Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
  • A61P25/36—Opioid-abuse
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• Opioids are substances that act on opioid receptors to produce morphine-like analgesic effects in humans and other mammals. Opioids are most often used medically to relieve pain, and by people addicted to opioids. Opioids include opiates, an older term that refers to such drugs derived from opium, including morphine itself.
• opioids are semi- synthetic and synthetic drugs such as hydrocodone (aka dihydrocodeinone, 4,5 ⁇ -epoxy-3- methoxy-17-methylmorphinan-6-one); oxycodone (aka dihydrohydroxycodeinone, 6-deoxy- 7,8-dihydro-14-hydroxy-3-O-methyl-6-oxomorphine); fentanyl (CAS #437-38-7; also known as fentanil, or N-(1-(2-phenethyl)-4-piperidinyl-N-phenyl-propanamide, or 1-(2- Phenylethyl)-4-(N-propananilido)piperidine); carfentanil (CAS #59708-52-0; Methyl 1-(2- phenylethyl)-4-[phenyl(propanoyl)amino]piperidine-4-carboxyl- ate); acetyl fentanyl (N-(1- QBI02PCT PCT International Patent
• Opioids produce their pharmacological effects through activation of G protein- coupled receptors (GPCRs).
• GPCRs G protein- coupled receptors
• opioid receptors There are four distinct genes coding for opioid receptors: the mu-, kappa-, and delta-opioid receptors (MOR, KOR, and DOR, respectively) and the opioid- like receptor1 (ORL-1) or the nociceptin receptor (NOP).
• MOR mu-, kappa-, and delta-opioid receptors
• ORL-1 opioid- like receptor1
• NOP nociceptin receptor
• the generation of genetic knockout mice has demonstrated that the majority of clinically used opioids including morphine produce their pharmacological effects primarily by activating the MOR.
• the MOR is widely distributed and expressed in neurons in the brain, spinal cord, and the periphery (Gutstein and Akil 2001).
• fentanyl While effective in reducing patient pain and discomfort, many of the opioid substances, acting upon the opioid receptors are also highly addictive, and may be lethal at relatively low doses. For example, two milligrams of fentanyl can be lethal depending on a person’s body size, tolerance and past usage.
• DEA Drug Enforcement Agency
• illicit fentanyl primarily manufactured in foreign clandestine labs and smuggled into the United States through Mexico, is being distributed across the United States and is being sold on the illegal drug market. Fentanyl is being mixed in with other illicit drugs to increase the potency of the drug, sold as powders and nasal sprays, and increasingly pressed into pills made to look like legitimate prescription opioids.
• counterfeit pills Because there is no official oversight or quality control, these counterfeit pills often contain lethal doses of fentanyl, with none of the promised drug. There is significant risk that illegal drugs have been intentionally contaminated with fentanyl. Because of its potency and low cost, drug dealers have been mixing fentanyl with other drugs including heroin, methamphetamine, and cocaine, increasing the likelihood of a fatal interaction. Analysis by the DEA of seized material has found counterfeit pills ranging from 0.02 to 5.1 milligrams (more than twice the lethal dose) of fentanyl per tablet. According to the Center for Disease Control (CDC), synthetic opioids (like fentanyl) are the primary driver of the growing number of overdose deaths in the United States.
• CDC Center for Disease Control
• Nanobodies are roughly 1/10 th the size of conventional antibodies, with a molecular weight of about 15 kDa, while retaining comparable antigen binding affinities.
• the small size of nanobodies enables cheaper production, easier bacterial expression and surface display, and expanded applications where ABP size is relevant to delivery (e.g., drug delivery).
• Nanobodies are also remarkably stable to environmental conditions, in some cases with melting temperatures observed above 80°C and refolding after denaturation. (Goldman et al., “Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview,” Front Immunol.2017 Jul 25;8:865. doi: 10.3389/fimmu.2017.00865. eCollection 2017).
• the present invention provides, inter alia, robust assay tools for the detection of fentanyl, carfentanil, and other analytes, including with effective continuous sample monitoring systems and methods of the invention.
• the present invention relates to an isolated recombinant antigen- binding protein that specifically binds fentanyl or carfentanil, comprising a V HH domain comprising a set of three complementarity determining regions (CDR): CDR1, CDR2, and CDR3, wherein each CDR comprises an amino acid sequence, wherein the set of three CDRs is selected from the group consisting of: [00015] (a) SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; [00016] (b) SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:44; QBI02PCT PCT International Patent Application [00017] (c) SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; [00018] (d) SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; [00019] (e) SEQ ID NO:17, SEQ ID NO:17, SEQ ID NO:17, SEQ ID NO:
• the present invention relates to an isolated nucleic acid, comprising a nucleotide sequence encoding the inventive recombinant antigen-binding protein, to an expression vector comprising the nucleic acid, and to a host cell, in culture, comprising the expression vector.
• Such host cells can be used in a method of producing an antigen-binding protein that specifically binds to fentanyl and/or carfentanil.
• VHH domain antibodies or “nanobodies” can be used as diagnostic tools, or in a pharmaceutical composition as a therapeutic in the treatment of a patient addicted to opiates (e.g., fentanyl) or the event of opiate overdose.
• the present invention is directed to the inventive recombinant antigen-binding proteins being expressed on the surface of a host cell, e.g., a microorganism.
• a host cell e.g., a microorganism.
• the inventive antigen-binding proteins can be used for real-time detection of fentanyl and/or carfentanil, employing synthetic biology to integrate fentanyl-binding nanobodies into gene circuits in microbial cells, such as E. coli, to develop biosensor strains that emit a signal in the presence of fentanyl.
• microbial cells such as E. coli
• biosensor strains that emit a signal in the presence of fentanyl.
• immunological assays such as lateral flow or agglutination assays.
• the present invention also relates to a continuous agglutination assay method for detecting an analyte of interest, which can be fentanyl and/or carfentanil, or a different analyte of interest.
• the continuous agglutination assay method includes the following steps: [00030] (a) mixing in a reaction vessel, a fluid aqueous suspension of host cells that display on their surfaces a plurality of recombinant antigen-binding proteins that specifically bind an analyte of interest, with a fluid aqueous sample, in a reaction mixture with an agglutinating agent comprising the analyte of interest, or an analyte conjugate, under conditions of temperature and pH that permit binding of the analyte or the analyte conjugate, by the antigen-binding protein and agglutination of the cells, in the presence of the analyte of interest; [00031] (b) measuring a parameter in a preselected portion of the reaction mixture over a continuous time course; [00032] (c) correlating the change in the measured parameter over the time course with the level of agglutination; and [00033] (d) normalizing the level of aggluti
• the continuous agglutination assay method can be practiced using an automated system for continuous agglutination assay, which the present invention also provides.
• the inventive automated system for continuous agglutination assay includes the following: [00034] (a) a first vessel configured to receive and to contain, a fluid aqueous suspension comprising host cells that display on their surfaces a plurality of recombinant antigen-binding proteins that specifically bind an analyte of interest, and, wherein the fluid aqueous suspension optionally comprises an antibiotic or bacteriostatic compound, a coagulant and/or an agglutination-enhancing additive; [00035] (b) an optional second vessel configured to receive and to contain a fluid aqueous sample to be analyzed; [00036] (c) a reaction vessel fluidly connected to the first vessel and to the optional second vessel by connecting lines, wherein the reaction vessel is configured to automatically receive via the connecting lines, a predetermined volume of the fluid aqueous suspension from the first vessel and
• the left panel shows ELISA results for 94 recovered nanobody proteins, targeted by an anti-alpaca V HH domain antibody.
• the net difference in absorbance (of 405 nm wavelength light) between a plate coated with fentanyl-BSA conjugate and unconjugated BSA is shown.
• the negative control well contains no nanobody and the positive control well was coated with a nanobody. Error bars represent standard deviation of VHH sequences present in two or more wells.
• the right panel shows the number of replicate wells containing each of the 28 unique VHH sequences from the 94 total colonies screened.
• Figure 3 shows normalized absorbance (of 600 nm wavelength light) of cell cultures after a 30-minute agglutination reaction, incubating cell cultures with 0.5 nM of fentanyl- BSA (“0.5 nM fen.-BSA”; white bars) and 200 ppb of either unbound fentanyl (“200 ppb fen.”; diagonal hatched bars) or unbound carfentanil (“200 ppb carfen.”; cross-hatched bars), as described in Example 2 herein.
