Lentiviral vector production process in a packed-bed bioreactor

Abstract

The present invention relates to the development of a method for the production of LVV vectors in packed-bed bioreactors by multiple-plasmid transient DNA transfection. More specifically, the present invention discloses and claims a process in which steps applied in perfusion or batch mode are combined to obtain an effective process. Furthermore, the present invention identifies an effective range of total DNA that can be successfully used in the transient transfection step in packed-bed bioreactors for the production of lentiviral vectors.

Description

[Technical Field]

[0001] The present invention relates to the field of large-scale production of lentiviral vectors by multiple plasmid DNA transient transfection of packaging cells performed in a packed-bed bioreactor system. [Background technology]

[0002] Lentiviral vectors, or lentiviruses (LVVs), are gene delivery vehicles primarily utilized in gene therapy applications. LVVs are known to offer numerous advantages, including the ability to (i) transduce dividing and non-dividing cells and (ii) permanently integrate into the host cell genome, resulting in sustained gene expression in the host cell and any progeny (Naldini et al. 2006). Furthermore, LVVs are considered relatively safe due to their low immunogenicity and toxicity, as well as their low rates of insertional mutagenesis and tumorigenicity compared with oncoretroviral vectors. LVVs can be pseudotyped with envelope proteins from other viruses, thereby conferring broad tropism or specificity for specific target cells (Chronin et al. 2005). For all these reasons, LVV is primarily used in various gene therapy applications, such as the treatment of several genetic disorders, including metachromatic leukodystrophy (Biffi et al. 2013), Wiskott-Aldrich syndrome (Aiuti et al. 2013), bthalassemia (Cavazzana-Calvo et al. 2010) or X-linked adrenoleukodystrophy (Cartier et al. 2009). All these clinical applications involve the use of CD34 transfectants infused into patients. +The ultimate goal is to correct genetic defects by delivering a gene of interest to hematopoietic stem cells. A further example of a clinical application in which LVV is used as a gene delivery vehicle is adoptive cell therapy, which uses T cells genetically engineered to express chimeric antigen receptors (CARs) or T cell receptors. Chimeric antigen receptors (CARs) are recombinant receptors that recognize specific proteins or antigens expressed on target cells. When subsequently expressed in T lymphocytes, called CAR-T cells, or other cells of the immune system, CARs can redirect a specific immune response against all cells expressing the antigen to which they bind. The most extensively explored clinical application of CARs is cancer immunotherapy, which involves the use of T cells or NK cells carrying CARs targeted against tumor antigens. This consists of the infusion of immune system cells, which are capable of generating a potent antitumor response against cells expressing the antigen targeted by the CAR (Sadelain et al., Cancer Discovery. 2013. 3(4):388-98).

[0003] Several CAR-T cell candidates are currently in clinical development, with varying success stories. The U.S. Food and Drug Administration (FDA) recently approved tisagenlecleucel (Kymriah®), the first CAR T-cell therapy indicated for the treatment of patients up to 25 years of age with refractory or second or subsequent relapsed B-cell precursor acute lymphoblastic leukemia (ALL).

[0004] The growing interest in the use of LVV has created a strong demand for large quantities of the vector for preclinical, toxicological and clinical studies, and ultimately for the production of market-approved drug products.

[0005] The most widely used method for the production of LVV involves the transient transfection of packaging cells (such as the 293 cell line and its derivatives 293T, 293E, or 293Vec) with multiple plasmids in monolayer culture using multi-tray systems such as T-flasks or cell factories. Various processes have been described that utilize various transfection reagents, including calcium phosphate (Segura et al. 2007). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 007873 [Non-patent literature]

[0007] [Non-Patent Document 1] Naldini et al. 2006 [Non-patent document 2] Chronin et al. 2005 [Non-patent document 3] Biffi et al. 2013 [Non-patent document 4] Aiuti et al. 2013 [Non-patent document 5] Cavazzana-Calvo et al. 2010 [Non-patent document 6] Cartier et al. 2009 [Non-Patent Document 7] Sadelain et al., Cancer Discovery. 2013. 3(4):388-98 [Non-patent document 8] Segura et al. 2007 [Non-Patent Document 9] Valkama et al. Optimization of lentiviral vector purification for scale-up in fix bed bioreactor Gene Therapy (2018) 25, 39-46 [Non-Patent Document 10] Mc Carron et al. Transient Lentiviral Vector Production Using a Packed Bed Bioreactor System (2019) Human Gene Therapy 30 (3) 93-101 [Non-Patent Document 11] Leinonen et al. Preclinical Proof of Concept, Analytical Development and Commercial Scale production of lentiviral vector in adherent cells (2019) Molecular Therapy 15, 63-71 Summary of the Invention

[0008] The present invention focuses on the development of a process for the large-scale production of LVV, which involves transient multiple plasmid transfection of cells in a packed-bed bioreactor. The inventors have found the conditions for carrying out a production process characterized by high productivity and quality of LVV, as well as reproducibility on both small and large scales, and an effective reduction in costs, taking into account the total process duration and, most importantly, material consumption.

[0009] The prior art discloses LVV production processes based on transient transfection of packaging cells carried out in packed bed bioreactors, but there is no suggestion of the essential technical features or process implementation according to the present invention.

[0010] For example, in Valkama et al. Optimization of lentiviral vector purification for scale-up in fixed bed bioreactor Gene Therapy (2018) 25, 39-46, the authors disclose a process for the production of LVVs by transient transfection of 293T cells in a packed bed bioreactor system, iCellis Nano. 2 Six runs of high compression and 2.67 m of surface area of ​​the iCellis Nano 2 The authors report the performance of four runs on the iCellis Nano low compaction, which has a surface area of ​​1000 nm. The amount of DNA used in transfection is reported to be one of the critical parameters for the yield of the process. Using PEI as the transfection reagent and a DNA:PEI ratio equal to 1:1, the authors report a transfection of 300-400 ng / cm. 2 We recommend using a total amount of DNA in the range of 100 ng / cm. Transfection efficiency is expected to be around 100 ng / cm. 2 It has been clearly documented that the use of DNA reduces the LVV production to only 5%. The entire run, except for 4 or 6 hours of transfection, is performed with perfusion of fresh medium into the bioreactor, i.e., during post-transfection, cell culture, and harvest. Perfusion harvest of recombinant LVV particles begins 24 hours after transfection and continues for the following 48 hours (i.e., the transfection and harvest steps require a total of 4 days). The definition of a harvest window between 24 and 72 hours after transfection stems from the observation that LVV productivity is low 6 hours after transfection. Perfusion is performed at a fixed or variable rate, and in some runs, a complete medium exchange is performed by perfusion 1 or 2 days after transfection.

[0011] WO 2018 / 007873 states that the 2 ~400ng / cm 2The optimized DNA amount for transfection was 100 ng / cm 2 This paper discloses a process substantially similar to that described in Valkama et al. (2018), including the finding that the DNA concentration is reduced to only 5% by using 100% DNA. WO 2018 / 007873 also discloses that to achieve optimal DNA concentration in the transfection mix (i.e., the mix obtained by combining DNA and PEI), a large volume of transfection mix is ​​required, which would require a complete exchange of the medium in the bioreactor. The inventors note that at large scales, a complete exchange of the medium (i.e., the discharge of almost the entire volume of cell culture medium present in the bioreactor) is not a practical process step because it takes time and can affect cell viability due to the fact that the agitation is closed during discharge and the cells in the upper carrier are not with the medium. To overcome this limitation, the transfection mix is ​​added to the bioreactor by recirculation.

[0012] In Mc Carron et al. Transient Lentiviral Vector Production Using a Packed Bed Bioreactor System (2019) Human Gene Therapy 30 (3) 93-101, the authors report that 18m 2We disclose a method for the production of LVV in a single-use packed-bed bioreactor, Bio-BLU (Eppendorf), with a surface area of ​​100 μm and a working volume of 3 L (i.e., a limited amount of medium is manipulated to drain and fill the bioreactor without requiring a long time). LVV production is performed by transient transfection of 293T cells with 4.79 mg of total DNA (DNA:PEI ratio 1:3) along with PEI transfection reagent. The entire production experiment is performed in batch mode (including cell culture, cell transfection, and harvesting). After transfection, the cell culture medium is removed from the system, and 3 L of fresh medium is added to the bioreactor. Harvesting of the cell culture medium containing recombinant LVV particles is performed by collecting the medium 48 and 72 hours after transfection (i.e., the transfection and harvesting steps require a total of 4 days). Sampling performed at 24, 48 and 72 hours post-transfection during this process showed that the highest productivity was obtained at 72 hours post-transfection, and the authors suggest evaluating productivity also at 72 hours post-transfection.

[0013] In Leinonen et al. Preclinical Proof of Concept, Analytical Development and Commercial Scale production of lentiviral vectors in adherent cells (2019) Molecular Therapy 15, 63-71, the authors reported that 2.67m 2 in the iCellis Nano bioreactor with a surface area of ​​100 m 2 Or 333m 2 We disclose a method for the production of LVV by transient transfection of 293T cells using an iCellis500 bioreactor with a surface of 300 ng / cm. 2 or 200 ng / m 2The transfection mix is ​​added to the bioreactor and incubated for 6 hours in the absence of perfusion. All runs are performed using perfusion or recirculation modes to supply fresh medium to the cells during all phases of the process except transfection. Perfusion (or recirculation) is applied during cell culture, 6 hours after transfection, and during LVV harvest. LVV harvest begins 24 hours after transfection and continues until 72 hours after transfection. Before the start of LVV harvest, i.e., 24 hours after transfection, a complete medium change is performed by perfusion using the same medium used after transfection until complete medium change. This step is reported to reduce productivity, but together with the addition of endonucleases into the bioreactor already during the production phase, it results in a positive impact on the purity of the final product with respect to DNA contamination in the final bulk containing LVV. When applied on a large scale, 333m 2 In a packed bed bioreactor with a surface area of ​​333 m, the process results in good physical titers, even higher than expected, and lower infectious virus titers than expected. 2 The total viral transfection units (TU) obtained using the process applied in a bioreactor with a surface area of ​​100 ml was obtained using a transfection-based process with a relatively high total DNA amount and without post-transfection medium exchange. 2 surface area (3.3-fold smaller bed size). Furthermore, the authors report that a single endonuclease treatment at the end of the run is not sufficient to provide DNA clearance. For this reason, the authors introduce one endonuclease treatment during the production phase in the bioreactor and a second treatment after harvest into the process to reduce DNA contamination.

[0014] To meet the growing demand for this type of gene delivery vehicle for the generation of genetically modified cell therapy products, there is a need to develop a highly reproducible manufacturing method that allows for obtaining LVVs in large quantities and with high quality. The present invention addresses this need.

[0015] Summary of the Invention The present invention relates to the development of a method for producing LVV vectors in a packed-bed bioreactor by transient transfection of multiple plasmid DNA. In particular, the inventors focused on the parameters and technical conditions for carrying out the process that resulted in a relatively low consumption of important reagents (such as DNA used in transfection) with respect to improved productivity (physical titer, infectious virus titer, and infectivity), final product quality (low DNA and host cell protein contamination), and final product quantity and functionality. Furthermore, the inventors found that by applying the claimed process, it was possible to reduce the time required for transfection and harvesting of LVV to a total of up to three days, compared with four days in the prior art processes. In particular, while the prior art discloses processes that are entirely run in batch or perfusion mode, the present invention discloses and claims a process in which both technical modes are applied and combined to obtain an effective process.

