Patent number 8778622 is assigned to
The following quote was obtained by the news editors from the background information supplied by the inventors: "Glycoproteins and glycoenzymes are proteins that contain a post-translational modification wherein oligosaccharide chains (known as glycans) are covalently attached to the protein's or enzyme's polypeptide side chains. This process, which is known as glycosylation, is one of the most abundant protein post-translational modifications. It is estimated that more than half of all cellular and secretory proteins are glycosylated. (Apweiler et al., 1999, Biochim. Biophys.
"Conventional techniques which are routinely employed for glycoprotein and glycoenzyme analysis include mass spectrometry, lectin affinity chromatography and western blotting. Although these conventional methods of analysis are generally accurate, they are time consuming, require purification of the protein, and some, such as mass spectrometry, require specific expertise and are technically challenging. (Wang et al., 2006, Glycobiol. Epub.; Qiu et al., 2005, Anal. Chem. 77:2802-2809; Qiu et al., 2005, Anal Chem. 77:7225-7231; Novotny et al., 2005,
"Traditionally, to provide a quantitative assessment of the glycan structure of a glycoprotein, lectin array platforms required the use of either a reliable glycoprotein specific antibody or direct conjugation of a fluorescent dye to the glycoprotein. These antibody-based detection strategies are limited by the fact that antibody recognition of a given glycoprotein or glycoenzyme may be blocked or reduced depending on the type of glycan structure linked to the protein or enzyme, thereby allowing recognition of only a subset of the total glycoprotein pool and not the range of potential glycan structures. Antibody-based recognition may also require multiple binding and wash steps, which can add time and complexity to an analysis. While these problems can be circumvented using direct labeling of the glycoprotein, direct labeling remains limited to pure preparations of material, since the labeling techniques do not discriminate among proteins. Accordingly, direct labeling cannot be used for 'dirty' or in-process samples. The utility of currently available methods for glycan analysis may be further limited because large quantities of highly purified materials may not readily be available from in-process test samples. Furthermore, purified material may only represent a subset of the initial glycoform population because the purification process is typically selective for certain glycan structures.
"The identification and characterization of protein and enzyme glycoforms is essential in the development of recombinant proteins and enzymes. For example, glycosylation of recombinantly-prepared enzymes must frequently be controlled during production to maintain the efficacy and safety of such recombinant enzymes, and cell culture conditions can affect the carbohydrate structures of glycoproteins. Further understanding of cell culture conditions that can impact the carbohydrate structures of recombinantly-prepared proteins or enzymes is also important for the development of an effective and robust recombinant production process.
"Improved methods and compositions are needed for the rapid, direct and systematic identification and evaluation of the glycan structures of a given protein or enzyme and their variant glycoforms. High throughput methods and compositions that are capable of efficiently assessing and distinguishing among a diverse range of glycosylation states or glycoforms would provide valuable information for drug discovery and disease therapeutics, provide valuable tools regarding ongoing research, and facilitate the optimization of recombinant production processes."
In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventor's summary information for this patent: "The present invention provides novel methods, assays and compositions for the accurate and rapid identification and/or detection of various glycoforms of enzymes. In particular, the present invention relies upon the intrinsic activity of the enzyme of interest to detect such enzyme's presence in a test sample. The methods, assays and compositions disclosed herein also provide novel strategies for analyzing the different glycoforms of unpurified proteins or enzymes in cell culture harvest test samples. Furthermore, the present invention provides the ability to detect and distinguish among different glycoforms or glycovariants of an enzyme in upstream harvest test samples, thereby facilitating the optimization of cell culture conditions that affect the viable glycoform content of recombinantly-prepared enzymes. The methods and kits of the present invention are advantageously capable of determining the presence of glycosylated enzymes in a test sample irrespective of the presence of additional cellular proteins, biological materials or other contaminants which may be present in that test sample.