• Absorbance for each strain is normalized to the no fentanyl-BSA condition (“No fen.-BSA”; gray bars). Error bars represent the standard deviation of triplicate tests.
• Figure 7a-d schematically illustrates an exemplary embodiment of the inventive continuous agglutination assay method for detecting an analyte of interest, as further described in Example 5 herein.
• Figure 7(a) left panel shows a schematic close-up representation of the agglutination reaction vessel (i.e., designated the “Cuvette” in Figure 7b).
• Figure 7b shows schematically an exemplary embodiment of the parts and instrumentation for the hardware involved in the inventive continuous agglutination assay method or system (or device), including the reaction vessel (“Cuvette”), pumps, and optional aqueous sample reservoir (shown schematically as “Water source”) and a reagent reservoir (shown here schematically as a single “Cell suspension” reservoir), although separate vessels QBI02PCT PCT International Patent Application or reservoirs for other diluents, buffers, and/or reagents can optionally be included in the assay system, with the entire assay system being under the control of the “Microcontroller,” which automatically directs the activity of the pumps, turbidity sensor apparatus, and data collection.
• the reaction vessel shown schematically as “Water source”
• a reagent reservoir shown here schematically as a single “Cell suspension” reservoir
• Figure 7d shows a representative response curve for the continuous competitive fentanyl assay extracted from the data in Figure 7c. Values were extracted after 30 minutes of the agglutination reaction, with an agglutination value representing the mean change in transmitted light of reactions containing 0 ppb fentanyl, and an agglutination value of 0 representing no change in transmitted light over the 30 minutes.
• Figure 8a-b shows representative data obtained from the inventive continuous agglutination assay method, in which the analyte of interest was a protein target (e.g., an antibody), as further described in Example 6 herein.
• Figure 8a shows an agglutination time series at varying concentrations of purified mouse IgG1, ⁇ isotype control antibody (BioLegend, Cat. No.400101). Each agglutination reaction was allowed to proceed for 45 minutes.
• Figure 8b shows bar plots of the mean agglutination at increasing antibody concentrations. Error bars represent the standard deviation of triplicate samples.
• FIG. 9 shows a schematic illustration of embodiment of a continuous, real-time system was also developed with pressurized gas directed into the headspace of the respective cell suspension reservoir(s) (“Vessel 1”) and the optional aqueous sample reservoir (“Vessel QBI02PCT PCT International Patent Application 2”), to control the flow of agglutinating agent and fluid aqueous sample into the reaction vessel.
• the arrows point in the direction of gas and fluid flow.
• Two electro-pneumatic gas pressure regulators (0.15-15 psi) for the gas plumbing system are shown.
• DETAILED DESCRIPTION OF EMBODIMENTS [00052]
• the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
• the isolated recombinant antigen-binding protein that specifically binds fentanyl or carfentanil is a single variable- domain antibody (“sdAb” or “nanobody” or “Nb,” terms used interchangeably herein), devoid of a light chain, specifically binding to fentanyl and/or carfentanil.
• the single domain antibody is derived from camelids. In the family of “camelids,” immunoglobulins devoid of light polypeptide chains are found.
• “Camelids” comprise old-world camelids (Camelus bactrianus and Camelus dromaderius) and new world camelids (for example, Lama paccos, Lama glama and Lama vicugna).
• the term “nanobody,” as used herein in its broadest sense, is not limited to a specific biological source or to a specific method of preparation.
• the antigen binding proteins of the present invention can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring V HH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized V HH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species and, in particular, from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain antibody” or “Dab” as described in the art, or by expression of a nucleic acid
• VHH sequences can generally be generated or obtained by suitably immunizing a species of camelid with fentanyl or carfentanil (i.e., so as to raise an immune response and/or heavy chain antibodies directed against fentanyl and/or fentanyl), by obtaining a suitable biological sample from the camelid (such as a blood sample, serum sample or sample of B-cells), and by generating VHH sequences directed against fentanyl (or carfentanil), starting from the sample, using any suitable technique known per se. Such techniques will be clear to the skilled person.
• an "isolated" protein e.g., an antibody protein
• an isolated protein is one that has been identified and separated from one or more components of its natural environment or of a culture medium in which it has been secreted by a producing cell.
• the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural or culture medium environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.
• Contaminant components of its natural environment or medium are materials that would interfere with diagnostic or therapeutic uses for the protein, e.g., an antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous (e.g., polynucleotides, lipids, carbohydrates) solutes.
• target refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen binding protein (including, e.g., an antibody or immunologically functional fragment of an antibody), and additionally capable of being used in an animal to produce antibodies capable of binding to that antigen.
• a selective binding agent such as an antigen binding protein (including, e.g., an antibody or immunologically functional fragment of an antibody), and additionally capable of being used in an animal to produce antibodies capable of binding to that antigen.
• Promoter and enhancer elements have been isolated from a QBI02PCT PCT International Patent Application variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis, et al., Science 236:1237 (1987)).
• in operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
• the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
• a control sequence in a vector that is "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.
• protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.
• the terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be expressed recombinantly using known protein engineering QBI02PCT PCT International Patent Application techniques.
• proteins can be derivatized as described herein and by other well- known organic chemistry techniques.
• purify or “purifying” a protein means increasing the degree of purity of the desired protein from a composition or solution comprising the protein of interest (i.e., the “POI,” and one or more contaminants by removing (completely or partially) at least one contaminant from the composition or solution.
• an "isolated protein” or, interchangeably, “protein isolate,” constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample.
• the isolated protein of interest will be “purified”: (1) to greater than 95% by weight of protein, and most preferably, more than 99% by weight, or (2) to homogeneity by SDS-PAGE, or other suitable technique, under reducing or nonreducing conditions, optionally using a stain, e.g., Coomassie blue or silver stain.
• An isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the protein's natural environment will not be present.
• the isolated or purified protein of interest e.g., a purified protein drug substance
• the isolated or purified protein of interest will be prepared by at least one purification step, which can include cell lysis (with or without centrifugation), filtration, and/or affinity chromatography.
• a "variant" of a polypeptide e.g., an immunoglobulin, or an antibody
• variants can include fusion proteins.
• fusion protein indicates that the protein includes polypeptide components derived from more than one parental protein or polypeptide.
• a fusion protein is expressed from a “fusion gene” in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence from a different protein.
• the fusion gene can then be expressed by a recombinant host cell as a single protein. Fusion proteins incorporating an antibody or an antigen-binding portion thereof are known.
• a "secreted” protein refers to those proteins capable of being directed to the endoplasmic reticulum (ER), secretory vesicles, or the extracellular space as a result of a secretory signal peptide sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage. In some other embodiments, the antibody protein of interest can be synthesized by the host cell as a secreted protein, which can then be further purified from the extracellular space and/or medium.
• soluble when in reference to a protein produced by recombinant DNA technology in a host cell is a protein that exists in aqueous solution; if the protein contains a twin-arginine signal amino acid sequence the soluble protein is exported to the periplasmic space in gram negative bacterial hosts, or is secreted into the culture medium by eukaryotic host cells capable of secretion, or by bacterial host possessing the appropriate genes (e.g., the kil gene).
• a soluble protein is a protein which is not found in an inclusion body inside the host cell.
• a soluble protein is a protein which is not found integrated in cellular membranes, or, in vitro, is dissolved, or is capable of being dissolved in an aqueous buffer under physiological conditions without forming significant amounts of insoluble aggregates (i.e., forms aggregates less than 10%, and typically less than about 5%, of total protein) when it is suspended without other proteins in an aqueous buffer of interest under physiological conditions, such buffer not containing an ionic detergent or chaotropic agent, such as sodium dodecyl sulfate (SDS), urea, guanidinium hydrochloride, or lithium perchlorate.
• SDS sodium dodecyl sulfate
• urea guanidinium hydrochloride
• lithium perchlorate lithium perchlorate
• an insoluble protein is one which exists in QBI02PCT PCT International Patent Application denatured form inside cytoplasmic granules (called an inclusion body) in the host cell, or again depending on the context, an insoluble protein is one which is present in cell membranes, including but not limited to, cytoplasmic membranes, mitochondrial membranes, chloroplast membranes, endoplasmic reticulum membranes, etc., or in an in vitro aqueous buffer under physiological conditions forms significant amounts of insoluble aggregates (i.e., forms aggregates equal to or more than about 10% of total protein) when it is suspended without other proteins (at physiologically compatible temperature) in an aqueous buffer of interest under physiological conditions, such buffer not containing an ionic detergent or chaotropic agent, such as sodium dodecyl sulfate (SDS), urea, guanidinium hydrochloride, or lithium perchlorate.