[0016] According to the present invention, the method comprises the following steps: (i) Inoculating cells (ii) culturing the cells for proliferation for at least 1 day; (iii) transfecting the cells by adding a transfection mix containing DNA transfecting particles obtained by mixing multiple plasmid DNAs and PEI to the bioreactor and incubating the cells with the DNA transfecting particles in the absence of perfusion or recirculation of fresh medium. (iv) exchanging the medium in a batch manner by draining the cell culture medium containing the DNA transfecting particles from the bioreactor and adding fresh medium to the cell culture. (v) immediately after the addition of fresh medium at time point iv, initiating the harvesting of the recombinant lentiviral vector by withdrawing the cell culture medium from the bioreactor in a perfusion manner. A method is provided for the production of lentiviral vectors in a packed-bed bioreactor by multiple plasmid DNA transient transfection, comprising:

[0017] Furthermore, in accordance with a further aspect of the present invention, the present inventors have identified an ideal range of total DNA to be used for multiple plasmid transient transfection in a process for producing LVVs carried out in a packed-bed bioreactor. The use of DNA in such a range makes it possible to obtain LVVs with good quality and productivity that is comparable to or even higher than that observed using processes disclosed in the prior art that use larger amounts of total DNA in transfection, with a subsequent significant reduction in costs.

[0018] Therefore, according to the present invention, the surface area of ​​the packed bed is 45 ng / cm 2 ~185ng / cm 2 The present invention provides a method for the production of lentiviral vectors in a packed bed bioreactor, comprising transiently transfecting cells with multiple plasmid DNAs and a PEI transfection reagent in a total amount of

[0019] Embodiment According to the present invention, in a first aspect, a method for producing a cellulose acetate solution comprising the steps of: (i) Inoculating cells (ii) culturing the cells for proliferation for at least 1 day; (iii) transfecting the cells by adding a transfection mix containing DNA transfecting particles obtained by mixing multiple plasmid DNAs and PEI to the bioreactor and incubating the cells with the DNA transfecting particles in the absence of perfusion or recirculation of fresh medium. (iv) exchanging the medium in a batch manner by draining the cell culture medium containing the DNA transfecting particles from the bioreactor and adding fresh medium to the cell culture. (v) immediately after the addition of fresh medium at time point iv, initiating the harvesting of the recombinant lentiviral vector by withdrawing the cell culture medium from the bioreactor in a perfusion manner. A method is provided for the production of lentiviral vectors in a packed-bed bioreactor by multiple plasmid DNA transient transfection, comprising:

[0020] In one embodiment, the steps of transfection (iii), post-transfection medium change (iv) and harvest by perfusion (v) are performed over a period of 3 days.

[0021] In another embodiment, the method further comprises a complete exchange of the medium between step ii and step iii.

[0022] In a further embodiment, the cells in step ii are cultured for growth in a perfusion mode. Preferably, the perfusion mode of culture is at a rate of at least 0.1 mL / cm of packed bed surface area. 2 This is done by feeding fresh medium into the bioreactor by the end of the growth phase in a total amount of 100 ml.

[0023] In a preferred embodiment, the cells in step ii are cultured for growth in a recirculation mode. More preferably, the recirculation mode culture is at a rate of at least 0.1 mL / cm of packed bed surface area. 2 The total volume is 100 ml, using fresh medium as a reservoir.

[0024] In a preferred configuration of the method of the present invention, the cells in step iii are incubated with the DNA transfecting particles for up to 18 hours, more preferably for 6 to 18 hours, and in a more preferred embodiment for 8 hours.

[0025] Preferably, in one embodiment, the cells in step iii are at a density of 45 ng / cm of packed bed surface area. 2 ~185ng / cm 2 The cells are transfected with multiple plasmid DNAs in a total amount of 1000 kJ / ml.

[0026] In another embodiment, the cells in step iii are present at a density of 45 ng / cm of packed bed surface area. 2 ~150ng / cm 2 The cells are transfected with multiple plasmid DNAs in a total amount of 1000 kJ / ml.

[0027] In a further embodiment, the cells in step iii are at a concentration of 45 ng / cm of packed bed surface area. 2 ~100ng / cm 2 The cells are transfected with multiple plasmid DNAs in a total amount of 1000 kJ / ml.

[0028] In a more preferred embodiment, the transfection mix in step iii is prepared with a ratio between the amount of DNA and the amount of PEI corresponding to 1:1.

[0029] In a preferred configuration of the present invention, the harvesting of the recombinant lentiviral vector in step v is performed by applying perfusion for at least 39 hours.

[0030] In another aspect of the present invention, the harvesting of the recombinant lentiviral vector in step v is performed by applying perfusion for 48 hours.

[0031] In one embodiment, harvesting the recombinant lentiviral vector in step v is performed by applying perfusion with the same medium used in step v.

[0032] In another embodiment, the collection of recombinant lentiviral vectors in step v is performed by applying perfusion with cell culture medium containing fetal bovine serum (FBS) in an amount of 2.5% to 10% FBS.

[0033] In a further embodiment, the harvesting of the recombinant lentiviral vector in step v is performed by applying perfusion with cell culture medium without FBS.

[0034] In another embodiment of the present invention, the packed bed surface area is 45 ng / cm 2 ~185ng / cm 2 The present invention provides a method for the production of lentiviral vectors in a packed bed bioreactor, comprising transiently transfecting cells with multiple plasmid DNAs and a PEI transfection reagent in a total amount of

[0035] Preferably, the present invention comprises the following steps: (a) Inoculating cells (b) culturing the cells for proliferation for at least one day; (c) 45 ng / cm for the surface area of ​​the packed bed 2 ~185ng / cm 2 Transfecting the cells with multiple plasmid DNAs and PEI transfection reagent in a total amount of (d) recovering the cell culture supernatant containing the recombinant lentiviral vector. The present invention provides a method for the production of lentiviral vectors in a packed-bed bioreactor by multiple plasmid DNA transient transfection, comprising:

[0036] In another embodiment, transfection is performed at a dose of 45 ng / cm of packed bed surface area. 2 ~150ng / cm 2 This is performed using multiple plasmid DNAs in a total amount of 1000 kJ / min.

[0037] In a further embodiment, transfection is at a rate of 45 ng / cm of packed bed surface area. 2 ~100ng / cm 2 This is performed using multiple plasmid DNAs in a total amount of 1000 kJ / min.

[0038] In a more preferred embodiment, the transfection is carried out with a ratio between the amount of DNA and the amount of PEI corresponding to 1:1.

[0039] In another embodiment, the method further comprises a complete medium exchange between culturing for expansion in step b and transfection in step c.

[0040] In a further embodiment, the cells are cultured for expansion in step b above in a perfusion mode. Preferably, the perfusion mode of culture is at a rate of at least 0.1 mL / cm of packed bed surface area. 2 This is done by feeding fresh medium into the bioreactor by the end of the growth phase in a total amount of 100 ml.

[0041] In a preferred embodiment, the cells are cultured for growth in step b above in a recirculation mode. Preferably, the recirculation mode of culture is at least 0.1 mL / cm of packed bed surface area. 2 The total volume is 100 ml, using fresh medium as a reservoir.

[0042] In a preferred embodiment of the method of the present invention, the cells are transfected in step c above for up to 18 hours, more preferably for 6 to 18 hours, and in a most preferred embodiment, the cells are transfected for 8 hours.

[0043] In one embodiment, the method further comprises exchanging culture medium between transfection in step c and the harvesting step in step d above, wherein such exchange of culture medium can be performed in a perfusion or batch manner, more preferably in a batch manner.

[0044] In another embodiment, the harvesting step in step d above is carried out in a batch or perfusion mode.

[0045] In one aspect of the present invention, there is provided a method wherein the harvesting step in step d above is performed in a batch mode by collecting cell culture medium from the bioreactor at 24 hours and 48 hours after the post-transfection medium change.

[0046] In another aspect of the present invention, there is provided a method wherein the recovery step in step d above is carried out in a perfusion mode applied for at least 39 hours after the post-transfection medium change.

[0047] In another aspect, the method is provided wherein the recovery step in step d above is performed in a perfusion mode applied for 48 hours after the post-transfection medium change.

[0048] In a more preferred configuration of the present invention, a method is provided in which the harvesting step in step d is performed in a perfusion mode, and the steps from transfection (step c) to the harvesting step (step d) are performed in 3 days. [Brief explanation of the drawings]

[0049] [Figure 1] FIG. 1 shows a flow chart depicting the process performed in the experiment disclosed in Example 1. [Figure 2] FIG. 1 shows a flow chart depicting the process performed in the experiment disclosed in Example 2. (*) Recirculation was applied on days 1-3 for run PDE_B18172 and on days 0-3 for run PDE_B18187; (**) Condition applied only to run PDE_B18172. (***) Condition applied to run PDE_B19013 (medium change 6 hours post-transfection), except for bio2 in run PDE_B19087. DETAILED DESCRIPTION OF THE INVENTION

[0050] Detailed Description of the Invention Various preferred features and embodiments of the present invention will now be described by way of non-limiting exemplary embodiments.

[0051] The present invention provides a method for producing lentiviral vectors (LVVs) in a packed-bed bioreactor by transient transfection of multiple plasmid DNAs. The method of the present invention is particularly focused on the production of LVVs in large quantities and with high quality on a large scale using automated devices. The inventors have found optimal conditions for obtaining a bulk LVV product with high physical and infectious viral titers and an optimal impurity profile with high reproducibility.

[0052] According to the present invention, the method comprises the following steps: (i) Inoculating cells (ii) culturing the cells for proliferation for at least 1 day; (iii) transfecting the cells by adding a transfection mix containing DNA transfecting particles obtained by mixing multiple plasmid DNAs and PEI to the bioreactor and incubating the cells with the DNA transfecting particles in the absence of perfusion or recirculation of fresh medium. (iv) exchanging the medium in a batch manner by draining the cell culture medium containing the DNA transfecting particles from the bioreactor and adding fresh medium to the cell culture. (vi) immediately after the addition of fresh medium at time point iv, initiating the harvesting of the recombinant lentiviral vector by withdrawing the cell culture medium from the bioreactor in a perfusion manner; A method is provided for the production of lentiviral vectors in a packed-bed bioreactor by multiple plasmid DNA transient transfection, comprising:

[0053] Lentiviral vector (LVV) The present invention relates to the production of LVV, a gene delivery vehicle derived from lentivirus. A detailed list of lentiviruses can be found in Coffin et al. ("Retroviruses," 1997 Cold Spring Harbour Laboratory Press, Eds: JM Coffin, SM Hughes, HE Varmus, pp. 758-763). Briefly, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototypical "slow virus," visna-maedi virus (VMV), as well as the closely related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0054] In a preferred embodiment, the lentiviral vector is derived from an HIV lentivirus, more preferably, the lentiviral vector is derived from an HIV-1 lentivirus.

