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WO2025085682A1 - Évaluation de niveaux de transcrit d'arnm à l'aide d'une ddpcr pour une évaluation précoce d'un clone et d'une seule cellule dans le développement d'une lignée cellulaire - Google Patents

Évaluation de niveaux de transcrit d'arnm à l'aide d'une ddpcr pour une évaluation précoce d'un clone et d'une seule cellule dans le développement d'une lignée cellulaire Download PDF

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WO2025085682A1
WO2025085682A1 PCT/US2024/051847 US2024051847W WO2025085682A1 WO 2025085682 A1 WO2025085682 A1 WO 2025085682A1 US 2024051847 W US2024051847 W US 2024051847W WO 2025085682 A1 WO2025085682 A1 WO 2025085682A1
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cells
chains
cell
antibody
protein
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Eugenia ZAH
Kayley COX
Edwige Gros
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Amgen Inc
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Amgen Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present application relates generally to methods for selecting single-cell clones and pools of cells which produce high amounts of a protein of interest. More specifically, the application relates to the use of digital PCR techniques, such as droplet digital PCR (ddPCR), for measuring mRNA transcript levels to select high expressing single-cell clones and pools of cells.
  • digital PCR techniques such as droplet digital PCR (ddPCR)
  • ddPCR droplet digital PCR
  • Biologicales are used worldwide in a variety of applications, such as therapeutics and diagnostics.
  • Mammalian cell lines are tire predominant expression systems for these biologies, with Chinese hamster ovary (CHO) cells being the predominate cellular factory .
  • CHO Chinese hamster ovary
  • CLD Cell line development
  • the present disclosure relates to a method of facilitating selection of a single cell clone or pools of cells for manufacturing an antigen-binding protein with two to four different antibody chains comprising: a) passaging a single cell clone or pool of cells expressing the antibody chains so that the cells have at least 85% viability and have a doubling time less than 40 hours for at least two consecutive passages, b) extracting mRNA from the cells; c) reverse transcribing the mRNA to generate cDNA; d) performing ddPCR to quantify the transcript levels of the_antibody chains and e) eliminating single cell clones or pool of cells having at least the lowest 30* percentile of the sum of the transcript levels of all the antibody chains.
  • At least one of the antibody chains is a heavy chain or heavy chain- ScFv and/or at least one of the antibody chains is a light chain.
  • the single cell clone or pool of cells expresses an antigen-binding protein with two different antibody chains.
  • the single cell clone or pool of cells expresses an antigen-binding protein with one heavy chain (HC) or heavy chain-scFv (HC-scFv) and one light chain (LC).
  • HC heavy chain
  • HC-scFv heavy chain-scFv
  • LC light chain
  • the method further comprises eliminating cell clones or pools of cells expressing an antigen-binding protein with one heavy chain (or HC-scFv) and one light chain w here the ratio of the transcript levels for HC:LC (or HC-scFv:LC) or LC-HC (or LC -HC-scFv) is less than 0.2 or greater than 1.5. This eliminates cell clones or pools of cells having a ratio at either extreme (i.e., too low or too high).
  • the single cell clone or pool of cells expresses an antigen binding protein with three or four different antibody chains.
  • the cells express an antigen-binding protein with two heavy chains (for example, HC 1 and HC2 or HC1 and HC2-scFv) and one light chain (LC).
  • tire method further comprises eliminating cell clones or pools of cells where the ratio of the transcript levels for HCLHC2 or HC2:HC1 is less than 0.2 or greater than 1.5 or the ratio of HCl:HC2-scFv or HC2-scFv:HCl is less than 0.2 or greater than 1.5.
  • the cells express an antigen-binding protein with two heavy chains (HC 1 and HC2) and two light chains (LC1 and LC2).
  • the method further comprises eliminating cell clones or pools of cells w here the ratio of the transcript levels for HC1 :HC2 or HC2:HC 1 is less than 0.2 or greater than 1.5.
  • eliminating single cell clones or pools of cells eliminates those having at least the lowest 50* percentile of the sum of the transcript levels of all the chains.
  • the method further comprises I) subjecting the remaining single cell clones or pools of cells to a fed batch culture for at least an additional 8 days while maintaining the cell viability above 80%.
  • the ddPCR is one-step ddPCR or two-step ddPCR.
  • the cells are selected from the group consisting of CHO. HEK293, NSO or Sp2/0 cells. In one aspect of this embodiment, the cells are CHO cells. In a sub-aspect of this embodiment, the CHO cells are DHFR- (dihydrofolate reductase minus) or GSKO (glutamine synthetase knock out).
  • the cells have been generated by using single cell printing.
  • the present disclosure also relates to a method of facilitating selection of a single cell clone or pools of cells expressing an antigen-binding protein with one or more antibody heavy chains and one or more antibody light chains for stable growth comprising: a) passaging the cells until the cells have a viability greater than 80% and have a doubling time less than 35 to 40 hours for at least two passages, b) extracting mRNA from the cells; c) reverse transcribing the mRNA to generate cDNA; d) performing ddPCR to quantify the transcript levels of the one or more heavy chains and the one or more light chains; and e) eliminating cell clones or pool of cells having at least the lowest 30 11 percentile of the sum of the transcript levels of all antibody chains.
  • the doubling time has less than 50% deviation between the at least tw o passages
  • Figs.1 A-C Transcript levels vs. Titer (Fed Batch) in various antibody modalities.
  • Fed batch transcript expression stuns are represented by grey bars and normalized bulk titer is represented by black bars for (A) a 4-chain heteroIgG.
  • B a 3-chain asymmetric antibody with HC-only binder (AmAb-HCAb or [Fab* VH] -heteroFc), and
  • C 3-chain asymmetric ClmAb. Normalized titer values were obtained by dividing each bulk titer value by the sample set’s lowest titer value. Top pool performers selected based on titer and product qualify attributes are marked by black stars.
  • Figs. 2A-C Transcript vs. Titer (Norm Passage) in various antibody modalities.
  • Nonn-passage transcript expression sum is represented by light-grey bars
  • fed batch expression sum is represented by grey bars
  • the normalized bulk fed batch titer is represented by black bars for (A) a 4- chain heteroIgG, (B) AmAb-HCAb, and (C) a 3-chain asymmetric ClmAb.
  • Normalized titer values were obtained by dividing each bulk titer value by the sample set’s lowest titer value. Top pool performers (marked by black stars), selected based on titer and product quality attributes, rank at the top two-thirds most range of transcript expression.
  • Figs. 3A-B Spearman matrix correlations between 3-chain AmAb-HCAb molecule transcript/chain ratio vs. product quality (where HC2 is the shorter heavy chain of the 3-chain AmAb- HCAb). Spearman analysis was performed to identify correlations between transcript expression and fed batch titer and product quality attributes from (A) fed batch and (B) norm-pass culture time points.
  • Fig. 4 Transcripts evaluated for bispecific molecules with different molecular structures (B2HmAb, C2HmAb, B2mAb, and C2mAb) with similar binding properties from norm-passage timepoint.
