News Column

Researchers Submit Patent Application, "Competition-Based Detection Assays", for Approval

August 25, 2014



By a News Reporter-Staff News Editor at Pharma Business Week -- From Washington, D.C., NewsRx journalists report that a patent application by the inventor Roseman, Daniel S. (Framingham, MA), filed on June 8, 2012, was made available online on August 14, 2014 (see also Pharmaceutical Companies).

The patent's assignee is Shire Human Genetic Therapies, Inc.

News editors obtained the following quote 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. Acta 1473: 4-8). Although mammalian glycoprotein oligosaccharides, for example, are constructed from a limited number of monosaccharides, their structural diversity is vast due to complex branching patterns. Glycoproteins, therefore, represent a diverse group of modifications, and variants of glycoproteins or glycoenzymes (which are known as glycoforms) can impact protein or enzyme activity or function. The ability to evaluate and distinguish specific glycan structures during the preparation of recombinant enzymes can accordingly provide valuable information relating to recombinant enzyme development and further optimization of the desired glycoform content of such recombinant enzymes.

"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, J. Sep. Sci. 28:1956-1968). Accordingly, these issues make the routine use of such technologies impractical for high-throughput monitoring of enzyme glycosylation, especially during process development and manufacturing. Such technologies may also present challenges to a typical research laboratory attempting to study the impact of glycosylation on the biological properties of proteins and enzymes.

"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, as well as determining the relative differences in the amount of glycans associated with such 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."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventor's summary information for this patent application: "The present invention provides novel methods, assays and compositions for the accurate and rapid identification and/or detection of various glycoforms of enzymes and the relative amount of glycan associated with such glycoforms. In particular, the present invention relies upon the ability of an enzyme of interest to competitively inhibit the binding of a ligand to detect such enzyme's presence in a test sample, as well as to determine the relative amount of glycan associated with such enzyme. 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. The methods, assays and compositions disclosed herein also provide novel strategies for analyzing the relative amounts of glycan associated with such different glycoforms. 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. Even further, the present invention provides the ability to detect and distinguish among the relative amount of glycan associated with various glycoforms or glycovariants of an enzyme in upstream harvest test samples, thereby further 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, as well as determining the relative amount of glycan associated with those glycosylated enzymes, 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) and the relative amount of glycan associated with the 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.' Some embodiments 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 extent to which the bound enzyme inhibits binding of a ligand to the capture agent. The extent to which the bound enzyme inhibits binding of the ligand to the capture agent is indicative of the relative amount of glycan associated with the enzyme in the test sample.

"Also disclosed are methods for detecting the presence of an enzyme (e.g., a recombinantly prepared enzyme) and the relative amount of glycan associated with the 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 ligand for the capture agent. In accordance with the present invention, the extent to which such bound enzyme competitively inhibits binding of the at least one ligand to the capture agent is indicative of the relative amount of glycan associated with the glycosylated enzyme of interest in the test sample. Conversely, the absence of competitive inhibition 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 ligand bound to the capture agent by contacting such ligand with a substrate, for example, a substrate which has known reactivity with the ligand. In accordance with the methods of the present invention, the presence of intrinsic enzymatic activity is indicative of the presence of ligand bound to the capture agent. Alternatively, the absence of intrinsic enzymatic activity may be indicative of the absence of such ligand bound to the capture agent in the test sample.

"In one embodiment, the methods, assays and kits of the present invention contemplate determining intrinsic enzymatic activity by contacting ligand bound capture agent with a substrate which is known to predictably react with the ligand of interest. For example, if the ligand is agalsidase alfa the selected substrate may be 4-nitrophenyl-.alpha.-D-galactopyranoside, if the ligand is galactocerebrosidase the selected substrate may be 4-nitrophenyl-.beta.-D-galactopyranoside, and if the ligand is aryl sulfatase A the selected substrate may be p-nitrocatechol sulfate. The presence or absence of intrinsic enzymatic activity of the bound ligand may be determined by means which are known to those of ordinary skill in the art. In one embodiment a quantitative assessment of the conversion of substrate to product may be indicative of intrinsic enzymatic activity of the ligand. For example, in one embodiment, following contacting an enzyme (e.g., ligand) 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 of the ligand. 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) and the relative amount of glycan associated with the glycosylated enzymes 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 ligand which is competitive with such glycosylated enzyme for binding the capture agent. 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 ligand which is known to compete with such enzyme for binding to the capture agent. 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 ligands (each of which compete with a particular enzyme whose presence is suspected in a test sample for binding to the one or more capture agents) to determine the extent to which such enzymes competitively inhibit binding of the ligands to the capture agents. Preferably, the selected ligand is known to predictably bind to, or react with, the selected capture agent, and in particular, to compete with the enzyme of interest for binding the capture agent. For example, if the enzyme is idursulfase the selected ligand may be agalsidase alfa, if the enzyme is heparan N-sulfatase the selected ligand may be agalsidase alfa, if the enzyme is aryl sulfatase A the selected ligand may be agalsidase alfa. Based upon the binding specificity or reactivity of the test sample with the ligand, one having ordinary skill in the art may use routine means to assess the extent to which the enzyme competitively inhibits the binding of the ligand to the capture agent (e.g., by detecting the presence or absence of intrinsic enzymatic activity of the ligand bound to the capture agent, e.g., by contacting the ligand bound to the capture agent with a substrate known to react with the ligand, e.g., by quantitatively determining 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.