[0055] An exemplary lentiviral vector suitable for use in the methods of the present invention comprises at least the following portions of the lentiviral genome: (a) a 5' long terminal repeat (LTR); (b) a packaging sequence (psi); (c) a Rev response element (RRE); (d) a promoter operably linked to a gene of interest; and (e) a 3' long terminal repeat (LTR). In a preferred embodiment, the U3 region of the 5' LTR is replaced with a heterologous promoter selected from the group consisting of the cytomegalovirus (CMV) promoter, the Rous sarcoma virus (RSV) promoter, or the Simian Virus 40 (SV40) promoter; thus, lentiviral transcription is Tat-independent. In a further preferred embodiment, the 3' LTR sequence contains a deletion of the U3 region (i.e., the vector is a self-inactivating vector or SIN vector). The lentiviral vector may further comprise a lentiviral central polypurine tract (cPPT) and a woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE).

[0056] Packed-bed bioreactor The process according to the present invention uses a packed-bed bioreactor for the production of LVV. A packed-bed bioreactor is a device consisting of a vessel containing, saturated with, or filled with a carrier material used as a support for cell immobilization. The carrier is composed of organic and inorganic materials on which the cells are entrapped, thereby allowing them to grow adherently to very high densities. A culture medium conditioned with an appropriate amount of O and the required pH conditions is circulated through the fixed bed to supply nutrients and O and allow cell growth. Various packed-bed bioreactors are available that operate in essentially two possible configurations: (1) medium circulates from the conditioning vessel to the fixed bed and back (e.g., CellCube system, Corning); or (2) the fixed bed is integrated into the bioreactor, and the medium conditioned in the bioreactor circulates through the bed (e.g., iCellis, Pall). In a preferred embodiment, the process according to the invention can be carried out in an iCELLis® single-use fixed-bed bioreactor system, as shown in the Examples. The iCELLis bioreactor system is an automated, single-use fixed-bed bioreactor for adherent cell growth in a controlled environment. The bioreactor comes in two formats: the iCELLis Nano (0.53 m), which is used for development studies, and the iCELLis 1000 (0.53 m) which has a variety of possible surface areas for cell growth. 2 ~4m 2 (unit of cultivation area) and 66 to 500 m 2 The iCellis 500 is available for large-scale production with a culture area unit of 1000 kJ / L. Cells grow in polyester macrocarriers housed in a fixed bed, while the cell culture medium flows through the fixed bed from bottom to top. At the top, the medium falls as a thin film down the outer wall, where it picks up O2 and returns to the reservoir. As used herein, the term "culture area" or "surface area" refers to the total surface of the packed bed of the bioreactor on which cells adhere and are cultured.

[0057] Multiple Plasmid Transient Transfection According to the present invention, LVV is produced in a packed-bed bioreactor by transient transfection of cells with multiple plasmid DNA; i.e., in the method of the present invention, at least two expression plasmids carrying two different genes are transiently co-transfected into a cell line (a so-called packaging cell line). According to a preferred embodiment of the present invention, cells are co-transfected with at least: a packaging plasmid carrying the lentiviral Gag / Pol genes, a plasmid carrying a gene encoding the envelope protein of interest, and a transfer plasmid carrying the essential lentiviral genomic elements and the gene of interest as disclosed above. In another preferred embodiment, the lentiviral regulatory protein Rev can be additionally expressed in trans on a fourth separate plasmid. In a further embodiment of the present invention, the lentiviral protein Tat can be additionally expressed on a fifth separate plasmid. Examples of suitable env genes include, but are not limited to, VSV-G env, MLV 4070 env, RD114 env, RD114-TR, RD114pro, baculovirus 5 GP64 env, GALV, or envelope proteins derived from measles virus.

[0058] In a preferred embodiment, in the method of the present invention, multi-plasmid DNA transfection is performed using a third-generation LVV viral vector system, which is composed of four plasmids: one plasmid carrying the lentiviral gag / pol genes, one plasmid carrying the lentiviral rev gene, one plasmid carrying a gene encoding an envelope protein, preferably a gene encoding the VSV-G envelope protein, and one plasmid carrying a transfer vector containing the required portion of the lentiviral genome and the exogenous gene of interest.

[0059] Inoculation of cells into bioreactor The production method according to the present invention involves the inoculation of cells, i.e. the introduction of cells into a bioreactor.

[0060] Cells were grown at 1000 cells / cm of bioreactor culture area. 2 ~10000 cells / cm 2 In another embodiment, the cells can be inoculated into the bioreactor at a rate of 2000 cells / cm. 2 ~8000 cells / cm 2 , preferably 4000 cells / cm 2 ~6000 cells / cm 2 and in a more preferred embodiment, 5000 cells / cm 2 are inoculated into a bioreactor. To obtain an appropriate amount, the thawed cells can first be expanded in cell culture outside the bioreactor. In one embodiment, the cells are thawed, expanded to an appropriate amount through adherent cell culture, and then detached from the support and inoculated into a bioreactor in an appropriate amount.

[0061] The prior art discloses the possibility of using suspension-adapted cell lines in packed-bed bioreactors as a result of the use of factors that promote cell adhesion during cell culture in the bioreactor (WO 2016 / 048556). Thus, in another embodiment according to the present invention, a suspension-adapted cell line is thawed, grown to a suitable quantity through cell culture runs in suspension, and subsequently inoculated into a bioreactor in the presence of factors that promote cell adhesion.

[0062] Any cell line known to function as a packaging cell line for the production of viral vectors can be used in the present invention. In one embodiment, the packaging cell line of interest for use in the present invention is selected from HEK293, HEK293T, HEK293E, HEK293FT, 293Vec, TE671, HT1080, or HeLa cell lines. In a preferred embodiment, the packaging cell line is HEK293T.

[0063] Cell proliferation According to the method of the present invention, in addition to inoculation, the cells are cultured for growth in a bioreactor for at least one day.

[0064] Those skilled in the art can use any of the cell culture operating conditions known in the art and applicable to the packed-bed bioreactor for use in this step to supply nutrient-containing cell culture medium to the cells. For example, the iCELLis® single-use fixed-bed bioreactor system allows cell culture to be operated in three different modes: (1) according to the "batch mode," cells are cultured with a fixed volume of cell culture medium in the bioreactor for a required period until medium consumption (i.e., consumption of nutrients necessary for cell growth and accumulation of cellular metabolic products), and when medium consumption is reached, it is necessary to completely drain the spent medium from the bioreactor and refill it with fresh medium; (2) according to the "perfusion mode," fresh medium is supplied to the bioreactor at a constant flow rate during cell culture. (3) In the "recirculation mode," cells are cultured in a constant volume of cell culture medium in a bioreactor, and the bioreactor is connected to an external vessel that serves as a medium reservoir, from which additional culture medium is added to the bioreactor at a constant flow rate (similar to the perfusion mode) and removed at the same flow rate and transferred back to the reservoir. Cultivation in the recirculation mode allows for an increase in the total volume of medium used for cell culture. Each of the above operating modes, or a combination thereof, can be utilized in the methods of the present invention to culture cells for expansion.

[0065] In one embodiment, a method according to the present invention is provided in which cells are cultured for expansion for at least one day by operating a bioreactor in a batch mode. More preferably, when cell culture for expansion is performed in a batch mode, a complete medium exchange is performed at the end of the expansion phase and before transduction by draining the total volume of spent cell culture medium from the bioreactor and then filling the bioreactor with fresh medium. Alternatively, the post-culture medium exchange can be performed in a perfusion mode by supplying a fixed amount of fresh medium corresponding to twice the volume of the cell culture medium present in the bioreactor. In another embodiment, when cell culture for expansion is performed in a batch mode, a post-culture medium exchange can be performed in a perfusion mode at the end of the expansion phase and before transduction by supplying a fixed amount of fresh medium corresponding to the same volume of cell culture medium present in the bioreactor.

[0066] In another embodiment, there is provided a method according to the invention, wherein cells are cultured for growth for at least 1 day by operating the bioreactor in perfusion mode. In a preferred configuration of the method of the invention, cell culture in perfusion mode is performed at a rate of at least 0.1 mL / cm of packed bed surface area. 2 This is achieved by feeding a total amount of fresh medium of 100 ml by the end of the growth phase.

[0067] In a preferred embodiment, a method according to the invention is provided in which cells are cultured for growth for at least one day by operating the bioreactor in a recirculation mode. In a preferred configuration of the method of the invention, cell culture in a recirculation mode is performed at a rate of at least 0.1 mL / cm per packed bed. 2 A total of 100 ml of additional medium is used as a reservoir.

[0068] The goal of the cell expansion phase is to achieve an optimal cell concentration on the day of transfection. In one embodiment, 0.9 x 10 cells per bioreactor surface area are used. 5 cells / cm 2 ~2×10 5 cells / cm 2The cells are grown until a concentration of 0.7 g / L to 1 g / L is obtained. Cell counting in a packed-bed bioreactor system is not always practical or possible, therefore, after inoculation, cell growth can be monitored by measuring appropriate metabolic parameters such as lactate accumulation or glucose consumption. In a preferred embodiment, cells are cultured for growth until a lactate accumulation of 0.7 g / L to 1 g / L is obtained in the bioreactor.

[0069] In a further preferred embodiment, cells are inoculated into the bioreactor on day 0 and subsequently cultured for up to day 4, or most preferably for up to day 3.

[0070] In one embodiment, cells are inoculated into a bioreactor on day 0 and subsequently cultured by running the cell culture in a batch mode. In a preferred embodiment, cells are inoculated into a bioreactor on day 0 and subsequently cultured in a batch mode until day 4, or most preferably until day 3. Preferably, at the end of the culture for expansion in a batch mode, the exhausted cell culture medium is replaced before transduction. In one embodiment, the exhausted cell culture medium is replaced by withdrawing it from the bioreactor and adding fresh medium. In another embodiment, the exhausted cell culture medium is replaced in a perfusion mode by supplying, at a fixed rate, an amount of fresh medium corresponding to at least the same volume of cell culture medium present in the bioreactor.

[0071] In another embodiment, cells are inoculated into the bioreactor on day 0 and subsequently cultured in perfusion mode until day 4, or more preferably until day 3. Perfusion can be initiated at least 4 hours after cell inoculation.

[0072] In a preferred embodiment, cells are inoculated into the bioreactor on day 0 and subsequently cultured in a recirculation mode until day 4, or more preferably until day 3. Perfusion can be initiated at least 4 hours after cell inoculation.

[0073] Numerous basal culture media are known in the art that may be suitable for the methods of the present invention, including, but not limited to, EMEM (Eagle's Minimum Essential Medium), BEM (Eagle's Basal Medium), DMEM (Dulbecco's Modified Eagle's Medium), Glasgow's Minimum Essential Medium, M199 Basal Medium, HAM's F-10, HAM's F-12, Iscove's DMEM, RPMI, Leibovitz's L15, MCDB, RPMI-1640, IMDM, and X-Vivo10. TM , X-Vivo15 TM and X-Vivo20 TM Preferably, the cell culture medium can be supplemented with other factors known to promote cell growth, such as glutamine or glutamax, fetal bovine serum (FBS), or alternatives that can be readily determined by one of skill in the art.

[0074] Transfection step In the methods of the present invention, cells are transfected with two or more plasmids adapted for the production of lentiviral vectors (ie, multi-plasmid DNA transfection).