  • Sum of heavy chain (HC) and light chain (LC) transcripts is represented by light-grey bars
  • normalized bulk fed batch titer is represented by black bars.
  • Each bar represents the average of four pools with identical vector configurations, each with three replicate expression values; error bars represent the standard deviation.
  • Figs. 5A-D Clone Transcript Expression Analysis for 3-chain AmAb-HCAb using Quick RNA- Extract Method from norm-passage timepoint.
  • A Clone expression sum is represented by grey bars, normalized bulk titer is represented by black bars), with the top pool performer (marked by black star). Error bars represent the standard deviation of three replicates.
  • B Individual chain expression titer results; clone with the lowest titer marked by a light-grey star.
  • C Spearman correlation matrix providing HC1/HC2 ratio (where HC2 is the shorter heavy chain of the 3-chain AmAb-HCAb).
  • D HC1:HC2 ratio versus MP and LMW. Top clone marked by a black square. Normalized titer values were obtained by dividing each bulk titer value by the sample set’s lowest titer value.
  • the present invention is based, in part, on the discovery that droplet digital PCR (ddPCR) quantitation of mRNA levels in a particular cell correlates with titers of secreted proteins.
  • ddPCR droplet digital PCR
  • a two-step ddPCR separates the RT step and the PCR step in tw o different reaction vessels.
  • SEC size-exclusion chromatography
  • HMW high molecular weight
  • LMW low molecular weight
  • nr-CE non-reduced capillary electrophoresis
  • This assay can serve as a powerful tool to predict the top performing single-cell clones and/or pools and eliminate the poor performing single-cell clones and/or pools before reaching the fed batch production stage.
  • This assay has the potential to save time, cost, and resources since it would further enable next-generation high- throughput cell line development (CLD) workflows before reaching the fed batch production stage.
  • CLD cell line development
  • the invention finds particular utility in assessing transcript levels of a light chain and a heavy chain (from a product comprising both a heavy chain and light chain), or combinations of 3 or 4 heavy or light chains, in different vector configurations to find the best vector configurations.
  • Digital PCR is a nucleic acid amplification and detection method that is based on the dilution of template DNA into independent non-interacting partitions. See Sykes, et al., 1992. BioTechniques 13: 444-449. Following Poisson statistics with high dilutions of DNA template, each reaction is independently interrogated for the presence of a nucleic acid at single molecule sensitivity.
  • ddPCR droplet digital PCR
  • digital PCR has seen wider use as an analytical tool for research and clinical applications.
  • digital PCR can be used as a robust tool for analyzing copy number variations seen in the amplifications or deletions of specific genes, detecting mutations, and quantifying specific nucleic acids species.
  • a “protein of interest” includes naturally occurring proteins, recombinant proteins, and engineered proteins (e.g., proteins that do not occur in nature and which have been designed and/or created by humans).
  • a protein of interest can. but need not be. a protein that is known or suspected to be therapeutically relevant.
  • the term “heterologous” used in connection with a nucleic acid means having a nucleic acid not naturally occurring within a host cell. This can include mutated sequences, e.g,, sequences differing from the naturally occurring sequence. This can include sequences from other species.
  • a cell already containing a heterologous nucleic acid encoding a protein of interest, for example, by stable integration of an expression cassette, would be considered to contain a heterologous nucleic acid sequence.
  • a CHO cell or a derivative thereof e.g., a DHFR- or GS knockout
  • having a nucleic acid encoding an antigen binding protein would be considered to have a heterologous nucleic acid.
  • cell culture or “culture” or “Norm Passage” refers to the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or while attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, can be used.
  • cell culture medium also called “culture medium.” “cell culture media.” “tissue culture media,”) refers to any nutrient solution used for growing cells, e.g., animal or mammalian cells, and which generally provides at least one or more components from the following: an energy source (usually in the form of a carbohydrate such as glucose); one or more of all essential amino acids, and generally the twenty' basic amino acids, plus cysteine; vitamins and/or other organic compounds typically required at low concentrations; lipids or free fatty’ acids; and trace elements, e.g., inorganic compounds or naturally occurring elements that arc typically required at very low concentrations, usually in the micromolar range.
  • an energy source usually in the form of a carbohydrate such as glucose
  • one or more of all essential amino acids and generally the twenty' basic amino acids, plus cysteine
  • vitamins and/or other organic compounds typically required at low concentrations
  • lipids or free fatty’ acids lipids or free fatty’ acids
  • trace elements e.g., inorganic compounds
  • a “fed-batch culture” refers to a form of suspension culture and means a method of culturing cells in which additional components are provided to the culture at a time or times subsequent to the beginning of the culture process.
  • the provided components typically comprise nutritional supplements for the cells which have been depleted during the culturing process. Additionally, or alternatively, the additional components may include supplementary components (e.g., a cell-cycle inhibitory compound).
  • a fed-batch culture is typically stopped at some point and the cells and/or components in the medium are harvested and optionally purified.
  • Cell density refers to the number of cells in a given volume of culture medium.
  • “Viable cell density” refers to the number of live cells in a given volume of culture medium, as determined by standard viability assays (such as try pan blue dye exclusion method).
  • Cell viability means the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The tenn also refers to that portion of cells which are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time.
  • “Titer” means the total amount of a polypeptide or protein of interest (which may be a naturally occurring or recombinant protein of interest) produced by a cell culture in a given amount of medium volume. Titer can be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter (or other measure of volume) of medium.
  • Cumulative titer is the titer produced by the cells during the course of the culture, and can be determined, for example, by measuring daily titers and using those values to calculate the cumulative titer.
  • the term “host cell” is understood to include a cell that has been genetically engineered to express a polypeptide of interest. Genetically engineering a cell involves transfecting, transforming or transducing the cell with a nucleic acid encoding a recombinant polynucleotide molecule (a “gene of interest”), and/or otherwise altering (e.g., by homologous recombination and gene activation or fusion of a recombinant cell with a non-recombinant cell) so as to cause the host cell to express a desired recombinant polypeptide.
  • a recombinant polynucleotide molecule e.g., a recombinant polynucleotide molecule
  • transcript expression sum refers to the sum of transcript expression of the individual antibody chains.
  • a cell sample is obtained from, for example, a frozen sample from either a single-cell clone or a stable pool and either from Norm Passage or from a fed-batch process.
  • the cells are generated by single cell printing, for example, using commercially available equipment from vendors such as NamoCell (San Jose. CA).
  • the cell sample contains nucleic acids expressing a protein of interest.
  • the cell sample may be cultured in order to obtain sufficient numbers of cells for mRNA extraction.
  • single-cell clones and pools are cultured until they achieve a viability of 85% ( ⁇ 2-4 weeks) with an average doubling time less than 35-40 hours for at least two or three consecutive passages.
  • the cell sample is subjected to mRNA extraction. See, e.g.. Cheng et al., 2021, Anal. Methods 3:289-298; and Svec, 2013, Front. Oncol. 3:1-11.
  • the sample may be treated to disrupt or lyse cells, for example by treating the samples with one or more detergents and/or denaturing agents (e.g., guanidinium agents).
