pasteur pipette in the bottom of 25 x 89 mm Quick seal ultra-clear tubes (Beckman Coulter, France). The suspension was raised by successive addition of 3 ml of 15% iodixanol at IM NaCl, 4 ml of 40% iodixanol and 5 ml of 60% iodixanol. Iodixanol solutions were all diluted in 1XDPBS- MK buffer (1 M NaCl, Immol/L MgCL2, 2.5 mmol/1 KC1, IX PBS). Tubes were heat- sealed with the tube topper system and placed in the Ti70 rotor and ultra-centrifuged at 375,000xg (50,000 rpm) for 120 min in an Optima L100XP ultracentrifuge. The filled virions were collected using ultracentrifuge piercing from the side at the interface of 40% and 60% iodixanol gradient.

Tangential Flow Filtration and 0.2 u Filtration:

The collected fraction was then diluted five times with Phosphate Buffer Saline (PBS) containing 0.001 % Pluronic F-68, before diafiltration through centrifugal concentration and filtration. The diafiltration was performed five to seven times, followed by sterile filtration using a 0.22 p Acrodisc syringe filter (Pall Life Sciences).

The downstream purification process for the AAV8-Factor IX gene therapy product was consistent across several batches, and all the steps are comparable in terms of process parameters. The result showed more than 90% step recovery of capsid particles using tangential flow filtration (TFF) from the clarified cell culture lysate (as shown in figure 6) in almost all the batches, except in two batches there is loss due to manual intervention. The capsid particle recovery using AAVX Capture Select Affinity resin yielded more than 60% step recovery with reasonable purified fractions in all the batches (as shown in figure 7). The ultracentrifugation step enriches filled virion from the mixture of empty and filled virions in the affinity output. The enrichments of filled virions range from 40 to 60% in all the consistency batches. The process exhibits the improvement in encapsidation of the preparation from 10 % to 25% at harvest level to 40% to 60 % after purification (as shown in figure 8). The overall cumulative VG recovery ranges were around 15 to 29% of the purified product (as shown in figure 9). The drug product obtained from the pooling of the drug substance was further characterized for quality parameters. Basic characterization of the batches were performed. Final drug product obtained from the scale up of purification process was analyzed using SDS-PAGE to confirm VP1, VP2 and VP3 bands with no other impurities. All the process and analytical data were within the range of ± 2 SD. The present downstream process was capable of performing consistently with comparable quality at Drug Substance (DS) stage. The downstream process was consistent at 20L wave bioreactor scales which can also be scalable to large scale in GMP facilities. Encapsidation of virus particle ranges from 22.8 to 64% and increased more than 50% using one step ultracentrifuge lodixanol density gradient step. The present downstream purification process for AAV8-FIX gene therapy product was observed consistent for six batches and all the steps were comparable in terms of process parameters.

EXAMPLE 4: ANALYTICAL TECHNIQUES FOR QUANTIFICATION AND CHARACTERIZATION OF AAV8-FIX PARTICLE

In-process scAAV8-FIX vector preparation was purified and characterized by SDS- PAGE and western blot for purity and identity, capsid ELISA for virus particle quantification, qPCR for vector genome titre (gene specific qPCR) and transduction assay (liver cell line- HepG2) for function integrity. The drug product formulation was carried out in the formulation buffer (PBS with 0.001% Pluronic F-68 at pH 7.0 ± 0.3) by pooling different lots of drug substance to achieve the right batch size required for analytical testing, stability study, and PCT study. Three drug product batches were prepared from the drug substance batches and characterized by various analytical tools for titer, size, identity, purity, morphology, aggregation, and potency. qPCR Vector Genome (VG) Titer Assay:

Vector genome quantification was done by qPCR method (as described in Francois A, et al, 2018). Vector genome titer per millilitre (VG/mL) was calculated from the plasmid DNA standard curve after multiplication with appropriate dilution factor. All the batches were consistent with VG titre with a value of 8.17E+12 ± 3.94E+11 VG/mL. Capsid Particle (CP) Titer Assay:

Capsid particle concentration was determined using Progen AAV8 titration ELISA. CP titer was calculated from the standard curve after multiplication with the appropriate dilution factor. All the batches were consistent with CP titre. The intact and assembled CP titre of the drug product batches were consistent with CP titre with a value of 2.28E+13 ± 5.18E+12 CP/mL.

Dynamic Light Scattering (DLS):

Dynamic light scattering (DLS) was used to measure size and contaminants or aggregates. AAV8-FIX drug product samples were diluted to 10 fold in filtered IX PBS, and measurements were performed on a Zetasizer (Malvern Panalytical) cuvette at 25 °C temperature, using the particle size measurement to get both size and size distribution per population. Results showed the rAAV8 FIX particles were consistent with a single population majority with size distribution ranging from 28.4 nm to 29.4 nm and an average size of 28.8 ± 0.6 nm. No significant aggregates were detected, as shown in Figure 10A to 10C.