[0075] Various techniques known in the art can be used to introduce nucleic acid molecules into cells. Such techniques include chemically assisted transfection using compounds such as calcium phosphate, cationic lipids, cationic polymers, and liposome-mediated transfection. However, according to a preferred embodiment of the present invention, multiple plasmid DNA transient transfection is performed using polyethyleneimine (PEI) as a transfection reagent.

[0076] The production method according to the present invention comprises a transfection step in which transfection is initiated by adding a transfection mix to a bioreactor.

[0077] As used herein, the term transfection mix refers to a solution containing DNA transfecting particles formed as a result of mixing a transfection agent with DNA.

[0078] Preferably, the preparation of the transfection mix comprises the following steps: (a) preparing a solution containing DNA (solution a) (b) preparing a solution containing PEI (solution b) (c) Mixing the two solutions a and b and further incubating them to obtain a transfection mix containing DNA transfecting particles.

[0079] In a preferred embodiment, transfection is at a rate of 45 ng / cm of packed bed surface area. 2 ~185ng / cm 2 This is performed using a total amount of multiple plasmid DNA.

[0080] In another embodiment, transfection is performed at a dose of 45 ng / cm of packed bed surface area. 2 ~150ng / cm 2 This is performed using a total amount of multiple plasmid DNA.

[0081] In a further embodiment, transfection is at a rate of 45 ng / cm of packed bed surface area. 2 ~100ng / cm 2 This is performed using a total amount of multiple plasmid DNA.

[0082] One skilled in the art can use any of the various DNA:PEI ratios disclosed in transfection protocols known in the art, including manufacturer-recommended conditions. In the methods of the present invention, according to a preferred embodiment, transfection is carried out using a ratio of DNA amounts to PEI equal to 1:1.

[0083] According to the method of the present invention, transfection is carried out by adding a transfection mix containing DNA transfecting particles to a bioreactor and incubating the cells in the bioreactor with the DNA transfecting particles in the absence of perfusion or recirculation of fresh medium.

[0084] In one embodiment, the transfection mix is ​​added to the cell culture medium of the bioreactor, after removal, if necessary, of a volume of cell culture medium proportional to or equal to the volume of the transfection mix.

[0085] In the method of the present invention, during transfection, incubation of cells with DNA transfecting particles is performed in the absence of perfusion or recirculation of fresh medium, thereby maintaining a constant amount of DNA transfecting particles in the bioreactor. Agitation is maintained to allow for O2 supply and DNA particle circulation.

[0086] As shown in the Examples, the incubation time with DNA transfecting particles can affect the productivity and quality of the final vector preparation. Examples 2.3 and 2.4 show that the LVV obtained after 21 hours of incubation with DNA transfecting particles has reduced infectivity. Therefore, according to a preferred embodiment of the present invention, cells are incubated with DNA transfecting particles for up to 18 hours during transfection. In addition, as shown in Example 1.3, a decrease in productivity can be observed by reducing the incubation time with DNA transfecting particles to 6 hours. Although productivity under these conditions is still acceptable, further reductions in the incubation time are expected to be even less effective. Therefore, in a preferred embodiment, cells are incubated with DNA transfecting particles for 6 to 18 hours, more preferably for 8 hours.

[0087] Optimal total DNA amount for transfection As explained above, in the method of the present invention, cells are transfected with two or more plasmids encoding the required components necessary to generate a vector (multiple-plasmid DNA transfection). Various protocols disclosing different ratios of each target plasmid to be used for transfection are known in the art. Depending on the plasmid system used (e.g., the number of packaging plasmids used, the nature and length of the transfer vector plasmid), those skilled in the art can optimize the optimal ratio between the different plasmids required for LVV production. Once defined, for example, in a flask or well, such ratio should be applied to the total amount of target DNA to be used in the bioreactor system.

[0088] The present inventors have identified an ideal range of total multiple plasmid DNA to be used in the method of the present invention to ensure high productivity and reproducibility of the process.

[0089] In one embodiment, transfection is performed at a dose of 45 ng / cm of packed bed surface area. 2 ~185ng / cm 2 This is done using multiple plasmid DNAs with a total amount of DNA.

[0090] The inventors have surprisingly found that it was possible to obtain bulk products with comparable or even higher productivity using DNA in amounts ranging from 0.1 to 100% lower than those disclosed in the prior art.

[0091] Prior art (Valkama et al. 2018 and Leinonen et al. 2019) used 200ng / cm with PEI as the transfection reagent. 2 or 300ng / cm 2 or 400ng / cm 2(DNA:PEI ratio equal to 1:1). Furthermore, Valkama et al. 2018 and WO 2018 / 007873 disclose the use of 100 ng / cm of total DNA in transfection. 2 It is clearly stated that the transfection efficiency is reduced to 5% by using DNA in an amount of 363 ng / cm. Example 1.2 of the present invention shows that the transfection efficiency is reduced to 5% by using DNA in an amount of 363 ng / cm. 2 , 181ng / cm 2 , and 91 ng / cm 2 Table 4 shows the results obtained by carrying out the manufacturing method for the production of LVV viral vectors in a packed-bed bioreactor by multiple plasmid DNA transient transfection performed with 181 ng / cm 2 and 91 ng / cm 2 The bulk product obtained using this amount of total DNA in transfection was 363 ng / cm 2 This clearly shows that the results have higher productivity (higher physical titer) compared to runs obtained with 1.06 ml of total DNA. Such surprising activity is also demonstrated by the results of runs using 1.06 ml of total DNA as shown in Example 2.3 and associated Table 23. 2 This was also observed by applying the process to iCellis nano, which has a surface area of ​​91 ng / cm 2 The bulk product obtained with 181 ng / cm of total DNA 2 It was found that the productivity was comparable to that obtained by transfecting cells with 200 ng / cm of total DNA. Consistent data were obtained on a larger scale as shown in Example 3. Unlike what is reported in the prior art, the surface area was increased to 200 ng / cm. 2 It was possible to achieve comparable or even higher productivity by reducing the amount of total DNA used in transfection to less than 100 ng / cm of surface area, a result even lower than that recommended by the PEI manufacturer for single plasmid transfection. 2This is particularly important considering that the process of the present invention requires the co-transfection of different plasmids in cells to obtain LVV production.

[0092] Mc Carron et al. (2019) describe a packed-bed bioreactor manufacturing process using a five-plasmid lentiviral vector system, where co-transfection is at 26.5 ng / cm. 2 The transfection was performed using 1 ng / cm of total DNA and PEI transfection reagent (DNA:PEI ratio 1:3, i.e., an amount of PEI three times higher than that used in the examples). The resulting bulk product was 1 ng / cm 2 of p24 measured corresponds to 1 × 10 7 vp / cm 2 It has been noted that the yields are low, i.e., titers up to about 70 times lower than those observed using DNA in the range according to the present invention.

[0093] Therefore, according to the present invention, the surface area of ​​the packed bed is 45 ng / cm 2 ~185ng / cm 2

[0010] A method is provided for the production of lentiviral vectors in a packed bed bioreactor, comprising transiently transfecting cells with multiple plasmid DNAs and a PEI transfection reagent in a total amount of 1000 kJ / ml.

[0094] In a more preferred embodiment of the present invention, the method comprises the steps of: (a) Inoculating cells (b) culturing the cells for proliferation for at least one day; (c) 45 ng / cm for the surface area of ​​the packed bed 2 ~185ng / cm 2 Transfecting the cells with multiple plasmid DNAs and PEI transfection reagent in a total amount of (d) recovering the cell culture supernatant containing the recombinant lentiviral vector. A method is provided for the production of lentiviral vectors in a packed bed bioreactor by multi-plasmid transient DNA transfection, comprising:

[0095] In another embodiment, transfection is performed at a dose of 45 ng / cm of packed bed surface area. 2 ~150ng / cm 2 The procedure is carried out using multiple plasmid DNA in a total amount of 1000 kJ / min.

[0096] In a further embodiment, transfection is at a rate of 45 ng / cm of packed bed surface area. 2 ~100ng / cm 2 The procedure is carried out using multiple plasmid DNA in a total amount of 1000 kJ / min.

[0097] In a preferred embodiment, transfection is performed using a DNA:PEI ratio equal to 1:1.

[0098] In a preferred embodiment, the multiple plasmid DNA is composed of one plasmid carrying the lentiviral gag / pol genes, one plasmid carrying the lentiviral rev gene, one plasmid carrying a gene encoding an envelope protein, preferably a gene encoding the VSV-G envelope protein, and one plasmid carrying a transfer vector containing the required portion of the lentiviral genome and the exogenous gene of interest as disclosed above.

[0099] Post-transfection medium change As disclosed above, it is necessary to monitor and measure the transfection time, i.e., the time during which cells are incubated with transfecting particles, and to maintain a constant amount of transfectable DNA particles in the bioreactor during the incubation period. The manufacturing method according to the present invention includes exchanging the medium at the end of transfection (i.e., after transfection). Such a step is effective for removing DNA transfecting particles at the end of the incubation period, which may negatively affect the productivity of the process and the quality of the bulk product in terms of contaminants present. Medium exchange in a packed-bed bioreactor can be performed in a perfusion mode by supplying a sufficient amount of fresh medium at a fixed rate. For example, it is possible to supply a fixed amount of medium corresponding to the same or twice the total volume of medium used in the bioreactor during transfection. Alternatively, post-transfection medium exchange can be performed in a batch mode, i.e., by discharging the cell culture medium containing the DNA transfecting particles from the bioreactor and adding fresh medium to the cells. The post-transfection medium change step allows the cessation of transfection as a result of the removal of the DNA transfecting particles, and therefore allows the control of the transfection time, i.e., the time the cells are incubated with the transfecting particles, which, as mentioned above, can also affect the infectious viral titer of the bulk product.

[0100] In a preferred configuration, the method of the present invention includes batch-wise medium exchange by draining the cell culture medium containing the DNA transfecting particles from the bioreactor and adding fresh medium to the cells. In one embodiment, up to 98% of the cell culture medium containing the DNA transfecting particles is removed from the bioreactor. Alternatively, up to about 95%, or up to about 90%, or up to about 85%, or up to about 80%, or up to about 75%, or up to about 70% of the cell culture medium containing the DNA transfecting particles can be removed from the bioreactor.

[0101] Prior art discloses complete post-transfection medium exchange in a perfusion mode. Valkama et al. (2018) discloses a method in which perfusion is applied to a packed-bed bioreactor 4 or 6 hours after transfection, followed by a complete perfusion medium exchange 1 or 2 days after transfection during LVV collection by perfusion. Leinonen et al. (2019) discloses a medium exchange performed by applying perfusion after transfection until a complete medium exchange is performed 24 hours after transfection, before the start of LVV particle collection by perfusion. Mc Carron et al. (2019) discloses a batch-based post-transfection medium exchange followed by batch-based LVV collection. The prior art does not disclose a batch-based post-transfection medium exchange combined with a perfusion-based LVV collection.

