DESC:RELATED APPLICATIONS
This application is related to Indian provisional application IN202421024307 filed 27th Mar. 2024 and is incorporated herein in its entirety
FIELD OF THE INVENTION
The present invention relates to a method for enhancing productivity of clones for enhancing expression of gene of interest.
BACKGROUND OF THE INVENTION
Generation of recombinant mammalian cell lines for production of pharmaceutical protein is driven by various factors like the gene transfer vector, the host cell line and the transfection method. An efficient combination of selection, screening and gene amplification leads to stably and highly producing clones ready to be propagated in scalable reactors. There will be influence of the integration locus on stability and productivity of the clones in order to obtain an additional criterion for clone selection since the majority of random integration sites leads to low, medium and high level expression.
Different factors that might be related to the integration site have been suggested to influence the expression level: assumes a ‘‘position effect’’, which is either caused by rearrangement or incorporation of recombinant genes into regions with nearby endogenous promoters and enhancers which support gene expression. Furthermore, various DNA elements can modulate the position effect: insulators, MAR elements, locus control regions and ubiquitous chromatin opening elements may be important in controlling expression levels and stability of recombinant protein production. The integration of the target gene within heterochromatic regions of DNA, which arise due to the dense packaging of DNA and associated proteins, are generally considered to be transcriptionally inactive.
The expression vector primarily used for gene expression in mammalian systems is a DNA plasmid, which exists extra-chromosomally and is designed to contain various elements that enhance transcription and translation. Typically, the plasmid will contain strong promoter and enhancer elements upstream of the gene of interest to drive its high expression. The function of a promoter is to be bound by transcription factors to initiate transcription.
In the present invention we have Puromycin containing vector which is CET1019HD-puro (procuring from Merck) and Glutamine synthetase containing vector which is pD2535nt-HDP (procuring from Horizon).
WO2012077128 discloses co-transfection of two different vector constructs, wherein (a) the first vector construct comprises the gene of POI and a DHFR amplification gene; and (b) the second vector construct comprises the same gene of POI and a selection marker other than DHFR.
WO200194571 discloses an expression vector comprising an expression unit containing a first selective marker gene and an expression unit containing a second selective marker gene which differs from the first selective marker gene.
The present invention focus on efficient process for developing a stable cell line for high level expression of recombinant protein using super transfection strategy.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing protein of interest and first selection marker; and ii) second transfection of cell with second vector containing protein of interest and second selection marker.
Another object of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing antibody and puromycin selection marker; and ii) second transfection of cell with second vector containing antibody and glutamine synthetase selection marker.
Another object of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing Pembrolizumab and puromycin selection marker; and ii) second transfection of cell with second vector containing Pembrolizumab and glutamine synthetase selection marker.
Another object of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing Daratumumab and puromycin selection marker; and ii) second transfection of cell with second vector containing Daratumumab and glutamine synthetase selection marker.
Another object of the present invention is to provide a method for enhancing productivity of clones comprising: i) post first transfection selection of minipools with puromycin; and ii) post second transfection selection of minipools with methionine sulfoximine (MSX) and without glutamine.
Another object of the present invention is to provide a method for enhancing productivity of clones comprising following steps:
i. first transfection of cell with first vector containing antibody and puromycin selection marker;
ii. post first transfection selection of minipools with puromycin;
iii. second transfection of cell with second vector containing antibody and glutamine synthetase selection marker;
iv. post second transfection selection of minipools with methionine sulfoximine (MSX) and without glutamine; and
v. post minipool selection minipool evaluated using fed-batch which shows 3 to 4 fold increase in average volumetric titre compare to first set of transfection.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing protein of interest and first selection marker; and ii) second transfection of cell with second vector containing protein of interest and second selection marker.
Another aspect of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing antibody and puromycin selection marker; and ii) second transfection of cell with second vector containing antibody and glutamine synthetase selection marker.
Another aspect of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing Pembrolizumab and puromycin selection marker; and ii) second transfection of cell with second vector containing Pembrolizumab and glutamine synthetase selection marker.
Another aspect of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing Daratumumab and puromycin selection marker; and ii) second transfection of cell with second vector containing Daratumumab and glutamine synthetase selection marker.