Size Exclusion HPLC (SE-HPLC):

SE-HPLC was performed to determine the homogeneity of AAV8-FIX preparations and to detect the presence of aggregates. AAV8-FIX drug product samples at 1E+11 CP concentration were loaded on to SEC column (SEC SRT-500) held at 25 °C with isocratic elution. The mobile phase contained IX phosphate buffer saline (pH 7.4). The separation was carried out with the isocratic flow of 100 % mobile phase with 0.5 mL/min flow rate, and eluted proteins were detected by UV absorbance at 220 nm. The drug product batches were consistent with a single peak contributing for (89 % to 91%) of intact monomeric AAV8 capsid particles, as shown in Figure 10D to 10F. Analytical Ultracentrifugation (AUC):

Analytical Ultracentrifugation (AUC) as a technique was used to characterize the homogeneous distribution of empty and filled capsids of AAV8-FIX preparations. AUC analysis was carried out in AUC Optima (Beckman Coulter). The percentages of fully packaged virions, intermediate particles, and empty particles were determined by analyzing 150 scans and was analysed by the Sedfit continuous-size C(S) distribution model by correlating the sedimentation coefficient to the molecular weight of each species. The relative percentages of capsid species with a sedimentation coefficient of 100S represent capsids harboring the full 4,400- nucleotide vector genome; the sedimentation coefficient of 62S represents empty capsids, and the sedimentation coefficient of 84S represents capsids harboring a fragmented genome. The AUC profile of one representative AAV8-FIX batch showed two distinct peaks with presence of genome-filled AAV8 capsids at sedimentation coefficient of - 100 S and empty AAV8 capsid population at sedimentation coefficient of - 60S (as shown in figure 10G).

Reversed-phase HPLC (RP-HPLC):

AAV8-FIX drug substance samples were analyzed by RP-HPLC to determine the presence of AAV capsid proteins VP1, VP2, VP3, and other protein impurities present in the product. The drug product batches were consistent for peak profile comprising three peaks representing VP2, VP1, and predominating VP3 (as shown in figure 11A to 11C).

Thermal Shift Assay:

Thermal shift assay was performed for the drug product batches to determine thermal stability using Sypro dye that has differential binding affinity that changes as the function of protein unfolding during incremental temperature excursions AAV8 Capsid melting point (Tm) is the temperature at which 50% of the capsid is unfolded and bound with dye and is determined by Thermal Shift assay. The capsid thermal stability or the melting temperature is defines as the function of relative fluorescence units (RFU) versus temperature (T [°C]). For AAV8-FIX DP batches Tm ranged from 71.8 ± 0.1 °C confirming that the preparations are consistent and are of specific identity of AAV8 vector in drug product batches (as shown in figure 1 ID to 1 IF)

SDS-PAGE Using Coomassie Staining:

The purity and identity of AAV8-FIX was determined by detecting the presence of AAV capsid proteins VP1, VP2, and VP3, their relative molecular weights, and stoichiometry. AAV8-FIX drug product samples were treated under reducing conditions and loaded onto 10% Mini-PROTEAN TGX stain-free precast gels at 2E+11 capsid particles per well, and electrophoresis was carried out. The gels were stained with colloidal Coomassie stain. The AAV8 VP1, VP2, and VP3 capsid protein bands were visualized and analyzed for their stoichiometry and molecular weight, and non-vector impurities. Purity of AAV8-FIX drug product revealed the presence of VP1, VP2, and predominating VP3 bands with relative molecular weights of ~89 kDa, ~75 kDa, and ~64 kDa. No non-vector impurities were detected with this assay. The drug product batches were consistent for vector purity of > 95 %, and the identity of viral proteins matched with their respective theoretical molecular weights (as shown in figure 11H).

Western Blotting:

The identity of AAV8-FIX was performed by western blotting with AAV monoclonal antibody. The AAV8 VP1, VP2, and VP3 capsid protein bands were visualized and analyzed. The identity of AAV8 in drug product batches was tested by immunological reaction with AAV monoclonal antibody in western blotting and consistent for VP1, VP2, and VP3 (as shown in figure Figure 11G). In Vitro Potency by Transduction Assay:

The vector potency of AAV8-FIX preparation is a cell culture-based in-vitro transduction assay on the HepG2 cell line. The FIX transgene expression levels in the transduced cell culture supernatants were measured by FIX antigen ELISA and functional activity was measured by chromogenic and clotting assays. The drug product batches were consistent for FIX antigen expression with FIX concentration with a value of 9.5 % ± 3.4 % in the in-vitro system (as shown in figure 12B).