• SDS sodium dodecyl sulfate
• urea guanidinium hydroch
• oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides may be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides may be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides may be used, for example, as PCR primers, cloning primers or hybridization probes.
• a "polynucleotide sequence” or “nucleotide sequence” or “nucleic acid sequence,” as used interchangeably herein, is the primary sequence of nucleotide residues in a polynucleotide, including of an oligonucleotide, a DNA, and RNA, a nucleic acid, or a character string representing the primary sequence of nucleotide residues, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence can be determined.
• a “reporter protein” as described herein, refers to a protein that is detected which is indicative of transcription or translation from a regulatory sequence of interest in a bacteria, cell culture or animal.
• a reporter gene is a gene that is attached to a regulatory sequence of another gene. These can be used to indicate whether a certain gene is expressed in the presence of an analyte.
• a reporter proteins can be green fluorescent protein, luciferase (which can catalyze a reaction with luciferin to produce light, and red fluorescent protein.
• E.coli lacZ gene which encodes beta-galactosidase which can cause bacteria to appear in a blue color when grown in a medium that contains the substrate X-gal.
• microelectrodes may be functionalized by coating them with a thin film (for example, Prussian blue) to increase sensitivity and selectivity. They may also be coated with a protectant (for example, Nafion) to prevent fouling.
• Microelectrodes may be QBI02PCT PCT International Patent Application positioned in the same fluidic channel as the cells or in an adjacent fluidic channel, separated by a thin barrier of PDMS. The latter sensing methodology may limit chemical fouling of the microelectrode surface over long measurement durations and is feasible due to the ability of H2O2 to diffuse through PDMS.
• an electrochemical reaction product is product that can produce a detectable electric current.
• the microfluidic devices comprise microelectrodes integrated into the microfluidic device.
• the microelectrodes may be functionalized by coating them with a thin film (e.g. Prussian blue) to increase sensitivity and selectivity.
• the microelectrodes are coated with a protectant (e.g. Nafion) to prevent fouling.
• the microelectrodes are positioned in the same fluidic channel as the cells or in an adjacent fluidic channel, separated by a thin barrier of PDMS.
• the enzymatic assay QBI02PCT PCT International Patent Application product is beta-galactosidase enzyme.
• the detectable agent is detected by addition of X-gal or LuGal.
• vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.
• expression vector or "expression construct” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of the operably linked coding sequence in a particular host cell.
• An expression vector can 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.
• a QBI02PCT PCT International Patent Application single expression vector can be used to express the different subunits of the protein of interest.
• the term "host cell” means a cell that has been transformed, or is capable of being transformed, with a nucleic acid and thereby expresses a gene or coding sequence of interest.
• the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present. Any of a large number of available and well-known host cells may be used in the practice of this invention to obtain the antigen-binding proteins of the invention, including mammalian cells, insect cells, microbial cells, or plant cells.
• Cell e.g., NS0 or sp2/0 mouse myeloma cells.
• Cell e.g., NS0 or sp2/0 mouse myeloma cells.
• Cell “Cell,” “cell line,” and “cell culture” are often used interchangeably and all such designations herein include cellular progeny.
• a cell “derived” from a CHO cell is a cellular progeny of a Chinese Hamster Ovary cell, which may be removed from the original primary cell parent by any number of generations, and which can also include a transformant progeny cell. Transformants and transformed cells include the primary subject cell and cultures derived therefrom without regard for the number of transfers.
• serum refers to the fluid portion of the blood obtained after removal of the fibrin clot and blood cells.
• protein-free media that are either completely free of any protein or at least are free of any protein that is not recombinantly produced.
• FVIII Factor VIII
• Human serum albumin is commonly used as a serum-free culture supplement for the production of recombinant proteins.
• the albumin itself stabilizes the FVIII, and the impurities present in serum-derived albumin preparations may also contribute to the stabilizing effect of albumin.
• Examples include, a silicone antifoam agent, or a non-ionic surface-active agent such as a polypropylene glycol (e.g. Pluronic F-61, Pluronic F-68, Pluronic F-71 or Pluronic F-108).
• a useful commercially available anti-foaming agent is Ex-Cell ® Antifoam (Sigma-Aldrich, Inc., St. Louis, MO; Product No.59920C).
• the anti-foam agent is generally applied to protect the cells from the negative effects of aeration (“sparging"), since without the addition of a surface-active agent the rising and bursting air bubbles may damage those cells that are at the surface of the air bubbles.
• the following amino acid mixture is can be added to the above-mentioned medium: L-asparagine (0.001 to 1 g/L; preferably 0.01 to 0.05 g/L; particularly preferably 0.015 to 0.03 g/1), L-cysteine (0.001 to 1 g/L; preferably 0.01 to 0.05 g/L; particularly preferably 0.015 to 0.03 g/L), L-proline (0.001 to 1.5 g/L; preferably 0.01 to 0.07 g/L; particularly preferably 0.02 to 0.05 g/L), L-tryptophan (0.001 to 1 g/L; preferably 0.01 to 0.05 g/L; particularly preferably 0.015 to 0.03 g/L) and L- glutamine (0.05 to 10 g/L; preferably 0.1 to 1 g/L).
• L-asparagine 0.001 to 1 g/L; preferably 0.01 to 0.05 g/L; particularly preferably 0.015 to 0.03 g/1
• amino acids may be added to the medium individually or in combination.
• the combined addition of the amino acid mixture containing all of the above-mentioned amino acids is particularly preferred.
• a serum- and protein-free medium is used additionally containing a combination of the above-mentioned amino acid mixtures and purified, ultrafiltered soybean peptone hydrolysate.
• Nutrient supplements such as yeast hydrolysate or various plant-based hydrolysates can be included in the medium, if desired.
• the aqueous medium is liquid, such that the host cells are cultured in a cell suspension within the liquid medium. Alternate media capable of supporting CHO cell growth and productivity of antibody can be used interchangeably with the media used in the working example described herein.
• hydrolysate includes any digest of an animal derived or plant derived source material, or extracts derived from yeast, bacteria, or plants, e.g.,”soy hydrolysate,” which can be a highly purified soy hydrolysate, a purified soy hydrolysate or crude soy hydrolysate.
• the cell culture contemplated herein may be any cell culture independently of the kind and nature of the cultured cells and the growth phase of the cultured cells, e.g. adherent or non-adherent cells; growing, or growth-arrested cells.
• sterile refers to a substance that is free, or essentially free, of microbial and/or viral contamination.
• the "contaminant” means a material that is different from the desired components in a preparation being a cell culture medium or at least a component of a cell culture medium.
• any given set-point slight variations in temperature may occur. Typically, such variation may occur because heating and cooling elements are only activated after the temperature has deviated somewhat from the set-point. In that case, the set-point is X ⁇ Y and the heating or cooling element is activated when the temperature varies by ⁇ Z°C, as appropriate.
• the permissible degree of deviation of the temperature from the set- point before heating or cooling elements are activated may be programmed in the process control system. Temperature may be controlled to the nearest ⁇ 0.5°C, ⁇ 0.4°C, ⁇ 0.3°C, ⁇ 0.2°C, or even ⁇ 0.1°C by heating and cooling elements controlled by thermostats.
• thermometers used in cell culture equipment may have a variability of ⁇ 0.3°C, or ⁇ 0.2°C, or even ⁇ 0.1°C.
• the temperature set-point is set at a value within the range X ⁇ Y°C, and the tolerance of the temperature is ⁇ Z°C (i.e. a heater or cooler is activated when the temperature deviates by ⁇ Z°C, as appropriate) this can also be expressed as a set-point of (X- QBI02PCT PCT International Patent Application Y to X+Y) ⁇ Z°C.
• ⁇ Y°C. and ⁇ Z°C are envisaged.
• “Culturing at” or “maintaining at” a set point of a particular desired pH value means that the process control systems are set to that desired pH value, in other words that the set point of pH is the intended target pH.
• “Culturing at” or “maintaining at” a pH that is set at X ⁇ Y means that the set point is at a value of from X+Y to X-Y pH units.
• the set-point is at a value within the range pH X ⁇ 0.05, ⁇ 0.04, ⁇ 0.03, ⁇ 0.02 or ⁇ 0.01.
• Liquid acids e.g., HCl or H 3 PO 4
• Sodium carbonate is usually the source of added alkali used to maintain pH for mammalian cell culture, and NH 4 OH is often selected to add alkali in microbial culture.