"Disclosed herein are methods for detecting the presence of an enzyme (e.g., a recombinantly prepared enzyme) in a test sample, such methods comprising contacting the test sample with at least one capture agent (e.g., a lectin) under conditions appropriate for binding of glycosylated enzyme in the test sample to the capture agent, wherein upon binding of glycosylated enzyme to capture agent a complex is formed which is referred to herein as a 'bound enzyme.' Such methods also contemplate separation of the test sample from the bound enzyme produced by the previous step (e.g., using routine means such as washing) followed by detection of the intrinsic enzymatic activity of the bound enzyme. The presence of intrinsic enzymatic activity is indicative of the presence of enzyme in the test sample.
"Also disclosed are methods for detecting the presence of an enzyme (e.g., a recombinantly prepared enzyme) in a test sample, wherein such methods comprise the steps of contacting a test sample with at least one capture agent (e.g., a lectin) under conditions appropriate for binding of the glycosylated enzyme, and thereby forming a bound enzyme when glycosylated enzyme is present. The methods of the present invention also contemplate separating the bound enzyme from the test sample and contacting the bound enzyme with at least one substrate. In accordance with the present invention, the presence of intrinsic enzymatic activity of such bound enzyme is indicative of the presence of the glycosylated enzyme of interest in the test sample. Conversely, the absence of intrinsic enzymatic activity is indicative of the absence of the glycosylated enzyme of interest in the test sample. The methods disclosed herein provide the ability to optimize the desired glycoform content of one or more recombinant enzymes during recombinant preparation.
"In one embodiment, the methods of the present invention further comprise the step of fixing a capture agent (e.g., one or more lectins) onto a solid support (e.g., a microtiter plate or one or more populations of beads). In one embodiment, such solid support may comprise or be coated with avidin, streptavidin or a metal chelator such as a nickel chelate. If such solid support comprises avidin or streptavidin, the use of derivatized lectins (e.g., biotinylated lectins) are preferred. If such solid support comprises a nickel chelate, the use of six consecutive histidine residues (6His) as an affinity tag is preferred. For example, a capture agent may be a fusion protein which includes one or more histidine (HIS) residues (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least eight, at least ten, at least twelve, at least twenty, at least twenty five or more HIS residues) at either the N- or C-terminus as an affinity tag to facilitate fixing of that capture agent (i.e., the fusion protein) to a solid support.
"In one embodiment of the present invention the capture agent comprises one or more lectins. The lectins contemplated by the methods, assays and kits of the present invention include, for example, concanavalin A, wheat germ agglutinin, Jacalin, lentil lectin, peanut lectin, lens culinaris agglutinin, Griffonia (Bandeiraea) simplicifolia lectin II, Aleuria aurantia lectin,hippeastrum hybrid lectin, sambucus nigra lectin, maackia amurensis lectin II, ulex europaeus agglutinin I, lotus tetragonolobus lectin, galanthus nivalis lectin, euonymus europaeus lectin, ricinus communis agglutinin I, and any combinations thereof.
"In another embodiment of the present invention the capture agent comprises a receptor, or a binding fragment thereof, known to demonstrate affinity for or otherwise bind to one or more particular glycoforms of an enzyme. For example, mannose-6-phosphate (M6P) binds the mannose-6-phosphate receptor (M6PR), and in one embodiment a recombinant fusion protein comprising the M6PR or a binding domain thereof (e.g., M6PR domain 9) may serve as the capture agent. In one embodiment, the recombinant fusion protein capture agent may also comprise one or more histidine residues (e.g., 6His) to facilitate purification, capture and/or fixing of the capture agent to a solid support. In one embodiment of the present invention, the capture agent comprises the fusion protein M6PR(D9)6His.
"Also disclosed is a method of determining the intrinsic enzymatic activity of the bound enzyme by contacting such bound enzyme with a substrate, for example, a substrate which has known reactivity with the enzyme suspected of being present in the test sample. In accordance with the methods of the present invention, the presence of intrinsic enzymatic activity is indicative of the presence of enzyme in the test sample. Alternatively, the absence of intrinsic enzymatic activity may be indicative of the absence of such enzyme in the test sample.