  • Nucleic acids may also be extracted from samples, for example, after detergent treatment and/or denaturing. Total nucleic acid extraction may be performed using known techniques, for example by non-specific binding to a solid phase (e.g., silica). See, e.g., U.S. Pat. Nos.
  • kits are available from companies such as Qiagen, Inc. (Germantown, MD).
  • the Qiagen kit uses silica-based extraction based on size >200bp to select for mRNA. First, samples are treated with DNAse to get rid of DNA and RNases are inactivated. mRNA is then enriched using the RNAeasy silica column.
  • the mRNA is reverse transcribed into cDNA using commonly known methods involving a reverse transcriptase.
  • a cell sample can be subjected to a quick lysate kit for cDNA transcription without the need for RNA isolation.
  • An example of such a kit is SingleShot from Bio-Rad (Hercules, CA).
  • the methods described herein employ digital PCR methods.
  • a sample containing nucleic acids is separated into a large number of partitions before performing PCR.
  • Partitioning can be achieved in a variety of ways known in the art, for example, by use of micro well plates, capillaries, emulsions, arrays of miniaturized chambers or nucleic acid binding surfaces. Separation of the sample may involve distributing any suitable portion including up to the entire sample among the partitions.
  • Each partition includes a fluid volume that is isolated from the fluid volumes of other partitions.
  • the partitions may be isolated from one another by a fluid phase, such as a continuous phase of an emulsion, by a solid phase, such as at least one wall of a container, or a combination thereof.
  • the partitions may comprise droplets disposed in a continuous phase, such that the droplets and the continuous phase collectively form an emulsion.
  • the partitions may be formed by any suitable procedure, in any suitable maimer, and with any suitable properties.
  • the partitions may be formed with a fluid dispenser, such as a pipette, with a droplet generator, by agitation of the sample (e.g., shaking, stirring, sonication, etc.), and the like.
  • the partitions may be formed serially, in parallel, or in batch.
  • the partitions may have any suitable volume or volumes.
  • the partitions may be of substantially uniform volume or may have different volumes. Exemplary partitions having substantially the same volume are monodisperse droplets.
  • Exemplary volumes for the partitions include an average volume of less than about 100, 10 or 1 pL, less than about 100, 10, or 1 nL, or less than about 100, 10. or 1 pL, among others.
  • PCR is carried out in the partitions.
  • the partitions when formed, may be competent for performance of one or more reactions in the partitions.
  • one or more reagents may be added to the partitions after they are formed to render them competent for reaction.
  • the reagents may be added by any suitable mechanism, such as a fluid dispenser, fusion of droplets, or the like.
  • nucleic acids are quantified by counting the partitions that contain PCR amplicons for the target and/or reference polynucleotides. Partitioning of the sample allows quantification of the number of different molecules by assuming that the population of molecules follows a Poisson distribution.
  • Hindson et al. 2011, Anal. Chem. 83:8604-8610; Pohl and Shih, 2004, Expert Rev. Mol. Diagn. 4:41-47; Pekin et al., 2011, Lab Chip 11: 2156-2166; Pinheiro et al., 2012, Anal. Chem. 84:1003-1011; Day et al., 2013, Methods 59:101-107; herein incorporated by reference in their entireties.
  • Digital PCR may include any method, process, and/or protocol, using instruments and/or kits associated with performing such, that can discretely amplify and quantitate a nucleic acid(s) within individual partitions of a sample.
  • the individual partitions for a digital PCR may be generated by a microfluidic process, such as by using a microfluidic device, and/or by a droplet generating process.
  • Droplet digital PCR Generation of individual partitions by a microfluidic process, such as by using a microfluidic device, and/or a droplet generating process to provide a plurality of partitions in the form of droplets and performing nucleic acid amplification thereon has been more particularly described in the art as "droplet digital PCR.”
  • the droplets generated for droplet digital PCR may be provided in, for example, a water-in-oil emulsion.
  • nucleic acid amplification includes droplet digital PCR (ddPCRTM) using Bio-Rad's QX100TM or QX200TM Droplet Digital PCR systems, and analysis of nucleic acid amplification products produced by the same but is not limited thereto.
  • ddPCRTM droplet digital PCR
  • Reagents necessary for nucleic acid amplification may be provided to each of the plurality of partitions, and nucleic acid amplification may be performed on each of the plurality of partitions.
  • reagents for nucleic acid amplification may be bundled within a partition, e.g., an aqueous phase of an emulsion, e.g., a droplet.
  • Partitions containing nucleic acid amplification reagents/components may be merged/fused with partitions containing a sample/nucleic acid extracted from a sample, and nucleic acid amplification performed on the merged/fused partitions.
  • a microfluidic device can be used to merge nucleic acid amplification reagent-containing partitions with samplc/nuclcic acid extract-containing partitions such that every samplc/nuclcic acidcontaining partition includes nucleic acid amplification components/reagents.
  • the number of partitions provided is not particularly limited.
  • the number of different sample/polynucleotide-containing partitions can be about, more than about, less than about, or at least about 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70.000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000. 900,000, 1.000,000, 2,000,000, 3,000,000, 4.000.000, 5,000,000, 6,000,000, 7.000.000, 8,000,000, 9,000,000, or 10,000,000 partitions.
  • about 1000 to about 10.000, about 10,000 to about 100,000, about 10,000 to about 500,000, about 100.000 to about 500,000, about 100,000 to about 1,000,000, about 500,000 to about 1,000,000, about 1,000,000 to about 5,000,000, or about 1,000,000 to about 10,000,000 partitions may be generated in the methods as set forth herein.
  • an inner droplet (or partition) can be fused with an outer droplet (or partition) by heating/cooling to change temperature, applying pressure, altering composition (e.g., via a chemical additive), applying acoustic energy (e.g., via sonication), exposure to light (e.g., to stimulate a photochemical reaction), applying an electric field, or any combination thereof.
  • the inner droplet may fuse to the outer droplet spontaneously.
  • the treatment may be continuous or may vary temporally (e.g., pulsatile, shock, and/or repetitive treatment).
  • the treatment may provide a gradual or rapid change in an emulsion parameter, to effect steady state or transient initiation of droplet fusion.
  • the stability of the partitions, and their responsiveness to a treatment to induce droplet fusion may be determined during their formation by selection of an appropriate surfactant ty pe, surfactant concentration, critical micelle concentration, ionic strength, etc., for one or more phases of the inner/outer partition.
  • Fusion may occur spontaneously, such that no treatment, other than a sufficient time delay (or no delay), is necessary before processing fused droplets.
  • the inner/outer droplet may be treated to controllably induce fusion of droplets to form assay mixtures.
  • the emulsion resulting from the fusion may be processed.
  • Processing may include subjecting the emulsion to any condition or set of conditions under which at least one reaction of interest can occur (and/or is stopped), and for any suitable time period. Accordingly, processing may include maintaining the temperature of the emulsion near a predefined set point, varying the temperature of the emulsion between two or more predefined set points (such as thermally cycling the emulsion), exposing the emulsion to light, changing a pressure exerted on the emulsion, adding at least one chemical substance to the emulsion, applying an electric field to the emulsion, or any combination thereof, among others.