[0102] As shown in the Examples, it was surprisingly found that applying perfusion to perform post-transfection medium exchange is detrimental to the productivity (physical and infectious virus titers) of the bulk product. In particular, Example 1.5 and the associated Table 13 show that LVV produced using the method according to the present invention (draining cell culture medium from the bioreactor and adding fresh medium to the cells, followed by recovery by perfusion) have higher physical titers and higher infectious virus titers than those obtained using a process in which post-transfection medium exchange is performed by applying perfusion until complete medium exchange, as disclosed in the prior art. Furthermore, as reported in Table 14, post-transfection medium exchange in a batch format results in effective reduction of contaminants (plasmid DNA, total DNA, and HCP) in the final pool without treatment with endonucleases.

[0103] This technical effect is not only surprising in itself, but also completely unexpected, considering that the complete draining and subsequent filling of a bioreactor at large scale requires a certain time (approximately 50 minutes) during which cells remain free of cell culture medium. Such a technical limitation would be expected to cause problems for production, both as a logical consideration and in light of the teachings of the prior art. For example, WO 2018 / 007873 states that "...at large scale, a complete exchange of medium is not a practical process step because it takes time and may affect cell viability due to the fact that the agitation is closed during draining and the cells in the upper carrier are free of medium."

[0104] In contrast, the data in this application show no indication of possible adverse effects on productivity and infectious virus titer caused by complete batch removal of cell culture medium at large scale. In Example 1.5, one of the production experiments performed includes a 50-minute hold time after draining the cell culture medium during post-transfection medium exchange in a batch mode to mimic large-scale drain / fill timing. The results obtained by performing the experiment including a 50-minute hold time confirm the increase in productivity and infectious virus titer compared to production experiments including post-transfection medium exchange in a perfusion mode.

[0105] Furthermore, Example 3 shows the results obtained by carrying out the manufacturing method according to the present invention on a large scale. To evaluate the efficiency of the process on a large scale, small-scale (iCellisNano) experiments were performed in parallel with iCellis500 experiments, and the data regarding physical titer and infectious virus titer were directly compared. The productivity (as physical and infectious virus titer) and final product quality (in terms of the presence of contaminants) obtained on a large scale match or even better than those obtained in the parallel small-scale experiments (e.g., as shown in terms of physical and infectious virus titer and infectivity and the presence of contaminants shown in Example 3.2).

[0106] Collection of LVV recombinant particles Identifying the harvesting procedure and time for harvesting LVV recombinant particles is one of the most important goals for developing an effective manufacturing process. According to the method of the present invention, the harvesting of recombinant LVV is carried out by starting the collection of cell culture medium (also referred to herein as cell culture supernatant) containing recombinant LVV in a perfusion manner immediately after the batch-wise post-transfection medium change, as disclosed above.

[0107] The prior art discloses methods for the production of LVVs using packed-bed bioreactors in which all steps are performed in batch (e.g., McCarron et al. 2019) or by perfusion (e.g., Valkama et al. 2018 or Leinonen et al. 2019). In the method according to the present invention, the inventors have found that a combination of batch and perfusion steps, after transfection, leads to the successful implementation of an effective and reproducible manufacturing process.

[0108] In Mc Carron et al. (2019), recombinant LVV particles were harvested in batches after batch-based post-transfection medium changes. The cell culture medium in the bioreactor was removed for LVV harvest at 48 hours post-transfection and at 72 hours post-transfection, allowing for maximum accumulation of recombinant LVV particles in the cell culture medium. Increased productivity was observed at 72 hours post-transfection, and the authors suggested further evaluation of productivity at 72 hours post-transfection. The total time required for transfection and subsequent harvest was 4 days.

[0109] In Valkama et al. 2018 or Leinonen et al. 2019, harvesting was performed by perfusion 24 hours after transfection, and in both papers there is a clear indication of very low production of LVV 6 hours after transfection. Furthermore, in Leinonen et al. 2019, the authors state that post-transfection medium exchange (performed by perfusion) causes a decrease in productivity, and for this reason certain additives are added to the medium used for harvesting.

[0110] In the method according to the invention, the performance of a batch post-transfection medium exchange and the subsequent immediate start of LVV collection by harvesting by perfusion makes it possible to avoid the loss of productivity observed in the prior art.

[0111] In one embodiment according to the methods of the present invention, perfusion harvesting is performed for 48 hours after post-transfection medium change.

[0112] In a preferred embodiment according to the method of the present invention, perfusion harvesting is carried out for at least 39 hours after post-transfection medium change.

[0113] In a more preferred embodiment, a method according to the present invention is provided, wherein the steps of transfection, post-transfection medium exchange and harvesting by perfusion have a total duration of 3 days.

[0114] In particular, Example 2.4 shows the results obtained using an optimized version of the production method according to the present invention. The example compares the results obtained by performing transfection and subsequent harvest over a three-day period (8 hours of transfection followed by batch medium change and 39 hours of harvest by perfusion) with the results obtained by performing transfection and subsequent harvest over a four-day period (21 hours of transfection followed by batch medium change and 48 hours of harvest). As shown in Table 27, infectious virus titers and infectivity are relatively high when post-transfection medium change and final harvest are expected within one day, i.e., when transfection and subsequent harvest are performed over a three-day period. Furthermore, the pooled harvest obtained by performing transfection and harvest over a total of three days results in relatively low contamination, including total DNA that is undetectable or below the limits of the analytical method, without the use of endonuclease treatment.

[0115] Key Advantages of the Invention The present invention relates to a method for producing LVV in a packed-bed bioreactor, in which the final bulk product is characterized by high productivity in terms of physical titer, infectious virus titer, and infectivity, and low amounts of contaminants. In particular, the inventors selected a combination of operating conditions for performing certain steps of the process that positively impacts the productivity of the process. Surprisingly, post-transfection medium exchange performed in batch mode results in a positive impact on productivity in terms of physical titer and infectious virus titer when directly compared with post-transfection medium exchange performed in perfusion mode. As disclosed above, prior art (WO 2018 / 007873) suggests avoiding the discharge of cell culture medium from the bioreactor, especially at large scale. In contrast, the data presented in Example 3 demonstrate the effectiveness of the process performed at large scale and the reproducibility of results obtained at small scale without the negative impact of post-transfection medium exchange performed in batch mode. Example 3.2 demonstrates the effectiveness of the process performed at large scale and the reproducibility of results obtained at small scale without the negative impact of post-transfection medium exchange performed in batch mode, on the one hand, and on the other, using an iCellis 500 (large scale, surface area 133 m). 2 ), and the other is iCellis nano (small scale, 1.06m 2By performing two parallel production experiments at large scales, we demonstrate that productivity at large scales is higher, with infectious virus titers and infectivities of the bulk product at large scales being twice as high as those obtained at small scales. Furthermore, the combination of post-transfection medium exchange and LVV collection by perfusion harvesting makes it possible to obtain a final bulk product characterized by a very good purity profile in terms of total DNA and host cell protein content at small scales as well as at large scales. As shown in Example 2.4, total DNA and host cell protein contamination in the bulk preparation is minimal or even undetectable without any treatment with endonucleases. As shown in Example 3.2, this result is confirmed at large scales. This is an even more surprising effect considering that the prior art discloses the need for an initial endonuclease treatment in the bioreactor during the production phase of the process (i.e., during collection of the LVV) and a subsequent additional endonuclease treatment of the bulk preparation to achieve acceptable results in terms of purity.

[0116] Furthermore, the inventors identify an effective range of the target total DNA amount used for transfection in the bioreactor, which allows obtaining productivity comparable or even higher than that obtained with a larger amount of total DNA in transfection. Such effect has already been observed by running the process entirely in batch mode, with further improvement by applying a more effective combination of operation methods as disclosed above. Reducing the total amount of DNA while maintaining high productivity is an important advantage in the economics of the process, since large-scale production of LVV for pharmaceutical preparations requires the use of GMP-grade raw materials, and DNA plasmids are among the most expensive materials, thus having a significant positive impact on costs. For example, the data reported in Example 3.2 show that a 91 ng / cm 2 Large-scale production experiments on the iCellis 500 were performed using multiple plasmid DNA transfections at a total dose of 60 ng / cm2 100 ng / cm2, a bulk preparation with a physical titer of 100 ng / cm2, a two-fold higher infectious virus titer, and a lower impurity profile than that obtained using the same process on a smaller scale. The data demonstrate the reproducibility and effectiveness of the process on both a small and large scale. Furthermore, the data reported in Example 4 demonstrate that the total amount of multiple plasmid DNA used in transfection was 60 ng / cm2. 2 or 45 ng / cm 2 These results show that by further reducing the concentration to 0.1% the productivity in terms of physical titer is substantially maintained and good levels of infectious virus particles are observed. [Example]

[0117] Example 1: iCELLis nano 0.53 m 2 Description of lentiviral vector generation in Example 1.1: Materials and Methods Packaging cells and cell culture medium Lentiviral vectors are produced by HEK293T cells cultured in phenol red-free IMDM, 10% FBS and 2% L-glutamine or 2% Glutamax.

[0118] bioreactor During the feasibility study, all experiments described in this Example 1 were performed on iCELLis nano with the characteristics reported in Table 1.

[0119] [Table 1]

[0120] Plasmid The multiple-plasmid DNA system used is a third-generation LVV packaging system containing four separate plasmids: one plasmid carrying the gag-pol genes (pGag / Pol), one plasmid encoding rev (pREV), one plasmid carrying the VSV-g gene (pENV-VSV-G), and one transfer vector plasmid carrying the gene encoding the GFP protein (pTransfer-GFP).

[0121] Analysis method The following analytical methods were applied to evaluate process performance: Physical viral titer: This analytical method is an ELISA for quantifying the p24 HIV protein, which can be used to assess the concentration of physical particles. Infectious virus titer: This assay is based on the transduction of the reference CEM A3.01 cell line. GFP protein expression is assessed by FACS analysis. Residual host cell proteins: This assay is an ELISA to quantify residual proteins from the HEK-293T packaging cell line in LVV samples. Residual total DNA: This analytical method is based on a fluorescent dye that stains the residual DNA and allows its quantification.

[0122] Manufacturing Process Overview A flow chart of the process applied in the experiments disclosed in Example 1 is reported in Figure 1. Briefly, for each experiment, cells were grown in flasks in IMDM without phenol red and containing 10% FBS and 2% L-glutamine (or glutamax). After splitting, cells were inoculated into the bioreactor iCELLis nano (5000 cells / cm). 2After the growth phase, during which pH, ​​DO%, glucose, and lactate concentrations were monitored, the culture medium was replaced with fresh complete medium, and the transfection step was performed on day 3 or 4. After 18 hours, a new medium change was performed to remove any remaining plasmid DNA and PEI Pro. Two harvests were collected 48 and 72 hours after the transfection step. The lentiviral vectors from each harvest were filtered at 0.45 μm and stored below −65° C. in samples for testing. From run PDE_B17054 B in the experiment shown in this Example 1, harvesting was performed in a perfusion mode, and the LV vector was collected after 48 hours of perfusion.

[0123] Transfection step In all experiments disclosed in this Example 1, LV vectors were generated using the polyethyleneimine (PEI)-mediated transfection method, which is easy to use and has a high level of reproducibility and consistency. To identify the appropriate amount of plasmid DNA to be used in the PEI transfection protocol, we selected an amount that is functional in other cell culture systems (such as flasks) and consistent with amounts disclosed in the prior art (i.e., 300 ng / cm of surface area as disclosed in Valkama et al., 2018). 2 ~400ng / cm 2 or 200–300 ng / cm as disclosed in Leinonen et al. 2019 2 A feasibility study was performed starting from 100 μg of PEI. The amount of PEI used was set to maintain a 1:1 ratio between PEI and DNA.