• the cell culture supernatant typically has a CO2 concentration of 1 to 10% (v/v), for example 4.0-9.0% (v/v), 5.5-8.5% (v/v) or about 6-8% (v/v). Conventionally, CO 2 concentration is higher than this due to the CO2 produced by the cells not being removed from the cell culture supernatant.
• Maintaining the CO2 concentration at 10% or lower is reported to increase the yield of recombinant protein; it helps the dCO2 (or pCO2) to be kept low if the feed medium is degassed (for example by bubbling air through it) as well as the cell culture supernatant in the bioreactor being sparged.
• the feed medium is degassed (for example by bubbling air through it) as well as the cell culture supernatant in the bioreactor being sparged.
• a suitable in-line dCO2 (or pCO2) sensor and its use are described in Pattison et al (2000) Biotechnol. Prog.16:769-774.
• a suitable in-line pH sensor is Mettler Toledo InPro 3100/125/Pt100 (Mettler-Toledo Ingold, Inc., Bedford, Mass.).
• a suitable off-line system for measuring dCO 2 (or pCO 2 ), in addition to pH and pO 2 is the BioProfile pHOx (Nova Biomedical Corporation, Waltham MA). In this system, or dCO2 (or pCO2) is measured by potentiometric electrodes within the range 3-200 mmHg with an imprecision resolution of 5%.
• an in-line pH probe may drift over time, such as over periods of days or weeks, during which the cells are cultured. In that event, it may be beneficial to reset the in-line probe by using measurements obtained from a recently calibrated off-line probe.
• a suitable off-line probe is the BioProfile pHOx (Nova Biomedical Corporation, Waltham MA).
• Mammalian cell cultures, and many other kinds of microbial cells need oxygen for the cells to grow, or can grow fastest under aerobic conditions. Normally, this is provided by forcing oxygen into the culture through injection ports. It is also necessary to remove the CO2 that accumulates due to the respiration of the cells.
• the head space may be overlayed with CO2. Under such conditions, low levels of dCO2 (or pCO 2 ) can still be achieved. Overlaying the headspace with CO 2 may also be used to reduce the pH to the set-point, if the pH is too basic.
• the culturing of a plurality of mammalian host cells can be any conventional type of culture, such as batch, fed-batch, intensified fed-batch, or continuous.
• Suitable continuous cultures included repeated batch, chemostat, turbidostat or perfusion culture.
• the desired scale of the recombinant expression will be dependent on the type of expression system and the quantity of different theoretical antibody variants to be studied. As noted herein, typically, 100 milligrams of total antibody protein will suffice, requiring only a batch cell culture of 20 mL to 500 mL; while larger scale culture batches or continuous cell culture methods can be employed, larger volumes are typically not cost-effective.
• a batch culture starts with all the nutrients and cells that are needed, and the culture proceeds to completion, i.e. until the nutrients are exhausted or the culture is stopped for some reason.
• a fed-batch culture is a batch process in the sense that it starts with the cells and nutrients but it is then fed with further nutrients in a controlled way.
• the fed-batch strategy is typically used in bio-industrial processes to reach a high cell density in the bioreactor.
• the feed solution is usually highly concentrated to avoid dilution of the bioreactor.
• the controlled addition of the nutrient directly affects the growth rate of the culture and allows one to avoid overflow metabolism (formation of metabolic by-products) and oxygen limitation (anaerobiosis).
• the growth-limiting nutrient is glucose which is fed to the culture as a highly concentrated glucose syrup (for example 500-850 g/L).
• the culture is diluted with complete growth medium before the cells reach their maximum concentration.
• the amount and frequency of dilution varies widely and depends on the growth characteristics of the cell line and convenience of the culture process. The process can be repeated as many times as required and, unless cells and medium are discarded at subculture, the volume of culture will increase stepwise as each dilution is made.
• the increasing volume may be handled by having a reactor of sufficient size to allow dilutions within the vessel or by dividing the diluted culture into several vessels. The rationale of this type of culture is to maintain the cells in an exponentially growing state.
• Serial subculture is characterized in that the volume of culture is always increasing stepwise, there can be multiple harvests, the cells continue to grow and the process can continue for as long as desired.
• the extracted medium contains cells.
• the cells remaining in the cell culture vessel must grow to maintain a steady state.
• the growth rate is typically controlled by controlling the dilution rate i.e. the rate at which fresh medium is added.
• the cells are cultured at a sub-maximal growth rate, which is achieved by restricting the dilution rate.
• the growth rate is typically high.
• the dilution rate is set to permit the maximum growth rate that the cells can achieve at the given operating conditions, such as pH and temperature.
• QBI02PCT PCT International Patent Application [000136]
• culture vessels, reactors or chambers, of any of various capacities are used to grow suspensions of mammalian host cells, e.g., CHO cells.
• Each culture vessel is connected via inlets to an array of porous tangential flow filters which in turn are connected via outlets back to the culture vessel.
• the suspensions of host cells and growth medium are pumped through the array of porous tangential flow filters to concentrate the cell suspension.
• the extracted medium is depleted of cells, because most of the cells are retained in the culture vessel, for example, by being retained on a membrane through which the extracted medium flows.
• a membrane typically retains 100% of cells, and so a proportion are removed when the medium is extracted.
• sonic cell separation technology achieves separation of cells from the media matrix with high-frequency, resonant ultrasonic waves rather than using a physical barrier, unlike tangential-flow filtration (TFF) or alternating tangential flow filtration (ATF); the cells are held back using an acoustic field as the bioprocess fluid flows through an open channel.
• acoustic waves allows differentiation of particles of equal size, and thus the technology can be used for the separation of particles from the nano- to macro- scales.
• acoustic wave-based technology for cell harvesting applications may help enable continuous manufacturing, BioPharm International 30(9):30 (2017)).
• it may not be crucial to operate perfusion cultures at very high growth rates, as the majority of the cells are retained in the culture vessel.
• a suitable fully continuous process can have a perfusion bioreactor coupled to recombinant protein harvesting and protein purification steps, for example, a multi-column chromatography capture step, followed by flow-through virus inactivation, multi-column intermediate purification, a flow-through membrane adsorber polishing step, continuous virus filtration and a final ultrafiltration step operated in continuous mode.
• Electronic processing of the resulting signal produces an output which is an accurate measurement of the concentration of viable cells.
• the system is insensitive to cells with leaky membranes, the medium, gas bubbles and debris.
• cell viability can be measured by use of a vital dye (or vital stain) to stain small-aliquot samples of culture sampled periodically, and microscopically enumerated to determine viable cell count.
• a vital dye or vital stain
• Trypan blue is a vital dye commonly used for this purpose.
• Automated cell counters supplied by Beckman e.g., Vi-CellTM XR
• Examples include cell counting instruments made by other QBI02PCT PCT International Patent Application manufacturers, e.g., Nova Biomedical, Olympus, Thermo Fisher Scientific and Eppendorf.
• the culture is typically carried out in a bioreactor, which is usually a stainless steel, glass or plastic vessel of 0.01 (i.e., 10-mL) to 10000 (ten thousand) litres capacity, for example, 0.01, 0.015, 0.10, 0.25, 0.30, 0.35, 1, 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, 500, 1000, 2500, 5000 or 8000 liters.
• the vessel is usually rigid but flexible plastic bags or bioreactor liners can be used. These flexible plastic bioreactor bags and liners are generally of the “single use” type.
• Protein A gene expression is regulated by DNA supercoiling which is modified by the ArlS–ArlR two-component system of Staphylococcus aureus, Microbiology 150:3807-19 (2004)).
• Protein A (Spa gene product) is useful in biochemical research and industry because of its ability to bind immunoglobulins. Protein A is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It has been shown via crystallographic refinement that the primary binding site for Protein A is on the Fc region, between the CH2 and CH3 domains.
• Recombinant versions of Protein A commonly contain the five homologous antibody binding domains, but for purposes of the present invention can vary in other parts of the structure in order to facilitate covalent coupling to substrates, e.g., resins (such as, but not limited to, agarose).
• substrates e.g., resins (such as, but not limited to, agarose).
• Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al, EMBO J.5: 15671575 (1986)). Also available commercially (e.g., from Molecular Cloning Laboratories (MCLAB) or Protein Specialists (Prospec)), is recombinant Protein G, an immunoglobulin-binding protein derived from the cell wall of certain strains of beta- hemolytic streptococci.