  • test signals and code signals each may be detected as fluorescence signals, which may be distinguishable based on excitation wavelength (or spectrum), emission wavelength (or spectrum), and/or distinct positions in a fused droplet (e.g., code signals may be detectable as more localized than test signals with respect to fused droplets), among others.
  • the test signals and code signals may be detected as distinct optical characteristics, such as test signals detected as fluorescence and code signals detected as optical reflectance.
  • the test signals may be detected optically and the code signals electrically, or vice versa.
  • Partitions can be formed by any mode of separating that can be used for digital PCR.
  • a partition can be a microfluidic channel, a well on a nano- or microfluidic device or on a microtiter plate, or a reaction chamber in a microfluidic device.
  • a partition can be an area on an array surface.
  • a partition can be an aqueous phase of an emulsion (e g., a droplet).
  • the droplets described herein can include emulsion compositions (or mixtures of two or more immiscible fluids) described in U.S. Patent No. 7,622.280. and droplets generated by devices described in International Patent Application Publication No. WO 2010/036352.
  • the tern emulsion as used herein, can refer to a mixture of immiscible liquids (such as oil and water). Oil-phase and/or water-in-oil emulsions can allow for the compartmentalization of reaction mixtures within aqueous droplets.
  • the emulsions can include aqueous droplets within a continuous oil phase.
  • the emulsions provided herein are oil-in-water emulsions, wherein the droplets are oil droplets within a continuous aqueous phase.
  • the droplets provided herein can be used to prevent mixing between compartments, and each compartment can protect its contents from evaporation and coalescing with the contents of other compartments.
  • One or more enzymatic reactions can occur in a droplet.
  • splitting a sample into small reaction volumes as described herein can enable the use of reduced amounts of reagents, thereby lowering the material cost of the analysis. Reducing sample complexity by partitioning can also improve the dynamic range of detection, since higher-abundance molecules can be separated from low-abundance molecules in different compartments, thereby allowing lower-abundance molecules greater proportional access to reaction reagents, which in turn can enhance the detection of lower-abundance molecules.
  • a computer can be used to store and process the data.
  • a computerexecutable logic can be employed to perform such functions as grouping and/or analyzing the data.
  • a computer can be useful for displaying, storing, retrieving, or calculating diagnostic results from the molecular profiling; displaying, storing, retrieving, or calculating raw data; or displaying, storing, retrieving, or calculating any sample or patient information useful in the methods described herein.
  • a reference sequence that is present at two copies per diploid genome can be used, e g., a housekeeping gene (e.g., a gene that is required for the maintenance of basic cellular function). Dividing the concentration or amount of the target by the concentration or amount of the reference can yield an estimate of the number of target copies per genome.
  • a housekeeping gene e.g., a gene that is required for the maintenance of basic cellular function. Dividing the concentration or amount of the target by the concentration or amount of the reference can yield an estimate of the number of target copies per genome.
  • a housekeeping gene that can be used as reference in the methods described herein can include a gene that encodes a transcription factor, a transcription repressor, an RNA splicing gene, a translation factor, tRNA synthetase, RNA binding protein, ribosomal protein, RNA polymerase, protein processing protein, heat shock protein, histone, cell cycle regulator, apoptosis regulator, oncogene, DNA repair/replication gene, carbohydrate metabolism regulator, citric acid cycle regulator, lipid metabolism regulator, amino acid metabolism regulator, nucleotide synthesis regulator, NADH dehydrogenase, cytochrome C oxidase, ATPase, mitochondrial protein, lysosomal protein, proteosomal protein, ribonuclease, oxidase/reductase, cytoskeletal protein, cell adhesion protein, channel or transporter, receptor, kinase, growth factor, tissue necrosis factor, etc.
  • housekeeping genes that can include
  • Methods of detecting nucleic acids are well-known in the art and may include specific hybridization of the probe to nucleic acid sequences and, for example, detecting fluorescence emission from a fluorescently tagged or labeled oligonucleotide probe that hybridizes to a nucleic acid and/or a nucleic acid amplification product and gives off/releases fluorescence emission as a result of hybridization with the nucleic acid and/or the nucleic acid amplification product.
  • any measurement tool known in the art may be used to analyze nucleic acids present in a sample and/or subject as described above, e g., a spectrophotometer for absorption or calorimetric measurements, a fluorometer or flow cytometer for fluorescence measurements, a scintillation or gamma counter for radioactive measurements, and an automated cell counter, automated plate counter, or manual plate counter for cell number measurements.
  • a microwell reader can be used for fluorescence, absorbance or calorimetric measurements.
  • measurements to detect nucleic acids/nucleic acid amplification products may include analysis by digital PCR.
  • measurements to detect nucleic acids/nucleic acid amplification products may include analysis by droplet digital PCR using, for example, but not limited to, ddPCRTM using Bio-Rad's QX100TM or QX200TM Droplet Digital PCR systems.
  • Measurement and analy sis of amplification products may include analysis of ID and/or 2D plots of fluorescence amplitude exhibited in a droplet digital PCR amplification.
  • measurements to identify nucleic acids/nucleic acid amplification products may include analysis by digital PCR.
  • measurements to identify nucleic acids/nucleic acid amplification products may include analysis by droplet digital PCR using, for example, but not limited to, ddPCRTM using Bio-Rad's QX100TM or QX200TM Droplet Digital PCR systems.
  • Measurement and analysis of amplification products may include analysis of ID and/or 2D plots of fluorescence amplitude exhibited in a droplet digital PCR amplification.
  • amplification, detection, analysis and/or identification of nucleic acids/nucleic acid amplification products may be performed as described by Maggi et al., 2020. J. Microbiol. Methods. 176, 106022.
  • capturing and concentrating of nucleic acids and/or nucleic acid sequences using highly porous hydrogel particles can be accomplished using, for example, NANOTRAP® technology (Ceres Nanosciences, Inc. (Manassas, VA)), including NANOTRAP® particles and/or NANOTRAP® magnetic particles, such as are described in. for example, U.S. Patent Application Publication Nos. 2009/0087346, 2009/0148961, 2012/0164749, and 2014/0045274.
  • Nanoparticles for example, NANOTRAP® particles and NANOTRAP® magnetic particles, functionalized with affinity baits, affinity’ groups, and/or affinity ligands that have very’ high affinities for the particular nucleic acids and/or nucleic acid sequences that arc biomarkers for a particular fastidious microorganism may be used, for example, to capture the particular nucleic acids and/or nucleic acid sequences on the nanoparticles under conditions that the particular nucleic acids and/or nucleic acid sequences bind to/have high affinities for the nanoparticles, followed by separating/isolating the nanoparticles including the captured nucleic acids and/or nucleic acid sequences bound to the nanoparticles from the sample, and eluting the captured nucleic acids and/or nucleic acid sequences from the nanoparticles to provide an enrichment of/an enriched sample including the particular nucleic acids and/or nucleic acid sequences.
  • the enriched sample may be suitable further analysis, such as
  • the methods described herein can utilize at least tw o sets of primers for separately amplifying a region of interest in a target polynucleotide sequence and a reference polynucleotide sequence used for comparison.