"In one embodiment, the methods, assays and kits of the present invention contemplate determining intrinsic enzymatic activity by contacting bound enzyme with a substrate which is known to predictably react with the enzyme of interest. For example, if the enzyme is agalsidase alfa the selected substrate may be 4-nitrophenyl-.alpha.-D-galactopyranoside, if the enzyme is galactocerebrosidase the selected substrate may be 4-nitrophenyl-.beta.-D-galactopyranoside, and if the enzyme is aryl sulfatase A the selected substrate may be p-nitrocatechol sulfate. The presence or absence of intrinsic enzymatic activity of the bound enzyme may be determined by means which are known to those of ordinary skill in the art. In one embodiment a quantitative assessment of the depletion of substrate and/or the conversion of substrate to product may be indicative of intrinsic enzymatic activity. For example, in one embodiment substrate depletion of about 5%, 10%, 20%, 30%, 40%, 50% or more, or preferably about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more relative to the amount of substrate introduced may be indicative of intrinsic enzymatic activity. Alternatively, following contacting an enzyme with a substrate, a relative increase in the formation of a product, or the conversion of substrate to product, in each case of about 5%, 10%, 20%, 30%, 40%, 50% or more, or preferably about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% or more, may be indicative of intrinsic enzymatic activity. Substrates contemplated by the present invention include, for example, 4-nitrophenyl-.alpha.-D-galactopyranoside, 4-nitrophenyl-.beta.-D-galactopyranoside and para-nitrocatechol sulfate.
"Also disclosed herein are kits which are useful for detecting the presence of glycosylated enzymes (e.g., a recombinantly prepared glycosylated enzyme) in a test sample. Such kits comprise at least one capture agent (e.g., a lectin) capable of binding a glycosylated enzyme, and at least one substrate which is reactive with such glycosylated enzyme. In one embodiment the kits of the present invention comprise a solid support (e.g., a multiple well microtiter plate), onto which may be fixed a capture agent (e.g., the lectin sambucus nigra agglutinin).
"In one embodiment, the kits of the present invention comprise a capture agent which is known to bind or demonstrate affinity for the targeted glycoform of the enzyme of interest (e.g., the M6PR(D9)6His fusion protein), and a substrate which is known to react with such enzyme. In one embodiment, such kits may also comprise a means of separating or removing excess test sample from the solid support, for example by washing, or other routine means available to one of ordinary skill in the art.
"Also contemplated are kits which are capable of identifying multiple glycosylated enzymes and multiple glycoforms of those enzymes in the same test sample. For example, the kits of the present invention may comprise multiple capture agents (e.g., lectins) fixed onto one or more solid supports (e.g., populations of inert beads), and thus provide the capability of binding to multiple glycoforms of one or more enzymes in the same test sample. The kits of the present invention may also comprise one or more substrates (each of which correspond to a particular enzyme whose presence is suspected in a test sample) to determine such enzymes' intrinsic enzymatic activities. Preferably, the selected substrate is known to predictably bind to, or react with, the enzyme of interest. For example, if the enzyme is agalsidase alfa the selected substrate may be 4-nitrophenyl-.alpha.-D-galactopyranoside, if the enzyme is galactocerebrosidase the selected substrate may be 4-nitrophenyl-.beta.-D-galactopyranoside, and if the enzyme is aryl sulfatase A the selected substrate may be p-nitrocatechol sulfate. Based upon the binding specificity or reactivity of the test sample with the substrate, one having ordinary skill in the art may use routine means to assess the presence or absence of intrinsic enzymatic activity (e.g., by quantitatively determining substrate depletion and/or the conversion of substrate to product).
"The above discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention when taken in conjunction with the accompanying examples."
URL and more information on this patent, see: Roseman, Daniel S.. Enzymatic Activity-Based Detection. U.S. Patent Number 8778622, filed
Keywords for this news article include: Antibodies, Blood Proteins, Cyclic Amino Acids, Enzymes and Coenzymes, Essential Amino Acids, Genetics, Glycoconjugates, Glycoproteins, Histidine, Immunoglobulins, Immunology, Peptides, Pharmaceutical Companies,
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