[0124] Transfection Protocol: One hour before transfection, perform a medium change with pre-warmed complete medium. Prepare the DNA mix by diluting the appropriate amount of plasmid DNA in IMDM-free. Dilute the appropriate amount of PEI in IMDM free of PEI. Transfer the diluted PEI into the tube containing the diluted DNA and mix. Incubate the final mix (100 mL for one bioreactor) according to the manufacturer's instructions. Transfer the mix to the bioreactor.

[0125] Example 1.2: Amount of Total DNA In this first group of experiments, 0.53 m 2 For the performance of the production experiments in an iCellis Nano bioreactor with a surface area of ​​1000 sq. m, the following conditions were applied: ·Cell seeding density 5000c / cm 2 ; Batch medium change after transfection at 18 hours; Two harvests were performed at 48 and 72 hours after transfection.

[0126] The optimal amount of DNA for transfection was determined. For each experiment, transfection was performed 3 days after inoculation. For runs PDE_M16142, PDE_M16155 Bio1, and PDE_M16177, the optimal amount of DNA was 181 ng / cm. 2 To test different ratios, transfection was performed using double the amount of DNA (2x), i.e., 363 ng / cm. 2 (which corresponds to the amount of DNA disclosed in the prior art) was used in run PDE_M16121 Bio1, and half the amount (1 / 2), i.e., 91 ng / cm 2 was used in run PDE_M16155 Bio2. The various conditions used are summarized in Table 2.

[0127] [Table 2]

[0128] Briefly, in all experiments, 5000 cells / cm 2were inoculated into iCELLis nano. Samples were taken on days 3-6 to monitor cell growth and measure pH, lactate, and glucose concentrations. During all experiments, three microcarriers were removed from each bioreactor and lysed on day 3 prior to transfection; 1 cm 2 Nuclei counts were performed to estimate cell density relative to the total volume; the spent culture medium was removed and replaced with fresh medium. PEI-mediated transfection was performed according to the previously described protocol (Example 1.1). 18 hours after transfection, the spent culture medium was removed (drained from the bioreactor) and replaced with fresh medium for production. Two batch harvests were performed 24 and 48 hours after medium change.

[0129] 1 cm on the day of transfection 2 The cell number relative to the culture time and lactate produced (mg / mL) on days 3–6 are reported in Table 3.

[0130] [Table 3]

[0131] The results are summarized in Table 4.

[0132] [Table 4]

[0133] As shown in this result, 181ng / cm 2 and 91 ng / cm 2 The bulk product obtained using this amount of total DNA in transfection was 363 ng / cm 2 This results in higher productivity compared to runs obtained with 100 μg of total DNA. For this reason, in the following runs, 962 μg was used for the transfection step.

[0134] Example 1.3: Incubation of the transfection mix In the following group of experiments, a total amount of 962 μg of multi-plasmid DNA per bioreactor (i.e., 181 ng / cm 2 surface area) and a total amount of 962 μg of PEI PRO (1 μg / μL) was used to measure 0.53 m 2 Transfections were performed on day 3 in iCellis Nano bioreactors with a surface area of ​​1000 μg / mL. In run PDE_B17028 A, medium changes were performed 6 hours after the start of the transfection phase to determine whether earlier medium changes were associated with enhanced LVV productivity. To reduce the amount of FBS used in the process, run PDE_M17028B examined a reduction in the percentage of serum (2.5%) during the production phase.

[0135] The various conditions tested are shown in Table 5.

[0136] [Table 5]

[0137] 5000 cells / cm 2 were seeded onto iCELLis nano on day 0. Samples were taken on days 3-6 to monitor cell growth; on day 3 prior to transfection, 3 carriers were removed from each bioreactor and lysed; 1 cm 2 Nuclei counts were performed to estimate cell density for each run. The exhausted culture medium was removed and replaced with fresh growth medium, and Pei-mediated transfection was performed. 18 hours after the transfection step (6 hours for run PDE_M17028A), a batchwise exhausted medium exchange was performed. In run PDE_M17028 B, the exhausted medium was replaced with IMDM 2.5% FBS and 2% L-glutamine. As in the previous run, two batch harvests were performed 24 and 48 hours after the medium exchange.

[0138] The physical and infectious virus titers and infectivity results of the pooled harvests are summarized in Table 6.

[0139] [Table 6]

[0140] Results from run PDE_M17028 A showed that a relatively early medium change after transfection (6 h incubation of cells with the transfection mix) reduced vector production; reducing the percentage of FBS during the recovery phase (PDE_M17028 B) had no effect on production but reduced the viral titer and infectivity of the LV vector.

[0141] Example 1.4: Harvesting LVV by perfusion harvesting To increase the infectivity of the generated viral vectors and avoid drain / fill times during harvest of iCELLis 500, perfusion harvesting was evaluated and compared to batch harvesting in run PDE_B17133. Specifically, PDE_B17133 A was performed using two batch harvests, and in run PDE_B17133B, LV vectors were harvested in a perfusion mode. Both bioreactors followed the same process up to the transfection phase. Briefly, two bioreactors were transfected with 5000 cells / cm on day 0. 2 were inoculated. Three days later, a pre-transfection medium change with complete medium was performed; this was followed by transfection using the same protocol followed in the previous run. 18 hours later, a batch post-transfection medium change was performed with fresh complete medium. Samples were taken on days 3-6 to monitor cell growth; prior to transfection, three carriers were removed from each bioreactor and lysed for nuclei counting.

[0142] For PDE_B17133 A, supernatant was collected by perfusion using a fixed rate (rate of 13.5 mL / hr): fresh medium was transferred to the IN bottle connected to the bioreactor and perfused for 48 hours. During perfusion harvest, supernatant was collected in the OUT bottle. Samples from the OUT bottle were collected 24 and 48 hours after medium change, filtered at 0.45 μm, and stored below −65° C. At 48 hours, the supernatant inside the bioreactor and in the OUT bottle was pooled, filtered, and stored below −65° C. Samples from the pool were collected, filtered at 0.45 μm, and stored below −65° C.

[0143] For PDE_B17133 B, two harvests were collected in batches at 24 and 48 hours after transfection and medium change. Samples from each harvest were collected and stored below -65°C. At the end of the process, the two harvests were pooled, filtered, and stored below -65°C.

[0144] Table 7 summarizes the conditions used for the two bioreactors.

[0145] [Table 7]

[0146] The results are reported in Table 8.

[0147] [Table 8]

[0148] The results demonstrated that the physical and infectious virus titers and infectivities obtained from the two bioreactors were comparable. The conditions introduced (supernatant harvested in batch perfusion starting after post-transfection medium change) allowed maintaining the same level of productivity in batch harvests.

[0149] Example 1.5: Culture conditions before transfection To optimize scale-up on the iCellis 500 and avoid drain / fill times in all phases of the process, alternatives to batch, pre-transfection medium exchange were evaluated. In particular, runs PDE_B18034 and PDE_B18068 implemented perfusion and recirculation medium exchange. Table 9 shows the conditions investigated for each run.

[0150] [Table 9]

[0151] Briefly, each iCellis nano bioreactor was cultured at 5000 cells / cm. 2 was inoculated on day 3 and samples were taken on days 3-6 to monitor pH, lactate, and glucose.

[0152] On day 3, a pre-transfection medium change was performed as described below: PDE_B18034 A and PDE_B18068 B: Exhausted medium was replaced with fresh growth medium in a perfusion mode. The perfusion volume was 1300 mL. · PDE_B18034 B: Batch medium changes were performed; spent medium was removed and 650 mL of fresh growth medium was used to fill the bioreactor. PDE_B18068 C: Exhausted medium was replaced with fresh growth medium in a perfusion mode. The perfusion volume was 650 mL.

[0153] PDE_B18068A: In this run, no medium change was performed, but recirculation was initiated one day after inoculation with 650 mL of fresh medium until day 3.

[0154] PEI-mediated transfection was performed as described in Example 1.3. From the transfection phase to the end of the process, a 48-hour perfusion harvest was performed in three bioreactors. During the perfusion phase, samples were collected from the OUT bottle 24 and 48 hours after medium change to measure physical and infectious viral titers.

[0155] The combined pool from PDE_B18034 and PDE_B18068 was tested for physical and infectious viral titer.

[0156] The physical and infectious viral titers and infectivity results of the experiment are reported in Table 10.

[0157] [Table 10]

[0158] The results demonstrated that LV vector productivity was higher in runs in which cell culture for expansion was performed with medium exchange in a perfusion or recirculation mode compared to standard conditions (run PDE_B18034B, medium exchange performed in batch).

[0159] Example 1.5: Post-transfection medium change The goal of this group of experiments was to investigate the best technical solution for performing post-transfection medium exchange.

[0160] Specifically, in run PDE_B18080 A, post-transfection medium exchange was performed by perfusion to avoid draining and filling the vessel (labor-intensive and difficult in the iCellis 500, and not recommended in prior art such as WO 2018 / 007873). 18 hours after transfection, 1300 mL of complete fresh IMDM was perfused at a rate of 9 mL / min to exchange 90% of the medium.

[0161] In run PDE_B18080 B, a 50-minute hold time was introduced during the batch, post-transfection medium exchange to mimic the drain / fill timing of the iCELLis 500. 18 hours after the transfection phase, the spent medium was removed and the bioreactor was left empty for 50 minutes, leaving only 16 mL inside; this amount of medium corresponds to the volume that the iCellis 500 cannot recover during draining due to technical limitations of the system. After 50 minutes, the bioreactor was filled with new, fresh medium.

[0162] For PDE_B18080C, post-transfection medium exchange was performed using standard conditions (batch, no holding time) to compare with the introduced novel conditions.

[0163] To assess residual plasmid DNA, samples were taken from the three bioreactors before and after the post-transfection medium change. In bioreactor A, the vessel was sampled after half the volume perfusion.

[0164] The various medium exchange conditions are summarized in Table 11.

[0165] [Table 11]

[0166] During the other phases of the process, all three bioreactors followed the standard conditions set up in the previous run and reported in Table 12.

[0167] [Table 12]

[0168] The total pool of the three bioreactors was tested for physical and infectious virus titers, HCP, plasmid and total residual DNA.

[0169] The results are reported in Tables 13 and 14.

[0170] [Table 13]

[0171] [Table 14]

[0172] As the results show, it was surprisingly found that LV vector productivity was lower in bioreactor A (PDE_B18080 A), which was run with post-transfection medium exchange in perfusion mode, than in run PDE_B18080 C, which was run with post-transfection medium exchange performed in batch mode. The productivity of run PDE_B18080B, which uses post-transfection medium exchange performed in batch mode with a hold time, is also higher than that obtained in run PDE_B18080 A, which was run with post-transfection medium exchange performed in perfusion, and even higher than run PDE_B18080 C, while the infectious virus titer of run PDE_B18080B is the same as run PDE_B18080 C. The amounts of contaminants, HCP, plasmid, and total DNA are comparable between the two runs PDE_B18080 B and PDE_B18080 C. Considering large-scale scale-up on the iCellis 500, the batch media exchange with hold time on the iCellis nano is more consistent with runs performed on the large-scale iCellis 500 (as 50 minutes is the time required to drain and fill the bioreactor), and therefore more representative of the performance of large-scale runs.