  • Primers can be designed with a 5' tail sequence to allow discrimination of amplicons for the target polynucleotide and the reference polynucleotide.
  • a first set of primers having tails of a certain length can be used for amplification of the region of interest in the target polynucleotide sequence, and a second set of primers having tails of a different length from that of the first set of primers can be used for amplification of the reference polynucleotide sequence, such that amplifying the nucleic acids with the two sets of primers results in amplicons of the target polynucleotide and the reference polynucleotide having detectably different lengths.
  • a fluorescent DNA dye can be used for detection of the amplicons resulting from the PCR reaction.
  • Any DNA dye can be used that binds nonspecifically to DNA (i.e. binds DNA of any sequence), which pennits discrimination of the amplicons by length and quantitative measurement of the nucleic acids.
  • Exemplary DNA dyes that can be used include EvaGreen dye (EG) dye, SYBR green, SYBR green II, SYBR gold, Oxazole yellow (YO), YOYO, Thiazole orange (TO). PicoGreen (PG), and SYTO dyes.
  • Primers can be designed with a region that is complementary’ to a portion of the template nucleic acid to be amplified to allow initiation of the polymerization reaction.
  • Noncomplcmcntary nucleotides can be added to the 5' ends of primers to create the 5' tails.
  • the lengths of primers range between 7- 100 nucleotides in length, such as 15-60, 20-40, and so on, more typically in the range of between 20-40 nucleotides long, and any length between die stated ranges.
  • primers and amplification conditions should be selected to obtain this result.
  • the lengths of the primers can be extended or shortened at the 5' end or the 3' end to produce primers with desired melting temperatures.
  • the complimentary regions of primers designed for amplification of the target and reference sequences may be the same length, their non-complimentary tail sequences will generally have different lengths to allow discrimination of amplicons for the target and reference polynucleotides.
  • Primers with shorter tails can be designed with a higher GC content, whereas primers with longer tails can be designed with a higher AT content to approximately match melting temperatures for the primertemplate complexes for target and reference polynucleotides, preferably within 3 °C of each other.
  • the amplification efficiency of any pair of primers can be readily determined using routine techniques (see e.g., Furtado et al.. “Application of real-time quantitative PCR in the analysis of gene expression.” DNA amplification: Current Technologies and Applications. Wymondham. Norfolk, UK: Horizon Bioscience p. 131-145 (2004)).
  • Primers can readily be synthesized by standard teclmiques, e.g., solid phase synthesis via phosphoramidite chemistry , as disclosed in U.S. Pat. Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311; and Applied Biosystems User Bulletin No. 13 (1 Apr. 1987).
  • Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et al., Meth. Enzymol. (1979) 68:109.
  • Poly (A) or poly (C), or other non-complementary nucleotide extensions may be incorporated into oligonucleotides using these same methods.
  • Hexaethylene oxide extensions may be coupled to the oligonucleotides by methods known in the art. Cload et al., J. Am. Chem. Soc. (1991) 113:6324-6326; U.S. Pat. No. 4,914,210 to Levenson et al.; Durand et al., Nucleic Acids Res. (1990) 18:6353-6359; and Hom et al., Tet. Lett. (1986) 27:4705-4708.
  • one or more PCR additives or enhancing agents may be included to improve the yield of the amplification reaction, for example, by reducing secondary structure in a nucleic acid or mispriming events.
  • additives or enhancing agents include, but are not limited to, dimethyl sulfoxide (DMSO), N,N,N-trimethylglycine (betaine), formamide, glycerol, nonionic detergents (e.g., Triton X-100, Tween 20, and Nonidet P-40 (NP-40)), 7-deaza-2'-deoxy guanosine, bovine serum albumin, T4 gene 32 protein, polyethylene glycol, 1,2-propanediol, and tetramethylammonium chloride.
  • DMSO dimethyl sulfoxide
  • betaine N,N,N-trimethylglycine
  • formamide glycerol
  • nonionic detergents e.g., Triton X-100, Tween 20, and Nonidet P-40 (NP
  • primers designed with 5 '-tails may be used in polymerase chain reaction (PCR)-based techniques to distinguish amplicons resulting from different polynucleotide sequences based on length.
  • PCR can be used for amplifying a desired target nucleic acid sequence contained in a nucleic acid molecule or mixture of molecules.
  • a pair of primers is employed in excess to hybridize to the complementary strands of the target nucleic acid.
  • the primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves after dissociation from the original target strand.
  • New primers are then hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules.
  • the PCR method for amplifying target nucleic acid sequences in a sample is well known in the art and has been described in, e.g.. Iimis et al. (eds.) PCR Protocols (Academic Press, N Y 1990); Taylor (1991) Polymerase chain reaction: basic principles and automation, in PCR: A Practical Approach, McPherson et al. (eds.) IRL Press, Oxford; Saiki et al. (1986) Nature 324:163; as well as in U.S. Pat. Nos. 4,683,195, 4,683.202 and 4,889.818, all incorporated herein by reference in their entireties.
  • PCR uses relatively short oligonucleotide primers which flank the target nucleotide sequence to be amplified, oriented such that their 3' ends face each other, each primer extending toward the other.
  • the polynucleotide sample is extracted and denatured, preferably by heat, and hybridized with the first and second primers that are present in molar excess.
  • Polymerization is catalyzed in the presence of the four deoxyribonucleotide triphosphates (dNTPs — dATP, dGTP, dCTP and dTTP) using a primer- and template-dependent polynucleotide polymerizing agent, such as any enzyme capable of producing primer extension products, for example, E.
  • dNTPs deoxyribonucleotide triphosphates
  • thermostable DNA polymerases isolated from Thermus aquaticus (Taq), available from a variety of sources (for example, Perkin Elmer), Thermus thermophilus (United States Biochemicals), Bacillus stereothennophilus (Bio-Rad), or Thermococcus litoralis (“Vent” polymerase, New England Biolabs). This results in two “long products” which contain the respective primers at their 5' ends covalently linked to the newly synthesized complements of the original strands.
  • the reaction mixture is then returned to polymerizing conditions, e.g., by lowering the temperature, inactivating a denaturing agent, or adding more polymerase, and a second cycle is initiated.
  • the second cycle provides the two original strands, the two long products from the first cycle, two new long products replicated from the original strands, and two “short products” replicated from the long products.
  • the short products have the sequence of the target sequence with a primer at each end.
  • an additional two long products are produced, and a number of short products equal to the number of long and short products remaining at the end of the previous cycle.
  • the number of short products containing the target sequence grows exponentially with each cycle.
  • PCR is carried out with a commercially available thermal cycler, e.g.. Perkin Elmer.
  • the amplification products can be detected in solution or using solid supports.
  • the methods described herein can be used to quantitate mRNA transcript levels of heterologous sequences encoding a protein of interest. Gene copy number and mRNA transcript levels have been shown to be correlated with antibody production. See Jiang et al.. 2006. Biotechnology Progress 22:313- 318.
  • the protein of interest will be an antigen binding protein with a heavy chain and a light chain.