• the albumin and cell surface binding domains have been eliminated from Recombinant Protein G to reduce nonspecific binding, although the Fc binding domain is still present and, therefore, can be used to separate IgG from crude samples.
• the recombinant Protein G is produced in Escherichia coli using sequence from Streptococcus C1-C2-C3.
• the Protein G contains 200 amino acids (190- 384 and five additional residues not including methionine) having a molecular mass of 21.8kDa.
• the Protein-G migrates on SDS-PAGE around 32kDa.
• matrix resins, beads, nanoparticles, nanofibers, hydrogels, membranes, and monoliths, or any other physical matrix, bearing a QBI02PCT PCT International Patent Application relevant covalently bound chromatographic ligand (e.g., Protein A, Protein G, or other affinity chromatographic ligand, such as a target ligand, an antibody targeting an epitope tag, a charged moiety, or a hydrophobic moiety, etc.) for purposes of the inventive method.
• chromatographic ligand e.g., Protein A, Protein G, or other affinity chromatographic ligand, such as a target ligand, an antibody targeting an epitope tag, a charged moiety, or a hydrophobic moiety, etc.
• the matrix to which the affinity target ligand is attached is most often agarose, but other matrices are available.
• mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
• the Bakerbond ABXTM resin J. T. Baker, Phillipsburg, NJ
• An affinity chromatography matrix may be placed or packed into a column useful for the purification of proteins. Loading of the cell-free supernatant fraction onto the affinity chromatography matrix preferably occurs at about neutral pH.
• the specific target ligand of interest can be covalently conjugated to an affinity chromatography matrix, e.g., for nanobody purification purposes.
• the affinity matrix with the target ligand covalently attached should have sufficient binding capacity to account for the required mass sufficient to be detected by a mass spectrometer, if desired. This can be achieved with appropriately dense conjugation reactive moieties on the matrix (e.g., resin and/or resin bed size in the column).
• the stability of a particular conjugated affinity chromatography matrix needs to be considered with regards to the conjugated target ligand itself or the mode by which the ligand is attached to the matrix.
• Ligand affinity conjugation instability and degradation of the conjugated affinity chromatography matrix reagent during storage can result in decreased antibody yields and/or binding artifacts leading to difficult data analysis or misinterpretation.
• to bind or “binding” a molecule to an affinity chromatography matrix comprising a covalently-conjugated target moiety, e.g., Protein A or a Protein A matrix, or Protein G or a Protein G matrix, or a particular conjugated target ligand of interest, means exposing the molecule to the affinity chromatography target moiety, under appropriate conditions (e.g., pH and selected salt/buffer composition), such that the molecule is reversibly immobilized in, or on, the affinity chromatography matrix (e.g., a Protein A- or Protein G- conjugated or target ligand-conjugated) by virtue of its binding affinity to the target moiety QBI02PCT PCT International Patent Application under those conditions, regardless of the physical mechanism of affinity that may be involved.
• appropriate conditions e.g., pH and selected salt/buffer composition
• loading buffer or “equilibrium buffer” refers to the buffer, and salt or salts, which is mixed with a protein preparation (e.g., a batch or perfusion culture supernatant or filtrate, or an eluant pool containing the antibodies of interest) for loading the protein preparation onto an affinity chromatography matrix, e.g., Protein A- or Protein G- conjugated matrix or a specific target ligand-conjugated affinity chromatography matrix, as the case may be.
• an affinity chromatography matrix e.g., Protein A- or Protein G- conjugated matrix or a specific target ligand-conjugated affinity chromatography matrix, as the case may be.
• This buffer is also used to equilibrate the matrix before loading, and to wash after loading the protein.
• wash buffer is used herein to refer to the buffer that is passed over an affinity chromatography matrix, following loading of a protein preparation and prior to elution or after flow-through of the protein of interest.
• the wash buffer may serve to remove one or more contaminants without substantial elution of the desired protein or can be used to wash out a non-binding protein.
• elution buffer or “eluant” refers to the buffer used to elute the protein of interest (POI) reversibly bound to a matrix.
• solution refers to either a buffered or a non-buffered solution, including water.
• eluting a molecule (e.g.
• QBI02PCT PCT International Patent Application Impurities particularly HMW species, often bind more tightly than the Nb or other antigen- binding protein product and also can be separated from the main desired fraction by adjusting the elution conditions and pool collection criteria (Yigzaw, Y., et al., (2009) supra; Gagnon, P., et al., (1996) supra; Pabst, T. M., et al., (2009) Journal of Chromatography 1216, 7950- 7956).
• the molecular interaction under consideration dictates the type of elution methods that can be used.
• salt can be used to disrupt hydrophobic interactions whereas pH can disrupt ionic and hydrogen binds.
• elution methods besides ionic strength and pH can be used to disrupt the interaction between the antibody and ligand.
• a peptide specific for the antibody epitope on the target ligand can be used to compete with the on-rate and affinity binding properties of the antibody.
• a small organic molecule can be used in a similar fashion as a peptide.
• stress can be applied to the antibody pool prior to binding to the ligand affinity column. Thermal, chemical and/or pH stress can induce a conformational change or denaturation event resulting in aggregation of the antibody which can be removed via precipitation (centrifugation or ultrafiltration) or preparative SEC. This step will remove non-viable candidates from binding to the target affinity matrix.
• increasingly stringent buffer conditions means employing a gradient (a step gradient or a linear gradient) of an increasingly more challenging condition by which antibody variants can be distinguished from each other.
• Examples include, but are not limited to, a gradient (a step gradient or a linear gradient) of increasing ionic strength (typically with higher conductivity going up to about 40-150 mS), or a pH gradient (a step gradient or a linear gradient) approaching an extreme of lower or higher pH than the initial buffer condition, or a gradient (a step gradient or a linear gradient) of increasing concentration of a molecule that competes for binding to the target ligand, such as but not limited to, a small molecule or an oligopeptide.
• elution pool or “eluant pool” means the material eluted from a chromatography matrix, which material includes the recombinant protein of interest, e.g., an antibody of interest.
• the term “loading,” with respect to an affinity chromatography matrix, means loading a protein preparation (e.g., a batch or perfusion culture supernatant or filtrate, or an eluant pool containing the protein of interest) onto the affinity chromatography matrix.
• QBI02PCT PCT International Patent Application [000155]
• the term “washing,” with respect to an affinity chromatography matrix, means passing an appropriate buffer through or over the affinity chromatography matrix.
• "Under physiological conditions" with respect to incubating buffers and immunoglobulins, or other binding assay reagents means incubation under conditions of temperature, pH, and ionic strength, that permit a biochemical reaction, such as a non- covalent binding reaction, to occur.
• Physiologically acceptable salt of a composition of matter, for example a salt of a protein of interest, e.g., a fusion protein, or another immunoglobulin, such as an antibody, or any other protein of interest, or a salt of an amino acid, such as, but not limited to, a lysine, histidine, or proline salt, means any salt, or salts, that are known or later discovered to be pharmaceutically acceptable.
• an electronic computerized “controller” or “microcontroller” or “digital control unit,” terms used interchangeably herein, receives input digital signals from one or more sensors or detectors of the physical or chemical parameter to be controlled, and/or from a chronometer or clock, and, in comparison to a predetermined control setpoint (often assigned by the operator using a user QBI02PCT PCT International Patent Application interface), the controller determines the necessary output signal required to correct the input value in the direction of the setpoint and issues responsive digital instructions to a system or subsystem.
• the microcontroller is situated within the perimeter of a discrete (but optional) system housing, or, in other embodiments, the microcontroller is outside the perimeter of the optional housing, so long as the controller is able to communicate with the pumps, the valves (if present in a particular embodiment), and the sensors within the system or device, i.e., that the controller can receive measurement signals transmitted from the sensors, and the controller can transmit instructions to all the pumps and optional valves. Transmission and/or reception of such measurement signals or instruction signals, as the case may be, can be via electrical cables, wires, fiber optic cables, or can be via infrared or radio wave transmissions, and/or any other suitable medium.
• Switching can be manually controlled and regulated; however, “automatically switching” means that the switch in flow does not require manual input on the operator’s part, but is controlled instead by the controller or microcontroller, which performs the switch, initiating flow or ceasing flow, under predetermined criteria or set-points.
• a step of a method or process, or within a system or device is performed “fluidly,” or is “fluidly connected” to, or “fluidly receives” material from, another step of the process or from another component or vessel within the system, when material flows by pipe, tubing, or other closed conduit between steps or systems without manual loading or unloading.