Another aspect of the present invention is to provide a method for enhancing productivity of clones comprising: i) post first transfection selection of minipools with puromycin; and ii) post second transfection selection of minipools with methionine sulfoximine (MSX) and without glutamine.
Another aspect of the present invention is to provide a method for enhancing productivity of clones comprising following steps:
i. first transfection of cell with first vector containing antibody and puromycin selection marker;
ii. post first transfection selection of minipools with puromycin;
iii. second transfection of cell with second vector containing antibody and glutamine synthetase selection marker;
iv. post second transfection selection of minipools with methionine sulfoximine (MSX) and without glutamine; and
v. post minipool selection minipool evaluated using fed-batch which shows 3 to 4 fold increase in average volumetric titre compare to first set of transfection.
BRIEF DESCRIPTION OF DRAWING
Figure 1: First set of transfection of Pembrolizumab – fed-batch evaluation of minipools
Figure 2: Second set of transfection of Pembrolizumab (supertransfection) - fed-batch evaluation of minipools
Figure 3: First set of transfection of Daratumumab – fed-batch evaluation of minipools
Figure 4: Second set of transfection of Daratumumab (supertransfection) - fed-batch evaluation of minipools
DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description of embodiments of the invention. The embodiments are in such details as to clearly communicate the invention. However, the amount of details offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents of embodiments, and alternative falling within the spirit and scope of the present invention.
DEFINITION
The following definitions are provided to facilitate understanding of certain terms used throughout the specification.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.
The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.
The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of’ or “consisting essentially of’ language.
The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, modified antibodies, subunits of antibodies, antibody derivatives, artificial antibodies, combinations of antibodies with proteins and antibody fragments sufficiently long to display the desired biological activity, the monoclonal antibodies as used herein may be human antibodies.
In the present invention antibody is selected from Ranibizumab, Denosumab, Pembrolizumab, Vedolizumab, Aflibercept, Daratumumab, Trastuzumab, Secukinumab, Pertuzumab, Nivolumab, Golimumab, Dupilumab, Etanercept, Atezolizumab, Risankizumab, Bevacizumab, Dulaglutide or Rituximab.
The term "expression vector" as used herein refers to a DNA molecule, linear or circular, comprising a segment encoding a protein of interest (POI) operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences. A vector may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator. Replication of expression vectors in a host organism can be autonomous or through integration into the host genome.
The term "selection marker" as used herein refers to a marker that will have function to protect the cell from a selective agent that would otherwise kill the cell or prevent its growth. Selection markers are selected from zeocin, zeomycin, puromycin, Glutamine synthetase, neomycin phosphotransferase, hygromycin phosphotransferase, and phosphoinothricin acetyltransferase.
The term “transfection” as used herein refers to a process by which foreign nucleic acids are delivered into a eukaryotic cell to modify the host cell's genetic makeup.
The term “Super transfection” as used herein refers to a target cell, which is exposed to a high-efficiency nucleic acid delivery system that enhances uptake and expression of exogenous genetic material.
The main embodiment of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing protein of interest and first selection marker; and ii) second transfection of cell with second vector containing protein of interest and second selection marker.
Another embodiment of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing antibody and puromycin selection marker; and ii) second transfection of cell with second vector containing antibody and glutamine synthetase selection marker.
Another embodiment of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing Pembrolizumab and puromycin selection marker; and ii) second transfection of cell with second vector containing Pembrolizumab and glutamine synthetase selection marker.
Another embodiment of the present invention is to provide a method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing Daratumumab and puromycin selection marker; and ii) second transfection of cell with second vector containing Daratumumab and glutamine synthetase selection marker.
Another object of the present invention is to provide a method for enhancing productivity of clones comprising: i) post first transfection selection of minipools with puromycin; and ii) post second transfection selection of minipools with methionine sulfoximine (MSX) and without glutamine.
Another embodiment of the present invention is to provide a method for enhancing productivity of clones comprising following steps:
i. first transfection of cell with first vector containing antibody and puromycin selection marker;
ii. post first transfection selection of minipools with puromycin;
iii. second transfection of cell with second vector containing antibody and glutamine synthetase selection marker;
iv. post second transfection selection of minipools with methionine sulfoximine (MSX) and without glutamine; and
v. post minipool selection minipool evaluated using fed-batch which shows 3 to 4 fold increase in average volumetric titre compare to first set of transfection.