  • the mRNA transcript levels can be measured for both the heavy chain and light chain. In such embodiments, it may be desirable to obtain a ratio of 1 : 1 for the heavy chain and light chain in order to achieve optimal formation of antibody.
  • the protein of interest will be an antigen binding protein with two heavy chains (HC1 and HC2) and one light chain (LC). In yet other embodiments, the protein of interest will be an antigen binding protein with two heavy chains (HC1 and HC2) and two light chains (LC1 and LC2). For three- and four-chain molecules, any combination of ratios of transcript levels can be used to assess potential candidates, e.g., HC1:HC2. [0076] Due to possible issues with individual chain(s). such as antibody chains, that may affect product quality (e.g..).
  • a preferred workflow selects and propagates those cells producing product that meet process and product quality requirements.
  • cells expressing the lowest levels of transcripts are eliminated from further processing. For example, single-cell clones or pools of cells with the lowest 80 th , 70 th , 60 th , 50 th , 40 th , or 30 th percentile of transcript levels can be eliminated from the workflow.
  • the remaining single-cell clones or pools of cells are cultured by performing fed-batch for at least an additional 8. 9, or 10 days to assess the best performing e h single-cell clones or pools of cells.
  • the viability of the cells is above 80%.
  • an expression vector can be optimized to achieve the desired ratio of heavy chain to light chain through the use of different promoter combinations, signal sequences, enhancer elements, and the like.
  • Transcript expression is normalized to transcript expression of a reference housekeeping gene so that apparent differences in RNA levels in different samples are not distorted by differences in sample amounts. Expression of each chain can be summed in the same sample and compared across different samples to determine relative expression.
  • the methods described herein can be adapted for multiplex PCR, for example, to detect and/or quantify multiple target polynucleotides simultaneously.
  • a plurality of primer sets comprising forward and reverse primers, can be used in each reaction mixture, each set of primers directed to different target polynucleotide sequences and comprising 5' tails of detectably different lengths to allow' the amplicons produced from the different target polynucleotides to be distinguished based on the intensity' of their fluorescent signals after binding a nonspecific fluorescent DNA dye.
  • multiplexing is performed such that all species of various target polynucleotides of interest can be simultaneously detected and/or quantified from a single sample.
  • a plurality of primers sets is used to simultaneously detect and/quantify different target alleles at the same locus or at different target loci. In other embodiments, a plurality of primers sets is used to simultaneously quantify transcript level expression of different target polynucleotide sequences (e.g., LC, HC).
  • target polynucleotide sequences e.g., LC, HC
  • a specific embodiment is a method wherein at least one step is performed in multi -well plates as well as a method wherein at least step b) is performed in multi-well plates.
  • the multi-well plates can be 96-well plates or 384-well plates, preferably 384-well plates.
  • a further embodiment is a method wherein steps a) to e) are performed in 96-well plates and steps g) to j) are performed in 384-well plates.
  • sample tracking can be ensured by barcoded plates and barcode reader using methods well known in the art.
  • Expression of a protein of interest in a cell can be achieved by well-known methods, either transiently or by stable expression (Davis et a!., Basic Methods in Molecular Biology, 2 nd ed., Appleton & Lange, Norwalk. Conn., 1994; Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).
  • stable integration is commonly achieved by transiently introducing a heterologous polynucleotide or a vector containing the heterologous polynucleotide into the host cell, which facilitates the stable integration of said heterologous polynucleotide into the cell genome.
  • the heterologous polynucleotide is flanked by homology arms, i.e., sequences homologous to the region upstream and downstream to the integration site.
  • circular vectors may be linearized to facilitate integration into the cell genome.
  • Methods for the introduction of vectors into cells are well known in the art and include transfection with biological methods, such as viral deliver . with chemical methods, such as using cationic polymers, calcium phosphate, cationic lipids or cationic amino acids; with physical methods, such as electroporation or microinjection; or with mixed approaches, such as protoplast fusion.
  • a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods.
  • a specific method of stable integration uses recombinase mediated cassette exchange (RMCE; Bode and Baer, 2001. Curr Opin Biotechnol. 12:473-80, and Bode et al., 2000. Biol. Chem. 381:801- 813) for site-specific integration in the genome (also termed “targeted integration”).
  • Site-specific recombinases such as Flp and Cre mediate recombination betw een tw o copies of their target sequence termed FRT and loxP, respectively.
  • Insertion of RMCE into a specific site in the genome can be mediated by nucleases (e.g., zinc finger protein (ZFP), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)) that can be engineered to create single- and double-stranded breaks (SSBs/DSBs) in the genome.
  • ZFP zinc finger protein
  • TALEN transcription activator-like effector nuclease
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas9 CRISPR-associated protein 9
  • Homologous recombination requires the presence of a homologous sequence as a template (e.g., "donor” containing RMCE) to guide the cellular repair process and the results of the repair are error-free and predictable.
  • a template or "donor”
  • the cell In the absence of a template (or "donor") sequence for homologous recombination, the cell typically attempts to repair the DSB via the unpredictable and error-prone process of non-homologous end-joining (NHEJ).
  • NHEJ non-homologous end-joining
  • a vector may be any molecule or entity (e.g.. nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, virus capsid, virion, naked DNA, complexed DNA and the like) suitable for use to transfer and/or transport protein encoding information into a host cell and/or to a specific location and/or compartment within a host cell.
  • Vectors can include viral and non-viral vectors, non-episomal mammalian vectors. Vectors are often referred to as expression vectors, for example, recombinant expression vectors and cloning vectors.
  • the vector may be introduced into a host cell to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein.
  • the cloning vectors may contain sequence components generally including, w ithout limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • Vectors are useful for transformation of a host cell and contain nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto.
  • An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto. “Operably linked” means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions.
  • a control sequence e.g., a promoter
  • a vector that is “operably linked’’ to a protein coding sequence are arranged such that normal activity of the control sequence leads to transcription of the protein coding sequence resulting in recombinant expression of the encoded protein.
  • Vectors may be selected to be functional in the particular host cell employed (i.e. , the vector is compatible with the host cell machinery, permitting amplification and/or expression of the gene to occur).
  • vectors are used that employ protein-fragment complementation assays using protein reporters, such as dihydrofolate reductase (see, for example. U.S. Pat. No. 6,270,964).
  • protein reporters such as dihydrofolate reductase (see, for example. U.S. Pat. No. 6,270,964).
  • Suitable expression vectors are known in the art and are also commercially available.
  • vectors used in host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • sequences will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, transcriptional and translational control sequences, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, various pre- or pro-sequences to improve glycosylation or yield, a native or heterologous signal sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, internal ribosome entry site (IRES) sequences, an expression augmenting sequence element (EASE), tripartite leader (TP A) and VA gene RNAs from Adenovirus 2, a polylinker region for inserting the polynucleotide encoding the polypeptide to be expressed, and
  • Vector components may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (e g., from a species other than the host cell species or strain), hybrid i.e.. a combination of flanking sequences from more than one source), synthetic or native.