• fluid propeller means any suitable device for impelling fluid flow within a system, vessel, or connecting line, e.g., a pump head, a pump, a gas pressure regulator, a vacuum line, a vacuum pump, hydrostatic pressure, or gravity.
• a "stable" formulation such as one comprising a pharmaceutical composition of the present invention, is one in which the protein therein, e.g., a single domain antibody (or nanobody), essentially retains its physical stability and/or chemical stability and/or biological activity upon processing (e.g., ultrafiltration, diafiltration, other filtering steps, vial filling), transportation, and/or storage of the antibody drug substance and/or drug product.
• the physical, chemical and biological stability of the protein in a formulation embody the “stability” of the protein formulation, which is specific to the conditions under which the formulated drug product (DP) is stored.
• DP formulated drug product
• a drug product stored at subzero temperatures would be expected to have no significant change in either chemical, physical or biological activity while a drug product stored at 40°C would be expected to have changes in its physical, chemical and biological activity with the degree of change dependent on the time of storage for the drug substance or drug product.
• the configuration of the protein formulation can also influence the rate of change. For instance, aggregate formation is highly influenced by protein concentration with higher rates of aggregation observed with higher protein concentration.
• Excipients are also known to affect stability of the drug product with, for example, addition of salt increasing the rate of aggregation for some proteins while other excipients such as sucrose are known to decrease the rate of aggregation during storage. Instability is also greatly influenced by pH giving rise to both higher and lower rates of degradation depending on the type of modification and pH dependence. [000165] Various analytical techniques for measuring protein stability are available in the art and are reviewed, e.g., in Wang, W. (1999), Instability, stabilization and formulation of liquid protein pharmaceuticals, Int J Pharm 185:129-188. Stability can be measured at a selected temperature for a selected time period.
• the formulation can be kept at 40°C for 2 weeks to 1 month, at which time stability is measured.
• the formulation should be stable at 30°C for at least 1 month, or 40°C for at least a week, and/or stable at 2-8°C for at least two years.
• QBI02PCT PCT International Patent Application [000166]
• a protein "retains its physical stability" in a formulation if it shows minimal signs of changes to the secondary and/or tertiary structure (i.e., intrinsic structure), or aggregation, and/or precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering or by size exclusion chromatography, or other suitable methods.
• Physical instability of a protein i.e., loss of physical stability
• loss of physical stability can be caused by oligomerization resulting in dimer and higher order aggregates, subvisible, and visible particle formation, and precipitation.
• the degree of physical degradation can be ascertained using varying techniques depending on the type of degradant of interest. Dimers and higher order soluble aggregates can be quantified using size exclusion chromatography, while subvisible particles may be quantified using light scattering, light obscuration or other suitable techniques.
• a protein "retains its chemical stability" in a formulation, if the chemical stability at a given time is such that covalent bonds are not made or broken, resulting in changes to the primary structure of the protein component, e.g., antibody.
• Changes to the primary structure may result in modifications of the secondary and/or tertiary and/or quaternary structure of the protein and may result in formation of aggregates or reversal of aggregates already formed.
• Typical chemical modifications can include isomerization, deamidation, N-terminal cyclization, backbone hydrolysis, methionine oxidation, tryptophan oxidation, histidine oxidation, beta-elimination, disulfide formation, disulfide scrambling, disulfide cleavage, and other changes resulting in changes to the primary structure including D-amino acid formation.
• Chemical instability i.e., loss of chemical stability
• Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
• Chemical alteration may involve size modification (e.g. clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and/or matrix- assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example.
• Other types of chemical alteration include charge alteration (e.g.
• Loss of physical and/or chemical stability may result in changes to biological activity as either an increase or decrease of a biological activity of interest, depending on the modification and the protein being modified.
• a protein "retains its biological activity" in a QBI02PCT PCT International Patent Application buffered solution or formulation, if the biological activity of the protein at a given time is within about 30% of the biological activity exhibited at the time the formulation was prepared. Activity is considered decreased if the activity is less than 70% of its starting value.
• Biological assays may include both in vivo and in vitro based assays such as ligand binding, potency, cell proliferation or other surrogate measure of its biopharmaceutical activity.
• Quantification of immunoglobulin protein e.g., a single domain antibody or nanobody
• An antibody that specifically binds a domain of the nanobody or nanobodies of interest can therefore be useful for these purposes.
• the antigen-binding protein of the invention is an antibody.
• antibody or interchangeably “Ab,” is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies (including human, humanized or chimeric antibodies), monomeric, homodimeric, and heterodimeric antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), single domain antibodies (sdAbs), and antibody fragments that can bind antigen (e.g., Fab, Fab', F(ab')2, Fv, single chain antibodies, diabodies), comprising complementarity determining regions (CDRs) of the foregoing as long as they exhibit the desired biological activity. Multimers or aggregates of intact molecules and/or fragments, including chemically derivatized antibodies, are contemplated.
• antibody encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof.
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
• Monoclonal antibodies that are antigen binding proteins QBI02PCT PCT International Patent Application are highly specific binders, being directed against an individual antigenic site or epitope, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different epitopes.
• Nonlimiting examples of monoclonal antibodies include murine, rabbit, rat, chicken, chimeric, humanized, or human antibodies, fully assembled antibodies, multispecific antibodies (including bispecific antibodies), antibody fragments that can bind an antigen (including, Fab, Fab', F(ab)2, Fv, single chain antibodies, diabodies), maxibodies, nanobodies, and recombinant peptides comprising CDRs of the foregoing as long as they exhibit the desired biological activity, or variants or derivatives thereof.
• the modifier "monoclonal” indicates the character of the sdAb or antibody as being obtained from a substantially homogeneous population of sdAb or antibodies, and is not to be construed as requiring production of the sdAb or antibody by any particular method.
• monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567).
• each chain includes a "variable" (“V") 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.
• the variable region differs among different antibodies.
• the constant region is the same among different antibodies.
• Within the variable region of each heavy or light chain there are three hypervariable subregions that help determine the antibody's specificity for antigen in the case of an antibody that is an antigen binding protein.
• the variable domain residues between the hypervariable regions are called the framework residues and generally are somewhat homologous among different antibodies.
• Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains.
• Human light chains are classified as kappa ( ⁇ ) and lambda ( ⁇ ) light chains.
• variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
• J variable and constant regions
• D variable region of about 10 more amino acids.
• An “antibody” also encompasses a recombinantly made antibody, and antibodies that are glycosylated or lacking glycosylation.
• the term "light chain” or “immunoglobulin light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity.
• a full-length light chain includes a variable region domain, VL, and a constant region domain, C L .
• the variable region domain of the light chain is at the amino-terminus of the polypeptide.
• Light chains include kappa chains and lambda chains.
• the term "heavy chain” or “immunoglobulin heavy chain” includes a full- length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity.
• Heavy chains may be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
• IgG including IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
• IgG isotypes may have different effector functions (mediated by the Fc region), such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
• ADCC antibody-dependent cellular cytotoxicity
• CDC complement-dependent cytotoxicity
• an “Fc region”, or used interchangeably herein, "Fc domain” or “immunoglobulin Fc domain”, contains two heavy chain fragments, which in a full antibody comprise the C H1 and C H2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
• a "Fab' fragment” contains one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.
• QBI02PCT PCT International Patent Application [000187]
• a "F(ab') 2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
• a F(ab') 2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
• Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light- chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH VL dimer. A single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
• Single-chain antibodies are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region.
• Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Pat. No.4,946,778 and No.5,260,203, the disclosures of which are incorporated by reference in their entireties.
• Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain, and optionally comprising a polypeptide linker between the V H and V L domains that enables the Fv to form the desired structure for antigen binding (Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Nati. Acad. Sci.
• An "Fd” fragment consists of the VH and CH1 domains.
• the term “diabodies” refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites.
• Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad.
• an antigen binding protein e.g., an immunoglobulin protein, or an antibody or antibody fragment
• a target ligand or antigen of interest when it has a significantly higher binding affinity for, and consequently is capable of distinguishing, that target ligand or antigen, compared to its affinity for other unrelated proteins, under similar binding assay conditions.
• KD Equilibrium dissociation constant
• Kd Equilibrium dissociation constant
• Kd ([total binding sites] x [total ligand])/[PL].
• the antigen binding protein specifically binds antigen with "high affinity” when the equilibrium dissociation constant is 10 -9 M or lower, and with “very high affinity” when the Kd or KD is 10 -10 M or lower.