BRIEF DESCRIPTION OF THE DRAWINGS

"The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

"FIG. 1 schematically illustrates one embodiment of the present invention in which biotinylated lectins are bound to streptavidin-coated plates, to which are added test sample materials containing different preparations of a given glycosylated enzyme which are allowed to bind. Unbound test sample materials are then removed by a wash step, and specific detection of the bound enzyme is performed by the addition of the appropriate substrate and assay conditions.

"FIG. 2 illustrates binding of agalsidase alfa to immobilized wheat germ agglutinin (WGA) and concanavalin A (ConA) as determined by measuring the enzymatic activity of agalsidase alfa (Replagal.RTM.) based on the substrate 4-nitrophenyl-.alpha.-D-galactopyranoside.

"FIG. 3 illustrates binding of galactocerebrosidase (GalC) to immobilized wheat germ agglutinin (WGA), concanavalin A (ConA), and Sambucus nigra lectin (SNA) as determined by measuring enzymatic activity of GalC using the substrate 4-nitrophenyl-.beta.-D-galactopyranoside.

"FIG. 4 illustrates binding of galactocerebrosidase (GalC) treated with increasing concentrations of sialidase to immobilized Sambucus nigra lectin (SNA), as determined by measuring enzymatic activity of GalC using the substrate 4-nitrophenyl-.beta.-D-galactopyranoside.

"FIG. 5 illustrates linkage-specific binding of purified aryl sulfatase A (ARSA) containing sialic acid in either .alpha.-2, 6 or .alpha.-2, 3 linkages to Sambucus nigra lectin (SNA), as determined by measuring enzymatic activity of ARSA using the substrate p-nitrocatechol sulfate.

"FIG. 6 illustrates binding of galactocerebrosidase (GalC) cell culture from different harvest test samples to Sambucus nigra lectin (SNA), as determined by measuring enzymatic activity of GalC using the substrate 4-nitrophenyl-.beta.-D-galactopyranoside.

"FIG. 7 schematically illustrates one embodiment of the present invention in which the M6PR(D9)6His fusion protein is bound to a nickel chelate-coated 96-well plate, to which are added test samples containing different preparations of a given glycosylated enzyme which are allowed to bind. Unbound test sample material is then removed by a wash step, and specific detection of the bound enzyme is performed by the addition of the appropriate substrate and assay conditions.

"FIG. 8 illustrates detection differences in the amount of aryl sulfatase A (ARSA) associated M6P using ARSA lots with known amounts of M6P.

"FIG. 9 illustrates detection differences in the relative amounts of sialic acid associated with idursulfase, heparan N-sulfatase (HNS), and ARSA using agalsidase alfa lots with known amounts of sialic acid.

"FIG. 10 illustrates detection differences in the relative amounts of M6P associated with idursulfase, HNS, and ARSA using agalsidase alfa lots with known amounts of M6P."

For additional information on this patent application, see: Roseman, Daniel S. Competition-Based Detection Assays. Filed June 8, 2012 and posted August 14, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=3031&p=61&f=G&l=50&d=PG01&S1=20140807.PD.&OS=PD/20140807&RS=PD/20140807

Keywords for this news article include: Antibodies, Pharmaceutical Companies, Peptides, Histidine, Immunology, Technology, Streptavidin, Glycoproteins, Plant Lectins, Blood Proteins, Fusion Proteins, Glycoconjugates, Immunoglobulins, Bacterial Proteins, Cyclic Amino Acids, Enzymes and Coenzymes, Essential Amino Acids, Wheat Germ Agglutinins.

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