  • sequences of components useful in the vectors may be obtained by methods well known in the art, such as those previously identified by mapping and/or by restriction endonucleases. In addition, they can be obtained by polymerase chain reaction (PCR) and/or by screening a genomic library' with suitable probes.
  • a ribosome-binding site is usually necessary' for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes).
  • the element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.
  • An origin of replication aids in the amplification of the vector in a host cell. They may be included as part of commercially available prokaryotic vectors and may also be chemically synthesized based on a known sequence and ligated into the vector. Various viral origins (e.g.. SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV). or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells.
  • SV40 polyoma
  • adenovirus vesicular stomatitus virus
  • papillomaviruses such as HPV or BPV
  • Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes.
  • promoter and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV).
  • SV40 adenovirus 2
  • CMV human cytomegalovirus
  • the human CMV promoter/enhancer of immediate early gene 1 may be used. See e.g. Patterson et al., 1994, Applied Microbiol. Biotcchnol. 40:691-98.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell.
  • Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., 1978, Nature 273:113; Kaufman, 1990, Meth, in Enzymol. 185:487-511). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgll site located in the SV40 viral origin of replication site is included.
  • a transcription termination sequence is typically located 3' to the end of a polypeptide coding region and serves to terminate transcription.
  • a transcription termination sequence in prokary otic cells is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library' or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis known to those of skill in the art.
  • a selectable marker gene encoding a protein necessary' for the survival and growth of a host cell grown in a selective culture medium.
  • Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex or defined media.
  • Specific selectable markers arc the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
  • a neomycin resistance gene may also be used for selection in both prokaryotic and eukaryotic host cells.
  • selectable marker genes may be used to amplify the gene that w ill be expressed. Amplification is the process wherein genes that are required for production of a protein critical for growth or cell survival are reiterated in tandem within the chromosomes of successive generations of recombinant cells.
  • suitable selectable markers for mammalian cells include glutamine synthase (GS), dihydrofolate reductase (DHFR), and promoterless thymidine kinase genes.
  • GS glutamine synthase
  • DHFR dihydrofolate reductase
  • Mammalian cell transformants arc placed under selection pressure wherein only the transformants arc uniquely adapted to survive by virtue of the selectable marker gene present in the vector.
  • Selection pressure is imposed by culturing the transformed cells under conditions in w hich the concentration of selection agent in the medium is successively increased, thereby leading to the amplification of both the selectable marker gene and the DNA that encodes a protein of interest.
  • increased quantities of a polypeptide of interest are synthesized from the amplified DNA.
  • the final protein product may have, in the -1 position (relative to the first amino acid of the mature protein), one or more additional amino acids incident to expression, which may not have been totally removed.
  • the final protein product may have one or tw o amino acid residues found in the peptidase cleavage site, attached to the amino-terminus.
  • use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cuts at such area within the mature polypeptide.
  • Expression and cloning will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding a protein of interest. Promoters are untranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, uniformly transcribe a gene to which they are operably linked, that is, with little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known.
  • Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hcpatitis-B virus, and Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hcpatitis-B virus, and Simian Virus 40 (SV40).
  • adenovirus such as Adenovirus 2
  • bovine papilloma virus such as Adenovirus 2
  • bovine papilloma virus such as Adenovirus 2
  • elastase I gene control region that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al.,
  • Biol. 7: 1436-1444 the mouse mammary 7 tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495); the albumin gene control region that is active in liver (Pinkert et al., 1987, Genes and Devel. 1 :268-276); the alpha-feto-protein gene control region that is active in liver (Knunlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 253:53-58); the alpha 1-antitrypsin gene control region that is active in liver (Kelsey et al., 1987, Genes and Devel.
  • Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent, having been found at positions both 5' and 3' to the transcription unit.
  • enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus is used.
  • the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be positioned in the vector either 5' or 3' to a coding sequence, it is ty pically located at a site 5' from the promoter.
  • a sequence encoding an appropriate native or heterologous signal sequence can be incorporated into an expression vector, to promote extracellular secretion of the protein of interest.
  • the choice of signal peptide or leader depends on the type of host cells in which the protein of interest to be produced, and a heterologous signal sequence can replace the native signal sequence.
  • Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 described in U.S. Patent No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846.
  • Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., in Animal Cell Technology, pp. 529-534 (1997); U.S. Patent Nos. 6,312,951 Bl, 6,027,915, and 6,309,841 Bl) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., 1982, J. Biol. Chem. 257: 13475-13491).
  • EASE expression augmenting sequence element
  • TPL tripartite leader
  • VA gene RNAs from Adenovirus 2
  • IVS internal ribosome entry site
  • one or more vectors may be inserted into a suitable cell for amplification and/or polypeptide expression.
  • the transformation of an expression vector into a selected cell may be accomplished by well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, nucleofection, microinjection. DEAE -dextran mediated transfection, cationic lipids mediated delivery, liposome mediated transfection, microprojectile bombardment, receptor- mediated gene delivery , delivery mediated by polylysine, histone, chitosan, and peptides.
  • the method selected will in part be a function of the type of host cell to be used.
  • transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA.
  • a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques.
  • the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell or can be maintained transiently as an episomal element without being replicated or can replicate independently as a plasmid.
  • a cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell.
  • a number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al..
  • transduction refers to the process whereby foreign DNA is introduced into a cell via viral vector. See Jones et al.. (1998). Genetics: principles and analysis. Boston: Jones & Bartlett Publ.
  • a “protein of interest” includes naturally occurring proteins, recombinant proteins, and engineered proteins (e.g.. proteins that do not occur in nature and which have been designed and/or created by humans) or polypeptides. Polypeptides and proteins of interest can be of scientific or commercial interest. A protein of interest can, but need not be, a protein that is known or suspected to be therapeutically relevant.
  • Proteins of interest include, among other things, secreted proteins, non-secreted proteins, intracellular proteins or membrane-bound proteins. Polypeptides and proteins of interest can be produced by recombinant animal cell lines using cell culture methods and may be referred to as “recombinant proteins”. The expressed protein(s) may be produced intracellularly or secreted into the culture medium from which it can be recovered and/or collected.
  • isolated protein or “isolated recombinant protein” refers to a polypeptide or protein of interest, that is purified away from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. Proteins of interest include proteins that exert a therapeutic effect by binding a target, particularly a target among those listed below, including targets derived therefrom, targets related thereto, and modifications thereof.
  • Proteins of interest include “antigen-binding proteins”.
  • Antigen-binding protein refers to proteins or polypeptides that comprise an antigen-binding region or antigen-binding portion that has affinity for another molecule to which it binds (antigen).
  • Antigen-binding proteins encompass antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including singlechain variable fragments (scFvs), double-chain (divalent) scFvs, and IgGscFv (see, e.g., Orcutt et al., 2010, Protein Eng Des Sei 23:221-228), hetero-IgG (see, e.g., Liu et al., 2015, J Biol Chem 290:7535- 7562), muteins, and antibodies made by the XmAb® process (Xencor, Inc., Monrovia, CA).
  • antigen binding proteins include a human antibody, a humanized antibody; a chimeric antibody; a recombinant antibody; a single chain antibody; a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab’)2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an IgG2a antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof.