• a number of nanobodies are disclosed herein having different affinities to fentanyl, as well as to carfentanil. Differing sensitivities or affinities to a plurality or multiplicity of different opioids amongst nanobody species can be useful for distinguishing QBI02PCT PCT International Patent Application between each opioid species. For example, in a multiplexed assay, differing affinities to a single target (e.g., fentanyl and/or carfentanil) can also be useful.
• affinities to the target can be helpful to ascertain analyte information (such as concentration) from a single preparation of sample, whereas an inconvenient dilution series may need to be prepared if only one species of nanobody were to be used.
• relatively low-affinity nanobodies can include those in which relatively weak binding is desirable, for examples, when it is desirable to easily dissociate the nanobody-antigen pair in order to recycle the nanobodies.
• antigen-binding proteins e.g., nanobodies
• antigen-binding proteins with relatively lower fentanyl affinity can have other desirable properties, such as but not limited to, a relatively higher affinity to other opioid species, or an increased stability to environmental conditions (e.g., temperature or pH).
• identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared.
• two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences).
• the programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoff et al., in Atlas QBI02PCT PCT International Patent Application of Protein Sequence and Structure, vol.5, supp.3 (1978)] can be used in conjunction with the computer program.
• amino acid residues e.g., norleucine (Nle)
• Nle norleucine
• amino acid residues typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.
• the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
• the conservative amino acid substitutions of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein.
• the greatest local average hydrophilicity of a protein correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
• the substitution of amino acids whose hydrophilicity values are within ⁇ 2 is included, in certain embodiments, those that are within ⁇ 1 are included, and in certain embodiments, those within ⁇ 0.5 are included.
• a cDNA library may be constructed by reverse transcription of polyA+ mRNA, preferably membrane-associated mRNA, and the library screened using probes specific for human immunoglobulin polypeptide gene sequences.
• the polymerase chain reaction is used to amplify cDNAs (or portions of full-length cDNAs) encoding an immunoglobulin gene segment of interest (e.g., a light or heavy chain variable segment).
• nucleic acids encoding antibodies is a library of such nucleic acids generated, for example, through phage display technology.
• Polynucleotides encoding peptides of interest, e.g., variable region peptides with desired binding characteristics, can be identified by standard techniques such as panning. [000235] Sequencing of DNA is carried out using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, which is incorporated herein by reference).
• Embodiments of the present method of enhancing in vitro recombinant expression of a protein of interest by a mammalian host cell or the method of manufacturing a protein drug substance of interest can involve so-called “Next-generation” sequencing, as a preferred method for confirming the presence of all engineered DNA constructs prior to the transfection step(s). (See, e.g., Buermans, H. P.
• Another useful cloning method is the so-called “TOPO” method, in which a complete insert containing a 3' adenosine overhang (generated by Taq polymerase) is present, and Topoisomerase I ligates the insert into a TOPO vector.
• Another useful cloning method is degenerate or error-prone PCR exploiting degenerate primers and/or a thermally stable low-fidelity polymerase caused by the polymerase within certain reaction conditions. Fragments or inserts are then cloned into an expression vector.
• the above are merely examples of known cloning techniques, and the skilled practitioner knows how to employ any other suitable cloning techniques.
• Isolated DNA can be operably linked to control sequences or placed into expression vectors, which are then transfected into host cells that do not otherwise produce immunoglobulin protein, to direct the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is well known in the art.
• Nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably QBI02PCT PCT International Patent Application linked to a coding sequence if it is positioned so as to facilitate translation.
• operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites.
• Vector components can include one or more of the following: a signal sequence (that may, for example, direct secretion of the expressed protein by the recombinant host cells); an origin of replication, one or more selection marker or reporter genes (that may, for example, encode a fluorescent protein, such as a green fluorescent protein (GFP), an enhanced green fluorescent protein (EGFP), a red- shifted green fluorescent protein (rs-GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), a cyan fluorescent protein (e.g., CyOFP1), mini Singlet Oxygen Generator (miniSOG), a luminescent protein (e.g., luciferase), or the like, or may confer antibiotic or other drug resistance, or complement auxotrophic deficiencies of the host cells or supply critical nutrients not available in the medium, e.g., di
• the inventive method for manufacturing a purified protein of interest involves culturing protein-secreting mammalian cells.
• Such cultured mammalian cells are typically made by recombinant DNA technology involving transient or stable transfection, e.g., the pooled plasmid constructs (expression vectors) from the cloning step can be transfected into a plurality of host cells (e.g., mammalian, e.g., HEK 293 or CHO cell, or insect cells, or microbial host cells, e.g., bacterial cells, yeast cells, or algal or microalgal cells) for expression using a cationic lipid, polyethylenimine, Lipofectamine TM , or ExpiFectamine TM , or electroporation.
• host cells e.g., mammalian, e.g., HEK 293 or CHO cell, or insect cells, or microbial host cells, e.g., bacterial cells
• the transfectant or transformant cells will be provided with a recombinant expression cassette for a selectable marker, for example, but not limited to, one or more of the following: glutamine synthase, dihydrofolate reductase, puromycin-N acetyl transferase, blasticidin-S deaminase, hygromycin phosphotransferase, aminoglycoside phosphotransferase, nourseothircin N-acetyl transferase, or a protein that binds to zeocin.
• a selectable marker for example, but not limited to, one or more of the following: glutamine synthase, dihydrofolate reductase, puromycin-N acetyl transferase, blasticidin-S deaminase, hygromycin phosphotransferase, aminoglycoside phosphotransferase, nourseothircin N-acety
• the protein of interest is typically obtained by culturing the transfected or transformed host cells under physiological conditions allowing the cells to express recombinant proteins. Most conveniently, the expressed recombinant proteins are directly secreted into the extracellular culture medium (by employing appropriate secretory-directing signal peptides) and are harvested therefrom; otherwise additional steps will be needed to isolate the expressed antibodies from a cell extract.
• secretory signal peptide (SP) sequences are known in the art, and these can be added, adjacent or distal, to any of the sequences shown in Table 1, herein, for the purpose of facilitating secretion of the inventive antigen-binding protein.
• the host cell in culture, is a microbial cell or microorganism (used interchangeably herein), e.g., a bacterium, a cyanobacterium, a fungus, a microalga, or an alga.
• the microbial cell can be a bacterium, such as, but not limited to, Escherichia coli, Bacillus subtilis, Salmonella sp., Aliivibrio fischeri, Pseudomonas fluorescens, Bacillus sp., Cupriavidus metallidurans, Deinococcus radiodurans, and Staphylococcus aureus.
• an epitope tag peptide can be used to facilitate purification and/or detection of the antigen-binding protein of the invention.
• the tagging peptide is detectable by itself (e.g. fluorescent tags such as GFP) while in other cases the tagging peptide is detectable because it specifically binds a detectable molecule (in turn, the detectable molecule can be directly detectable, e.g. fluorescent, or it may be detected by specific binding to it of a detectable molecule, i.e. a scaffold of molecules may be required for detection).
• such a peptide is usually designed (or found) to have a high affinity to a readily, or even commercially, available antibody molecule.
• Such peptides are often derived from a species unrelated to the species where the polypeptides is intended to be used to avoid any cross reaction, especially during detection.
• the molecule binding the tagging peptide may be selected for its detectability and/or for ease of immobilization and/or recovery in purification processes.
• Common tagging peptide include HA-tag (a short peptide from human influenza hemagglutinin), Flag-tag, His- tag or hexa-histidine (comprising at least 6 histidine residues) and the Strep-tag (comprising QBI02PCT PCT International Patent Application eight amino acids and which is readily bound by commercially available Strep-tactin (an engineered streptavidin) and antibodies).
• the VHH-comprising polypeptide of the invention comprises a Strep-tag fused C-terminally to the V HH , particularly intercalated between the VHH and effector peptide.
• cysteine bond(s) may be added to the immunoglobulin to improve its stability (particularly where the immunoglobulin is an antibody fragment such as an Fv fragment).
• immunoglobulin variants are prepared with the intent to modify those amino acid residues which are directly involved in epitope binding in a starting sequence. In other embodiments, modification of residues which are not directly involved in epitope binding or residues not involved in epitope binding in any way, is desirable, for purposes discussed herein. Mutagenesis within any of the CDR regions and/or framework regions is contemplated. [000267] In order to determine which antigen binding protein amino acid residues are important for epitope recognition and binding, alanine scanning mutagenesis can be performed to produce substitution variants.