[0173] Example 2: iCELLis nano 1.06m 2 lentiviral vector generation in Example 2.1: Materials and Methods Packaging cells and cell culture medium The production of LVV vectors described in this Example 2.1 was carried out using the HEK293T cell line. HEK293T cells were thawed and grown in IMDM 10% FBS, 2% Glutamax.

[0174] bioreactor 0.53m 2 With the exception of the first experiment in Example 2.2, where one run was performed on a bioreactor iCellis Nano with a surface area of ​​1.06 m, all other runs described in Examples 2.2 and 2.3 were performed on a bioreactor with a surface area of ​​1.06 m. 2 The results were obtained using a bioreactor iCellis Nano with a surface area of ​​1000 Å. The characteristics of the two supports are reported in Table 15.

[0175] [Table 15]

[0176] Plasmid The multiple-plasmid DNA system used in the production of LVV vectors in iCELLis nano in this Example 2 is a third-generation LVV packaging system containing four separate plasmids: one plasmid carrying the gag-pol genes (pGag / Pol), one plasmid encoding rev (pREV), one plasmid carrying the VSV-g gene (pENV-VSV-G), and one transfer vector plasmid carrying the gene encoding the GFP protein (pTransfer-GFP).

[0177] Analysis method The following analytical methods were applied to evaluate the process execution: Physical viral titer: This analytical method is an ELISA for quantifying the p24 HIV protein, which can be used to assess the concentration of physical particles. Infectious virus titer: This assay is based on the transduction of the reference CEM A3.01 cell line. GFP protein expression is assessed by FACS analysis. Residual host cell proteins: This assay is an ELISA to quantify residual proteins from the HEK-293T packaging cell line in LVV samples. Residual total DNA: This analytical method is based on a fluorescent dye that stains the residual DNA and allows its quantification.

[0178] Manufacturing Process Overview A flow chart of the process applied in the experiments disclosed in Example 2 is reported in Figure 2. Briefly, for each experiment, cells were grown in T-flasks in IMDM without phenol red and containing 10% FBS and 2% Glutamax. After splitting, cells were inoculated into the bioreactor iCELLis nano (5000 cells / cm). 2 After the growth phase, during which pH, ​​DO%, glucose, and lactate concentrations were monitored, the culture medium was replaced with fresh complete medium (either by perfusion or through recirculation), and the transfection step was performed on day 3. After 21 hours, a batch medium change was performed to remove all residual plasmid DNA and PEI Pro. Harvesting was performed in the perfusion mode. Lentiviral vector supernatant was collected 48 hours after the post-transfection medium change. From run PDE_B18030, post-transfection medium change and harvesting were scheduled on days 3 and 5, respectively, to collect vector supernatant with higher infectivity. Lentiviral vector samples during perfusion harvesting were 0.45 μm filtered and stored below -65°C for testing.

[0179] Transfection step In all experiments reported in this Example 2, LVV vector production was performed using the polyethyleneimine (PEI)-mediated transfection method. Optimization studies were performed using the amount of μg of plasmid / mL previously tested in Example 1. Until run PDE_B18187, the DNA / cell ratio was maintained at 1.06 m 2In the bioreactor, the volume of the transfection mix and the amount of DNA and PEI were doubled (1924 μg total DNA). The amount of DNA / cell was then halved to 0.53 ml. 2 The same volume of transfection mix and DNA / mL ratio of iCELLis nano was used (962 μg total DNA). The amount of PEI used was set to maintain a 1:1 ratio between PEI and DNA.

[0180] Protocol for transfection on day 3: 1. One hour before transfection, perform a medium change (if provided) by perfusion with pre-warmed complete medium. 2. Prepare the total DNA mix for the bioreactor by diluting the appropriate amount of DNA in free IMDM. 3. Dilute the appropriate amount of PEI in IMDM free of PEI. 4. Transfer the diluted PEI into the tube containing the diluted DNA and mix. 5. Incubate the final mix according to the manufacturer's instructions. 6. Remove the appropriate volume from the bioreactor and transfer the mix to the bioreactor.

[0181] Example 2.2: iCELLis nano 1.06m 2 and iCELLis nano 0.53m 2 Manufacturing in The goals of this first set of experiments were to test: 1.06m 2 a bioreactor having an area of; · Recirculation during cell culture; -Reduced collection volume.

[0182] In all these experiments, 5000 cells / cm 2 were inoculated into iCELLis nano on day 0. After the growth phase in flask T225, 26.5 × 10 6 Cell(0.53m 2in bioreactor) and 53 × 10 6 Cell(1.06m 2 The control Petri dish contained 5.50 x 10 cells to monitor the transfection phase. 5 Cells / Petri were seeded in parallel.

[0183] Bioreactors were sampled during the process to measure pH, lactate, and glucose concentrations. For the first two runs, on day 3, three carriers were removed from each bioreactor and dissolved; 1 cm 2 Nuclei counts were performed to estimate cell density relative to the nuclei.

[0184] The recirculation mode during cell culture for growth was evaluated. For all experiments, PEI-mediated transfection was performed according to the protocol described in Example 2.1. 2 The transfection mix for iCELLis nano was prepared using 962 μg of total DNA (181 ng / cm) in a total volume of 100 mL. 2 The sample was prepared using 962 μg PEI (corresponding to a surface area of ​​1.06 m 2 For iCELLis nano, the volume of the transfection mix and the amounts of DNA and PEI were doubled. 21 hours after transfection, the spent culture medium was removed and replaced with fresh medium for production after a 50-minute holding period. Perfusion harvesting was performed up to 48 hours after the post-transfection medium change.

[0185] [Table 16]

[0186] To monitor growth rate, cells / cm 2 The number of seroconverters and the amount of lactate produced (mg / mL) were assessed and reported in Table 17.

[0187] [Table 17]

[0188] The viral titer results for P24 and TU are summarized in Table 18.

[0189] [Table 18]

[0190] The amounts of HCP and total DNA in the pooled harvests are reported in Table 19.

[0191] [Table 19]

[0192] This result is 1.06m 2 The infectious viral titer of the vector produced in the iCELLis bioreactor was 0.53 m 2 This demonstrates that the productivity of LVV vectors is higher than that produced in iCELLis nano. The recirculation mode does not cause any adverse effects on the cell productivity of LVV vectors and replaces medium exchange.

[0193] Example 2.3: iCELLis nano 1.06m 2 Manufacturing in In this Example 2.3, the conditions set in the previous run (Example 2.2) were maintained: 1.06m 2 iCELLis bioreactor; No medium change before transfection; · Recirculation during cell culture; -Perfusion harvesting.

[0194] 1.06m 2The transfection step and post-transfection medium exchange were investigated in bioreactors with a surface area of ​​1000 μg / cm². In run PDE_B18197, various ratios of DNA / n° and DNA / mL of cells were tested. In Bio1, the same transfection conditions used in Example 2.2 were applied (i.e., 1924 μg total DNA, i.e., 181 ng / cm² of surface area). 2 , 1924 μg PEI in 200 mL transfection mix volume). 2 Compared to previous experiments using bioreactors, half the volume of transfection mix (100 mL) was used. Therefore, the DNA / cell ratio was maintained, but the DNA and PEI concentrations were doubled to match the total volume. For Bio3, 0.53 mL was used. 2 The same volume (100 mL) and DNA (962 μg, i.e., 91 ng / cm of surface area) used for iCELLis nano were used. 2 ) and PEI (962 μg). In this way, the DNA / mL ratio was maintained, but the amounts of DNA and PEI were halved compared to the cell number.

[0195] The various conditions used are summarized in Table 20.

[0196] [Table 20]

[0197] Run PDE_B19013 replicated the conditions of PDE_B18197 Bio3, but tested different timings for the transfection step and subsequent medium change. In Bio1, the previous conditions were maintained (transfection at 11:30 AM on Monday and post-transfection medium change on Tuesday morning); in Bio2, both the transfection step and medium change were performed on Monday at 9:30 AM and 5:30 PM, respectively. Bio3 was performed similarly to Bio2, but with a 2-hour delay from Monday. In all three bioreactors, perfusion harvesting began after the post-transfection medium change.

[0198] Briefly, 5000 cells / cm 2 were seeded into iCELLis nano on day 0. Medium recirculation was performed during cell culture for transfection. Subsequent transfection steps and batch post-transfection medium exchange were performed as previously described. Perfusion harvesting was initiated after post-transfection medium exchange.

[0199] Samples were taken on days 3-6 (morning and afternoon) to monitor cell growth.

[0200] The experimental conditions are summarized in Table 21.

[0201] [Table 21]

[0202] To monitor growth rate, cells / cm 2 The number of seroconverters and the amount of lactate produced (mg / mL) were assessed and reported in Table 22.

[0203] [Table 22]

[0204] The physical and infectious virus titers and infectivity results of the pooled harvests are summarized in Table 23.

[0205] [Table 23]

[0206] The amounts of HCP and total DNA in the pooled harvests are reported in Table 24.

[0207] [Table 24]

[0208] The results show that LVV vector production for P24 in experiment PDE_B18197 Bio3 was the same as in Bio1 and higher than in Bio2, demonstrating that, surprisingly, it was possible to obtain the same or even higher productivity using half the total amount of DNA. Furthermore, this condition improved infectious virus titers, thus resulting in increased infectivity.

[0209] Reducing the transfection time from 21 hours to 8 hours in run PDE_B19013 does not significantly decrease the physical virus titer, but does increase infectivity. Notably, the results show that the infectivity of Bio2 and Bio3 on Wednesday was higher than in the other runs, where harvesting occurred by Thursday. The HCP and total DNA results for runs PDE_B19013 Bio2 and Bio3 were lower than in previous runs, demonstrating that the supernatants had relatively low impurity presence.

[0210] Example 2.4: Transfection and harvest in 3 days To confirm the results obtained, the conditions previously tested in Example 2.3 and selected as the best were repeated in the runs described in Table 25.

[0211] [Table 25]

[0212] As in the previous run, 5,000 cells / cm 2 were inoculated into iCELLis nano on day 0. Medium recirculation was performed with 1300 mL of fresh medium until day 3. PEI-mediated transfection was performed at 10:00 a.m., and a batch post-transfection medium change was performed 8 hours later, both on day 3. Perfusion harvesting began after the post-transfection medium change. Samples were collected on days 3 to 6 to monitor cell growth.

[0213] To monitor the growth rate, the amount of lactate produced (mg / mL) was assessed and is reported in Table 26.

[0214] [Table 26]

[0215] The physical and infectious virus titers and infectivity results of the final pool harvest are reported in Table 27.

[0216] [Table 27]

[0217] The amounts of HCP and total DNA in the pooled harvests are reported in Table 28.