  • bispecific T cell engager molecules BiTE®
  • bispecific T cell engager molecules having extensions such as half-life extensions, for example HLE BiTE ® molecules, Heterolg BiTE * and others, chimeric antigen receptors (CARs, CAR Ts), and T cell receptors (TCRs).
  • CARs CAR Ts
  • TCRs T cell receptors
  • BlmAb refers to [Fab-scFv*Fab]- heteroFc
  • BIHmAb refers to [Fab-VH*Fab]-heteroFc
  • B2mAb refers to a Fab-scFv-Fc
  • B2HmAb refers to Fab-VH-Fc
  • ClmAb refers to a Fab-heteroFc-[scFv*]
  • C2mAb refers to a IgG-scFv
  • C2HmAb refers to a IgG-VH.
  • the term “antigen binding protein” is used in its broadest sense and means a protein comprising a portion that binds to an antigen or target and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen.
  • the antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody -derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See.
  • PAMs peptide antibody mimetics
  • antibody heavy chain and antibody light chain include both standard full-length antibody heavy chains and light chains, as well as derivatives thereof that contain at the respective variable region (VL or VH).
  • antibody heavy chain fusion and “antibody heavy chain fusion protein” refer to a polypeptide that contains an antibody heavy chain covalently linked to one or more additional proteins or peptides.
  • an “antibody heavy chain fusion protein” can be an antibody heavy chain covalently linked to a cytokine.
  • the linkage may be direct, or via a peptide linker (e.g. a glycine-serine linker).
  • an antibody heavy chain fusion protein In an antibody heavy chain fusion protein, the antibody heavy chain may be linked to additional protein(s) at the N- terminus or the C-terminus of the heavy chain (or both locations).
  • the terms "antibody light chain fusion protein” and “antibody light chain fusion” have the same meaning as described immediately above for “antibody heavy 7 chain fusion protein”, except for an antibody light chain.
  • an “antibody fusion protein” refers to an antibody as provided herein which is covalently linked to one or more additional proteins or polypeptides (e.g. via a heavy chain or light chain of the antibody).
  • an antibody fusion protein contains at least an antibody heavy chain fusion protein or an antibody light chain fusion protein as one of the polypeptides of the antibody fusion protein.
  • an antibody fusion protein is a molecule that contains two antibody light chains, one antibody heavy chain, and one antibody heavy chain fusion protein, such that the additional protein is linked to one of the heavy chains of the antibody.
  • An antigen binding protein can have, for example, the structure of a naturally occurring immunoglobulin.
  • An "immunoglobulin” is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1. CDR1, FR2. CDR2. FR3, CDR3 and FR4. The assignment of amino acids to each domain can be done in accordance with the definitions of Rabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept, of Health and Human Services, PHS, NIH. NIH Publication no. 91-3242, (1991). As desired, the CDRs can also be redefined according to an alternative nomenclature scheme.
  • rheumatoid factors immunotoxins, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, MIC (MIC -a, MIC-B), ULBP 1-6, EPCAM, addressins, regulatory proteins, immunoadhesins, antigen-binding proteins, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and
  • CHO pools expressing three different types of molecules were evaluated: 4-chain heteroIgG, 3- chain AmAb, and 3-chain ClmAb.
  • 1 x 10 6 - 5 x 10 6 cells were collected during normal pool passaging and fed batch production with a criterion of >80% cell viability at harvest. Cells were spun down at 300 x g for 5 min and then a wash step in lx DPBS (Life Technologies) was performed to remove any residual media. Cell samples were frozen in either 1 mL cry opreservation media (cell culture media plus 10% DMSO) or 350 pL of RNeasy kit RLT buffer (QIAGEN) and stored at -80°C until ready for cDNA generation.
  • RNA quick extract kit BioRad SingleShot Cell Lysis
  • a reverse transcription step was performed using iScript cDNA synthesis kit (BIORAD) with an incubation time of 20 minutes at 46°C using a Thermal Cycler (Applied Biosystems).
  • the mRNA and cDNA were quantified and checked for quality by the 260/280 absorbance ratio using a NanoDrop spectrophotometer following mRNA isolation and cDNA synthesis.
  • Primer and probe sets specific to unique sequences in each heavy chain and light chain of each molecule were designed using PrimerExpress or Integrated DNA Technologies (IDT) PrimerQuest Tool and ordered from IDT.
  • ddPCR assay to quantity mRNA transcript levels Two-step RT-ddPCR was used to rapidly quantify mRNA transcript levels (e.g., each heavy chain and light chain of a multispecific antibody). Each reaction consisted of 0.2 ng/pL cDNA, 18 LIM of each primer and 5 u M of the probe for both the target and ChoBAct reference gene (Sigma- Aldrich) combined with Supermix for probes (no dUTP) (BIORAD) and water to reach a final volume of 20 pl per well. Droplets were generated using the AutoDG system (BIORAD) and PCR amplification was performed using the following thermal cycling conditions: 95°C for 10 minutes.
  • Protein expression and product transcript levels from FB pools are shown in Figure 1 and normpass pools are shown in Figure 2. Both protein expression and product transcript levels were found to positively correlate with titer results for (A) 4- chain heteroIgG, (B) a 3-chain molecule, and (C) an asymmetric ClmAb.
  • HC1/HC2 ratio is positively proportional to main peak, but inversely proportional to LMW (SEC) (where HC2 is the shorter of two HC in the 3-chain molecule).
  • Fig. 5D shows a strong correlation between HC1:HC2 ratio versus MP and LMW. Top clone falls at around the 0.6 HC1:HC2 ratio, marked by a black square.
  • This assay can be used to predict not only titers from pools and clones but also product quality attributes (PQA). For example for a 3-chain molecule, HC1/HC2 transcript ratio was analyzed for correlations with PQA and found to be positively proportional to %main peak and %high molecular species (HMW) (as measured by size-exclusion chromatography, SEC) and inversely proportional to %pre-peaks (as measured by non-reduced capillary electrophoresis. nr-CE) and %low molecular weight species (LMW, as measured by SEC) at both fed batch and norm-pass culture timepoints. See Figures 3A- B. Overall, this assay could sen e as a powerful tool to predict top performing pools before FB assessment with potential applications in clone screening as well.
  • HMW %main peak
  • SEC size-exclusion chromatography
  • nr-CE %low molecular weight species
  • LMW %low molecular weight

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Abstract

La présente divulgation concerne le domaine de la technologie de culture cellulaire. L'invention concerne un procédé pour permettre la sélection d'un clone cellulaire unique ou de groupes de cellules pour la fabrication d'une protéine de liaison à l'antigène avec deux à quatre chaînes d'anticorps différentes. Le procédé utilise la ddPCR pour la quantification de niveaux de transcription.
PCT/US2024/051847 2023-10-18 2024-10-17 Évaluation de niveaux de transcrit d'arnm à l'aide d'une ddpcr pour une évaluation précoce d'un clone et d'une seule cellule dans le développement d'une lignée cellulaire Pending WO2025085682A1 (fr)

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