FIX Antigen ELISA Assay:

Factor IX antigen in transduced cell culture supernatants was quantitatively determined with a kit-based ELISA (VisuLize™ FIX Antigen kit, Affinity Biologicals, FIX- AG). Subsequent expression of the transgene encoded hFIX antigen was quantitated by FIX antigen-specific ELISA (as percent antigen against the standardized and calibrated human plasma (as shown in figure 12A).

Chromogenic Assay for FIX Antigen Activity:

The chromogenic assay of Factor IX (FIX) activity was measured with the BIOPHEN FIX kit (Hyphen BioMed, 221806). An automated Coagulation Analyzer (Sysmex CS-1600) was used for this assay. The analysis was performed as per the instrument program, and the results were recorded at the end of the analysis. The drug product batches were consistent for FIX antigen expression with FIX antigen activity with a value of 9.2 % ± to 2.0 % when determined by chromogenic assay (as shown in figure 12B).

Clotting Assay for FIX Antigen Activity:

The Activated Partial Thromboplastin Time (aPTT) Assay was used for Factor IX (FIX) activity by evaluating the intrinsic/common pathway's overall integrity. An automated Coagulation analyzer (Sysmex CS-1600) was used for this assay. The analysis was performed as per the instrument program, and the results were recorded at the end of the analysis. The drug product batches were consistent for FIX antigen expression with FIX antigen activity with a value of l l.6 % ± 1.8 % as determined by APTT based clotting assay (as shown in figure 12B). Transmission Electron Microscopy (TEM) of Negatively Stained rAAV8 Particles:

The rAAV8 FIX were imaged in Transmission Electron Microscopy (TEM) to determine the quality of the preparation in terms of the filled particle and capsid assembly. The drug product batches were negatively stained with 2% uranyl acetate staining. The results showed difference in electron-density contrast between filled and empty particles. The images demonstrates the AAV particles are intact and homogeneous for all vector preparations (as shown figure 10H).

Claims

27 We Claim,

1. An adeno-associated virus (AAV) vector comprising transgene encoding Factor IX which provides long term sustained expression of FIX protein with increased activity and maintain the FIX expression level.

2. A method for the production of AAV vector comprising transgene encoding Factor IX, wherein said method comprising steps of:

(a) Vial thaw at seed density (>0.30) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days;

(b) Seed propagation in a working volume of 80-100 mL at seed density of (0.5-0.7) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days;

(c) Seed propagation in a working volume of 350 mL at seed density of (0.5-0.7) x 10⁶ cells/mL, at 37°C temperature, 125 RPM for 3 to 4 days;

(d) Production in a wave bioreactor;

(e) Production in a stirred tank bioreactor;

(f) Maintaining dissolved oxygen (DO) in the range of 30-50 % using Power per unit volume (P/V) in the range of 5-30 W/m³ and pH in the range of 6.8 to 7.3;

(g) Incubating transfected culture with enhancers under temperature shift conditions in the range of 30°C to 37°C; wherein post-transfection utilize pH rise as a surrogate indicator of glucose limitation and provide continuous feedback control for glucose delivery or specific feed; and h) Harvesting whole cell culture containing AAV comprising transgene encoding FIX.

3. The method according to claim 2, wherein said method comprises use of suspension and serum-free cell line.

4. The method according to claim 2, wherein said method comprises culturing cells in serum- free chemically defined media.

5. The method according to claim 2, wherein production in a wave bioreactor is at an inoculation density (0.4-0.6) x 10⁶ cells/mL, at 37°C temperature, 18-20 Rocks, 8-9° Angle, DO: 50%, pH 7.2 ± 0. 1 for 5 to 6 days. The method according to claim 2, wherein production in a stirred tank bioreactor is at scale of 10L to 200L at high density suspension culture, wherein said suspension culture having a cell density ranging from 1.5 x 10⁶ cells/mL to 2 x 10⁷ cells /mL at 37°C temperature. A method for purification of adeno-associated virus (AAV) vector comprising a transgene encoding FIX, wherein said method comprising steps of in-vitro cell lysis and clarification, affinity chromatography and density gradient centrifugation. The method according to claim 7, wherein affinity chromatography comprises equilibration with 20 mM Tris buffer containing 150 mM NaCl at pH 7.5, washing the column with high salt wash buffer (20 mM Tris, 500 mM NaCl, pH 7.0) and low pH wash buffer (100 mM Sodium acetate pH 5.0 and elution with low pH buffer (100 mM Glycine, pH 2.7). The method according to claim 7, wherein density gradient ultracentrifugation is performed with iodixanol gradient. A method for purification of adeno-associated virus (AAV) vector comprising a transgene encoding FIX, wherein said method comprising steps of:

(a) Harvesting whole cell culture containing AAV comprising transgene encoding FIX

(b) Cell Lysis and Clarification;

(c) Tangential Flow Filtration (TFF);

(d) Affinity Chromatography;

(e) Density Gradient Ultracentrifugation;

(f) Ultra-filtration and Diafiltration (UFDF); and

(g) 0.2 p Filtration.