In the present invention, post minipool selection minipools are allowed to grow for 14 days under fed-batch cell culture media.
The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also from the part of this specification and are intended to be included within the scope of it.
EXAMPLE
Experimental details
Gene of interest cloned into both the vectors CET1019HD-Puro and pD2535nt-HDP.
First set of transfection (Traditional transfection) was carried out in CHO cells with vector CET1019HD-Puro containing puromycin selection marker and kept under selection with puromycin for positive selection to get the best minipools. Post minipool selection to evaluate productivity minipools are allowed to grow for 14 days under fed-batch cell culture media for production run.
Minipool which shown highest productivity are taken forward for second set of transfection (Super transfection) with vector pD2535nt-HDP containing glutamine synthetase selection marker and kept under selection with methionine sulfoximine (MSX) and without glutamine for positive selection to get the best minipools. Post minipool selection to evaluate productivity minipools are allowed to grow for 14 days under fed-batch cell culture media. Top minipools were selected for single cell cloning process for production run.
EXAMPLE 1 TRANSFECTION USING PEMBROLIZUMAB AS GENE OF INTEREST
First set of transfection (Traditional transfection) using Pembrolizumab - minipool evaluated using fed-batch, as per the productivity profile average volumetric titre (g/L) ranging from 0.2 to 0.8g/L (Refer figure 1).
Second set of transfection (Super transfection) using Pembrolizumab - minipool evaluated using fed-batch, as per the productivity profile average volumetric titre (g/L) ranging from 1.75 to 2.8 g/L which has been increased by 3 to 4 fold in comparison with respect to first set of transfection minipools (Refer figure 2).
EXAMPLE 2 TRANSFECTION USING DARATUMUMAB AS GENE OF INTEREST
First set of transfection (Traditional transfection) using Daratumumab – minipool evaluated using fed-batch, as per the productivity profile average volumetric titre (g/L) ranging from 1.0 to 2.6g/L (Refer figure3).
Second set of transfection (supertransfection) using Daratumumab – minipool evaluated using fed-batch, as per the productivity profile average volumetric titre (g/L) ranging from 1.5 to 4.5 g/L which has been increased by 2 to 3 fold in comparison with respect to First set of transfection minipools. (Refer figure 4).
As per above study of Pembrolizumab and Daratumumab, it shows that supertransfection helping to increase the volumetric titre which in turn helps in totality for the product development.
Above supertransfection method were also performed using Nivolumab, Atezolizumab, Dupilumab, Secukinumab and Risankizumab molecules. ,CLAIMS:We Claim,
1. A method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing antibody and puromycin selection marker; and ii) second transfection of cell with second vector containing antibody and glutamine synthetase selection marker.
2. The method according to claim 1, wherein the antibody is selected from Ranibizumab, Denosumab, Pembrolizumab, Vedolizumab, Aflibercept, Daratumumab, Trastuzumab, Secukinumab, Pertuzumab, Nivolumab, Golimumab, Dupilumab, Etanercept, Atezolizumab, Risankizumab, Bevacizumab, Dulaglutide or Rituximab.
3. The method according to claim 1, wherein the method for enhancing productivity of clones comprising: i) post first transfection selection of minipools with puromycin; and ii) post second transfection selection of minipools with methionine sulfoximine (MSX) and without glutamine.
4. A method for enhancing productivity of clones comprising following steps:
i. first transfection of cell with first vector containing antibody and puromycin selection marker;
ii. post first transfection selection of minipools with puromycin;
iii. second transfection of cell with second vector containing antibody and glutamine synthetase selection marker; and
iv. post second transfection selection of minipools with methionine sulfoximine (MSX) and without glutamine.
v. post minipool selection minipool evaluated using fed-batch which shows 3 to 4 fold increase in average volumetric titre compare to first set of transfection.
5. The method according to any of the previous claims, wherein the antibody is selected from Pembrolizumab, Daratumumab, Dupilumab or Risankizumab.
6. A method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing Pembrolizumab and puromycin selection marker; and ii) second transfection of cell with second vector containing Pembrolizumab and glutamine synthetase selection marker.
7. A method for enhancing productivity of clones comprising: i) first transfection of cell with first vector containing Daratumumab and puromycin selection marker; and ii) second transfection of cell with second vector containing Daratumumab and glutamine synthetase selection marker.