• Jermutus et al, Proc Natl Acad Sci U S A., 98(l):75-80 (2001) showed that tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of scFvs; Fermer et al., Tumour Biol.2004 Jan- Apr;25(l-2):7-13 reported that use of phage display in combination with DNA shuffling raised affinity by almost three orders of magnitude.
• N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
• the tripeptide sequences asparagine-X- serine and asparagine -X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
• the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.
• N-linked glycosylation sites may be added to an immunoglobulin by altering the amino acid sequence such that it contains one or more of these tripeptide sequences.
• Homodimeric immunoglobulins or homodimeric antibodies may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993).
• an immunoglobulin can be QBI02PCT PCT International Patent Application engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-CancerDrug Design 3: 219-230 (1989).
• Modifications to increase serum half- life also may desirable, for example, by incorporation of or addition of a salvage receptor binding epitope (e.g., by mutation of the appropriate region or by incorporating the epitope into a peptide tag that is then fused to the immunoglobulin at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., W096/32478) or adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers.
• a salvage receptor binding epitope e.g., by mutation of the appropriate region or by incorporating the epitope into a peptide tag that is then fused to the immunoglobulin at either end or in the middle, e.g., by DNA or peptide synthesis
• Mutation of residues within Fc receptor binding sites can result in altered (i.e. increased or decreased) effector function, such as altered affinity for Fc receptors, altered ADCC or CDC activity, or altered half-life.
• potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative QBI02PCT PCT International Patent Application substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different subclass (e.g. replacing an IgGl residue with a corresponding IgG2 residue at that position).
• the isolated recombinant antigen-binding protein can include a VHH domain comprising an amino acid sequence selected from the group consisting of SEQ QBI02PCT PCT International Patent Application ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10 , SEQ ID NO:11, SEQ ID NO:15 , SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 , SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:33 , SEQ ID NO:37, SEQ ID NO:38 , SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54; or can comprise any one of the group consisting
• amino acid sequence variants of the immunoglobulin sequences disclosed herein will have an amino acid sequence having at least 60% amino acid sequence identity with the original or reference immunoglobulin domain (e.g., VHH) amino acid sequence.
• Amino acid variant sequences of a V HH domain sequence of the invention can also have at least 65%, or at least 70%, or at least 75% or at least 80% amino acid sequence identity, more preferably at least 85% sequence identity, even more preferably at least 90%) sequence identity, and most preferably at least 95% sequence identity, including for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity.
• terminal insertions include an immunoglobulin with an N-terminal methionyl residue or the immunoglobulin (including antibody or antibody fragment) fused to an epitope tag or a salvage receptor binding epitope, which can be useful in detection and/or purification of the inventive proteins of interest.
• Other insertional variants of the immunoglobulin or antibody molecule include the fusion to a polypeptide which increases the serum half-life of the immunoglobulin, e.g. at the N-terminus or C-terminus.
• the addition to the reaction vessel for mixing, of a fluid aqueous suspension of host cells that display on their surfaces a plurality of recombinant antigen-binding proteins, and/or a fluid aqueous sample, and/or an agglutinating agent comprising the analyte of interest is accomplished manually, e.g., with appropriately sized pipettes, micropipettes, or other liquid handling apparati. Between runs, vessels and tubing of the connecting lines can be cleaned or replaced manually, as well.
• Embodiment 1 An isolated recombinant antigen-binding protein that specifically binds fentanyl or carfentanil, comprising a VHH domain comprising a set of three complementarity determining regions (CDR): CDR1, CDR2, and CDR3, wherein each CDR comprises an amino acid sequence, wherein the set of three CDRs is selected from the group consisting of: (a) SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; (b) SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:44; (c) SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; (d) SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; (e) SEQ ID NO:17, SEQ ID NO:18, and S
• Embodiment 9 The host cell, in culture, according to any of Embodiments 5- 7, wherein the host cell is selected from the group consisting of Saccharomyces cerevisiae and Trichosporon cutaneum.
• Embodiment 10 The host cell, in culture, of Embodiment 5, wherein the host cell is a mammalian cell, an insect cell, or a plant cell.
• Embodiment 11 The host cell, in culture, of Embodiment 10, wherein the mammalian cell is derived from a Chinese Hamster Ovary (CHO) cell.
• Embodiment 15 A device for detecting the presence of fentanyl and/or carfentanil, comprising the host cell according to any of Embodiments 5-12.
• Embodiment 16 The device according to any of Embodiments 14-15, comprising a lateral flow immunodetection test strip, or an ELISA system or array, a radioimmunoassay (RIA) system, or an agglutination reaction-based assay system (such as but not limited to latex agglutination).
• a lateral flow immunodetection test strip or an ELISA system or array, a radioimmunoassay (RIA) system, or an agglutination reaction-based assay system (such as but not limited to latex agglutination).
• RIA radioimmunoassay
• agglutination reaction-based assay system such as but not limited to latex agglutination
• Embodiment 23 The continuous agglutination assay method according to Embodiment 22, wherein the step of measuring the level of agglutination in the reaction mixture is over a continuous time course of at least about 1 to 45 minutes.
• Embodiment 24 The continuous agglutination assay method according to any of Embodiments 22-23, wherein the step of measuring the level of agglutination in the reaction mixture is through a fixed distance in the preselected portion of the reaction mixture in the reaction vessel.
• Embodiment 25 The continuous agglutination assay method according to any of Embodiments 22-24, wherein the step of measuring the level of agglutination in the reaction mixture comprises measuring the turbidity of the reaction mixture.
• Embodiment 26 The continuous agglutination assay method according to any of Embodiments 22-25, wherein the step of measuring the level of agglutination in the reaction mixture comprises detecting the transmission, scattering, or absorbance of light.
• Embodiment 27 The continuous agglutination assay method according to any of Embodiments 22-26, wherein the preselected portion of the reaction mixture is in the upper half- to upper third- portion.
• Embodiment 35 The automated system for continuous agglutination assay according to any of Embodiments 32-34, wherein the reaction vessel, the first vessel, and/or the optional second vessel, further comprise a stirrer.
• Embodiment 36 The automated system for continuous agglutination assay according to any of Embodiments 32-35, wherein the plurality of fluid propellers comprise one or more pump heads.
• Phage-display library A phage-display library was created from the VHH sequences obtained in the two production bleeds.
• Peripheral blood mononuclear cells (PBMCs) were isolated from the production bleeds within 4 hours of drawing.
• RNA was isolated from the PBMCs, cDNA was transcribed from the isolated RNA, and the VHH sequences were inserted into phagemid DNA for expression in E. coli.
• Three rounds of library panning were performed with fentanyl-BSA immobilized on high-bind plastic ELISA plates. After panning, the enriched phagemid pool was transformed into E.
• This expression system enables the extracellular display of each VHH linked to an outer membrane anchored ⁇ -barrel domain and allows for the V HH s to be evaluated without purification.
• the function of the six VHH sequences was first validated by an agglutination assay in which E. coli displaying a V HH domain were incubated with a multivalent antigen, leading to extensive E. coli-antigen linking and the formation of large complexes that can be detected by eye. (See, Kylilis, N.
• the lysate was centrifuged at 1,900 x g for 15 minutes and the soluble protein was removed from the pelleted cell debris by pipetting and kept for affinity measurement.
• Microtiter plates were coated with varying concentrations of fentanyl-BSA conjugate at concentrations spanning 0- 100 nM, at 4°C overnight; the microtiter plates were then washed with PBS, and blocked with 1% casein in PBS (ThermoFisher Scientific, Cat. No.37582) for two hours at room temperature. Wells were washed with PBS and then incubated with cell lysate for at 37°C for 2 hours.
• This sample can include a batch water sample, or an inline plug-in to a continuously flowing aqueous stream, which can be received and contained by the optional second vessel, or which can be introduced directly into the reaction vessel via a sample inlet, as desired.
• inventive fentanyl assay to be functional on a range of water sources, including tap water, river water, algal pond water, and dairy manure lagoon water.
• Pretreatment or filtration of the aqueous sample can optionally be employed, depending on the pH, salinity, or solids content of the aqueous sample.
• a multi-parameter test can contain multiple cell suspension reservoirs (i.e., a plurality of the first vessel), each with a sensor strain sensitive to a different specific analyte, and this embodiment of the system is capable of drawing from each of different cell suspension reservoirs.
• the path switching can be automated, including the use of automated valves directed by the microcontroller.
• the microcontroller can be configured and programmed to also operate the valves and gas pressure regulators of the gas plumbing system.

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