[0218] [Table 28]

[0219] As the results show, infectious virus titers and infectivity are higher when post-transfection medium change and final harvest are allowed for up to 1 day, i.e., when transfection and harvest are performed over a 3-day period. Furthermore, the pooled harvest obtained with a total of 3 days of transfection and harvest results in a relatively low level of contamination, with total DNA either undetectable or below the limits of the analytical method.

[0220] Example 3: Production on iCellis 500 Example 3.1: 66m 2 Production on iCellis 500 with a surface area of 66m 2 iCellis500 with a surface area of ​​0.53m 2 Two parallel vector productions on the iCellis nano, which has a surface area of ​​1000 μg / cm, were performed as summarized in Table 29. The process was performed using the 293-T cell line and the third generation multiple plasmid DNA system used in Examples 1 and 2.

[0221] [Table 29]

[0222] Comparability between iCellis500 and iCellis nano for post-clarification pre-Benzonase samples is reported in Table 30.

[0223] [Table 30]

[0224] The results show that scaling up the process to the iCellis 500 maintains productivity and results in improved infectious virus titers and infectivity compared to data obtained using the iCellis nano. The impurity profiles of the two runs are comparable and acceptable.

[0225] Example 3.2: Surface area 133 m 2 Production on iCellis 500 with 133m 2 and iCellis500 with a surface area of ​​1.06 m 2 Two parallel vector productions on the iCellis nano, which has a surface area of ​​1000 μg / cm, were performed as summarized in Table 31. The process was performed using the 293-T cell line and the third generation multiple plasmid DNA system used in Examples 1 and 2.

[0226] [Table 31]

[0227] Comparability between iCellis500 and iCellis nano for pre-clarification samples is reported in Table 32.

[0228] [Table 32]

[0229] The results show that scaling up the process to the iCellis 500 maintains productivity and results in improved infectious virus titer and infectivity (2-fold higher infectious virus titer and infectivity) compared to data obtained using the iCellis nano. The impurity profiles of the bulk products obtained in the two runs were comparable, and the total DNA content of the large-scale and small-scale products was below the detection limit of the analytical method used, as was the case for samples collected before treatment with the endonuclease.

[0230] Example 4: Minimal total DNA amount The goal of this experiment is 45ng / cm 2 and 60 ng / cm 2 This is an evaluation of the productivity of the process obtained by transfection using a total amount of DNA.

[0231] Packaging cells and cell culture medium The production of LVV vectors described in this Example 4 was performed using the HEK293T cell line. HEK293T cells were thawed and grown in IMDM 10% FBS, 2% Glutamax.

[0232] bioreactor 1.06m 2 The experiment was carried out using a bioreactor iCellis Nano with a surface area of ​​1000 m.

[0233] [Table 33]

[0234] Plasmid For the production of LVV vectors in iCELLis nano in this Example 4, the multi-plasmid DNA system used is a third-generation LVV packaging system containing four separate plasmids: one plasmid carrying the gag-pol genes (pGag / Pol), one plasmid encoding rev (pREV), one plasmid carrying the VSV-g gene (pENV-VSV-G), and one transfer vector plasmid carrying the gene encoding the GFP protein (pTransfer-GFP).

[0235] Analysis method The following analytical methods were applied to evaluate the process execution: Physical viral titer: This analytical method is an ELISA for quantifying the p24 HIV protein, which can be used to assess the concentration of physical particles. Infectious virus titer: This assay is based on the transduction of the reference CEM A3.01 cell line. GFP protein expression is assessed by FACS analysis.

[0236] Manufacturing Process Overview The processes performed in this Example 4 are summarized in Table 34.

[0237] [Table 34]

[0238] Transfection step LVV vector production was performed using polyethyleneimine (PEI)-mediated transfection. Bio1 was administered at a dose of 45 ng / cm2 of surface area. 2 Transfection was performed with multiple plasmid DNA at a total dose of 60 ng / cm2 for Bio2. 2 Transfections were performed using multiple plasmid DNAs in a total amount of 1000 kJ / ml. In both Bio1 and Bio2, the amount of PEI used was set to maintain a 1:1 ratio between PEI and DNA.

[0239] Protocol for transfection on day 3: 1. Prepare the total DNA mix for the bioreactor by diluting the appropriate amount of DNA in free IMDM. 2. Dilute the appropriate amount of PEI in IMDM free of PEI. 3. Transfer the diluted PEI into the tube containing the diluted DNA and mix. 4. Incubate the final mix according to the manufacturer's instructions. 5. Remove the appropriate volume from the bioreactor and transfer the mix to the bioreactor.

[0240] The results obtained are shown in Table 35, which shows the physical particle and viral titers obtained for Bio1 and Bio2, and in column 3, the surface area at 90 ng / cm 2 The figures show the median values ​​of the same parameters obtained in the indicated number of runs ("n") performed by transfection using multiple plasmid DNAs in a total amount of 1000 kJ / ml.

[0241] [Table 35]

[0242] This result is 60ng / cm 2 and 45 ng / cm 2 These results demonstrate that productivity in terms of physical particles is substantially maintained by reducing the total amount of multiple plasmid DNA in transfection to 60 ng / cm. 2 or 45 ng / cm 2 Although a slight decrease is observed with the total multiple plasmid DNA, the data obtained still surprisingly demonstrate that transfection yields a surface area of ​​181 ng / cm 2 Using circle TU / cm 2 were 2.2E+06 and 1.7E+06, respectively (as shown in Table 23), revealing good levels of infectious viral particles, comparable to those observed in Example 2.3 for runs PDE_B18197 Bio1 and PDE_B18197 Bio2. The present invention encompasses, for example, the following embodiments: [1] A method for producing lentiviral vectors in a packed-bed bioreactor, comprising the steps of: (a) Inoculating cells (b) culturing the cells for proliferation for at least one day; (c) 45 ng / cm for the surface area of ​​the packed bed 2 ~100ng / cm 2 Transfecting the cells with multiple plasmid DNAs and PEI transfection reagent in a total amount of (d) recovering the cell culture supernatant containing the recombinant lentiviral vector. A method comprising: [2] The method according to [1], further comprising exchanging the medium at the end of transfection, wherein the medium exchange is carried out in a perfusion mode or a batch mode. [3] The method according to [2], wherein the batchwise medium exchange is carried out by discharging the cell culture medium containing the DNA transfecting particles from the bioreactor and adding fresh medium to the cell culture. [4] The method according to any one of [1] to [3], wherein the cell culture supernatant recovery step is carried out in a perfusion manner. [5] The method according to [3], wherein collection of the recombinant lentiviral vector is initiated by collecting the cell culture medium from the bioreactor in a perfusion manner immediately after adding the fresh medium. [6] The method according to any one of [1] to [5], wherein the cells are transfected for up to 18 hours. [7] The method according to [4] or [5], wherein the recovery step in a perfusion mode is carried out for at least 39 hours after the post-transfection medium change. [8] The method according to [4] or [5], wherein the recovery step in a perfusion mode is carried out for 39 hours after the post-transfection medium change. [9] The method according to any one of [1] to [8], further comprising a complete exchange of the medium between the culture step (b) for proliferation and the transfection step (c).

[10] The cells have a packed bed surface area of ​​at least 0.1 mL / cm 2 The method according to any one of [1] to [8], wherein the cells are cultured for growth in a perfusion mode by supplying fresh medium in a total amount of

[11] The cells are at least 0.1 mL / cm per packed bed.2 The method according to any one of [1] to [8], wherein the cells are cultured for growth in a recirculating manner using a total amount of fresh medium as a reservoir.

[12] The method according to any one of [1] to

[11] , wherein the transfection step (c) is carried out in the absence of perfusion or recirculation of fresh medium.

[13] The method according to any one of [1] to

[12] , wherein the transfection is carried out using a ratio of DNA amount to PEI amount corresponding to 1:1.

[14] The method according to any one of [1] to

[13] , wherein the cells are transfected in step (c) for 8 hours.

[15] The method according to any one of [1] to

[14] , wherein the cells are selected from HEK293, HEK293T, HEK293E, HEK293FT, 293Vec, TE671, HT1080, or HeLa cell lines.

[16] The method described in any of [1] to

[15] , wherein the multiple plasmid DNAs are composed of one type of plasmid carrying the lentiviral gag / pol genes, one type of plasmid carrying the lentiviral rev gene, one type of plasmid carrying a gene encoding an envelope protein, and one type of plasmid carrying a transfer vector containing a required portion of the lentiviral genome and the desired exogenous gene.

Claims

1. 1. A method for the production of lentiviral vectors in a packed bed bioreactor, comprising the steps of: (a) Inoculating cells (b) culturing the cells for proliferation for at least one day; (c) 45 ng / cm for the surface area of ​​the packed bed 2 ~100ng / cm 2 Transfecting the cells with multiple plasmid DNAs and PEI transfection reagent in a total amount of (d) recovering the cell culture supernatant containing the recombinant lentiviral vector. Including, A method wherein the transfection is carried out using a ratio of DNA amount to PEI amount corresponding to 1:

1.

2. 2. The method of claim 1, further comprising changing the medium at the end of transfection, wherein the medium change is performed in a perfusion or batch mode.

3. 3. The method of claim 2, wherein the batchwise medium exchange is performed by draining cell culture medium containing DNA transfecting particles from the bioreactor and adding fresh medium to the cell culture.

4. The method according to any one of claims 1 to 3, wherein the step of collecting the cell culture supernatant is carried out in a perfusion mode.

5. 4. The method of claim 3, wherein harvesting of the recombinant lentiviral vector is initiated by withdrawing the cell culture medium from the bioreactor in a perfusion manner immediately after the addition of fresh medium.

6. The method of any one of claims 1 to 5, wherein the cells are transfected for up to 18 hours.

7. 6. The method of claim 4 or 5, wherein the harvesting step in perfusion mode is carried out for at least 39 hours after post-transfection medium change.

8. 6. The method of claim 4 or 5, wherein the harvesting step in perfusion mode is carried out for 39 hours after post-transfection medium change.

9. The method of any one of claims 1 to 8, further comprising a complete exchange of medium between the growth culturing step (b) and the transfection step (c).

10. The cells are at least 0.1 mL / cm of packed bed surface area. 2 9. The method according to claim 1, wherein the cells are cultured for growth in a perfusion mode by supplying fresh medium to the bioreactor by the end of the growth phase in a total amount of

11. The cells are at least 0.1 mL / cm of packed bed 2 The method according to any one of claims 1 to 8, wherein the cells are cultured for growth in a recirculating manner using a total amount of fresh medium as a reservoir.

12. 12. The method of any one of claims 1 to 11, wherein the transfection step (c) is performed in the absence of perfusion or recirculation of fresh medium.

13. 13. The method of any one of claims 1 to 12, wherein the cells are transfected in step (c) for 8 hours.

14. 14. The method of any one of claims 1 to 13, wherein the cells are selected from HEK293, HEK293T, HEK293E, HEK293FT, 293Vec, TE671, HT1080 or HeLa cell lines.

15. 15. The method of any one of claims 1 to 14, wherein the multiple plasmid DNA is composed of one type of plasmid carrying the lentiviral gag / pol genes, one type of plasmid carrying the lentiviral rev gene, one type of plasmid carrying a gene encoding an envelope protein, and one type of plasmid carrying a transfer vector containing the required portion of the lentiviral genome and the exogenous gene of interest.

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