High mannose glycans

Abstract

Methods and compositions related to high mannose glycans are described.

Claims

1. A method of quantifying high mannose glycans on a therapeutic glycoprotein, the method comprising: (a) providing a sample of a therapeutic glycoprotein, wherein said therapeutic glycoprotein comprises glycoforms containing high mannose glycans, wherein said high mannose glycans are present in an abundance of less than 20% relative to the total glycan mass of the glycoprotein in said sample, (b) treating said sample of said therapeutic glycoprotein with Endoglycosidase F3, to produce a processed therapeutic glycoprotein comprising at least one high mannose glycan; and (c) quantifying said at least one high mannose glycan remaining on said processed therapeutic glycoprotein after step (b), wherein said at least one high mannose glycan is present in an abundance of less than 20% relative to the total glycan mass of the glycoprotein sample.

2. The method of claim 1, wherein said quantifying step (c) comprises performing capillary electrophoresis (CE), reverse phase LC-MS or targeted reverse phase-LC-MS on the said processed therapeutic glycoprotein.

3. The method of claim 1, further comprising determining one or more of: (i) the amount of high mannose glycans on said processed therapeutic glycoprotein relative to the total glycans on said therapeutic glycoprotein; (ii) one or more relative ratios of two high mannose species selected from Man4, Man5, Man6, Man7, Man8, and Man8, remaining on said processed therapeutic glycoprotein; (iii) the relative ratio of high mannose glycans remaining on said processed therapeutic glycoprotein to hybrid structures on said therapeutic glycoprotein, (iv) the relative ratio of high mannose glycans remaining on said processed therapeutic glycoprotein to complex structures remaining on said glycoprotein, (v) the relative ratio of high mannose glycans remaining on said processed therapeutic glycoprotein to fucosylated structures remaining on said processed therapeutic glycoprotein; (vi) the presence or abundance of modified high mannose glycans remaining on said processed therapeutic glycoprotein.

4. The method of claim 1, wherein said method further comprises separately quantifying at least two individual glycans remaining on said processed therapeutic glycoprotein.

5. The method of claim 1, wherein said therapeutic glycoprotein is an antibody or a receptor-Fc fusion.

6. The method of claim 5, wherein said antibody is a pharmaceutical preparation produced from a mammalian cell culture.

7. The method of claim 1, wherein said method is performed under good manufacturing practice (GMP) conditions.

8. The method of claim 1, further comprising comparing the quantified amount of high mannose glycans remaining on the processed therapeutic glycoprotein to a reference level or a quality criterion.

9. The method of claim 3, wherein said modified high mannose glycans remaining on said processed therapeutic glycoprotein are fucosylated high mannose glycans.

10. The method of claim 1, further comprising performing a buffer exchange to a buffer compatible with enzymatic digest and/or mass spectrometry (MS) analysis.

11. The method of claim 1, further comprising reducing and alkylating said processed therapeutic glycoprotein and/or performing a buffer exchange to a buffer compatible with mass spectrometry.

12. The method of claim 8, wherein said reference level is that of a control sample, a GMP standard, or a pharmaceutical standard.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a cartoon of the pathway for biosynthesis of high mannose and complex glycans. The monosaccharides that compose the N-glycan are illustrated as the following; Fucose (light grey triangle), GlcNAc (black square), Mannose (dark grey circle), and Galactose (light grey circle). The structures that represent the high mannose species are indicated as HM9, HM8, etc.

(2) FIG. 2 is a set of LC profiles of glycans harvested from a wild-type cell line (CHO) and a Lec1 mutant (MGAT1 null) cell line.

(3) FIG. 3 is a set of plots reflecting glycan levels modeled to reflect varying levels of MGAT1. Each plot refers to the level of the indicated glycan (% of starting) based on the level of MGAT1 expression (% of starting). These illustrate that an elevation of High mannose structures does not require complete abolition of the MGAT1 transferase

(4) FIG. 4 is a set of plots representing a linear analysis of the expression of the gene UGP-2 in a cell population as it correlates to Man5 content on a glycoprotein produced by the cell. Each dot on this plot indicates a particular clone of one of four listed transformed cell lines.

(5) FIG. 5 is a plot of increasing levels of MnC12 vs levels (% of total glyans) of Man5. The data represent duplicate determinants.

DETAILED DESCRIPTION

Definitions

(6) High Mannose as used herein refers to one or a multiple of N-glycan structures including HM4, HM5, HM6, HM7, HM8, and HM9 containing 3, 4, 5, 6, 7, 8, or 9 mannose residues respectively. Alternatively these may be called Man4, Man5, Man6, Man7, Man8, Man9 These structures are illustrated in FIG. 1 as indicated.

(7) A preparation of cells, as used herein, refers to an in vitro preparation of cells. In the case of cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

(8) The term genetically engineered, as used herein in reference to cells, is meant to encompass cells that express a particular gene product following introduction of a heterologous DNA molecule into the cell. The heterologous DNA can be a sequence encoding the gene product and/or including regulatory elements that control expression of a coding sequence (e.g., of an endogenous sequence) for the gene product. The DNA molecule may be introduced by gene targeting or homologous recombination, i.e., introduction of the DNA molecule at a particular genomic site.

(9) The disclosure of WO 2008/128227 is incorporated herein in its entirety. Various aspects of the invention are described in further detail below.

(10) Host Cells/Genetically Engineered Cells

(11) A host cell used to produce a glycoprotein described herein can be any cell containing cellular machinery to produce high mannose structures. For example, insect cells, plant cells, yeast, or mammalian cells (such as murine, human or CHO cells). CHO cells useful as host cells include cells of any strain of CHO, including CHO K1 (ATCC CCL-61), CHO pro3-, CHO DG44, CHO-S, CHO P12 or the dhfr-CHO cell line DUK-BII (Chassin et al., PNAS 77, 1980, 4216-4220). Murine cells useful as host cells include strains of NS0 or other hybridoma cell types, or similar rodent cells of BHK. Human cells useful as host cells include strains of PerC6, hybridoma cells, or retinal cells to name a few.

(12) Suitable mammalian cells include any normal mortal or normal or abnormal immortal animal or human cell, including: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) (Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese Hamster Ovary (CHO), e.g., DG44, DUKX-V11, GS-CHO (ATCC CCL 61, CRL 9096, CRL 1793 and CRL 9618); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243 251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse melanoma cells (NSO); mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather, et al., Annals N.Y. Acad. Sci. 383:44 46 (1982)); canine kidney cells (MDCK) (ATCC CCL 34 and CRL 6253), HEK 293 (ATCC CRL 1573), WI-38 cells (ATCC CCL 75) (ATCC: American Type Culture Collection, Rockville, Md.), MCF-7 cells, MDA-MB-438 cells, U87 cells, A127 cells, HL60 cells, A549 cells, SP10 cells, DOX cells, SHSY5Y cells, Jurkat cells, BCP-1 cells, GH3 cells, 9L cells, MC3T3 cells, C3H-10T1/2 cells, NIH-3T3 cells and C6/36 cells. The use of mammalian tissue cell culture to express polypeptides is discussed generally in Winnacker, FROM GENES TO CLONES (VCH Publishers, N.Y., N.Y., 1987).

(13) Exemplary plant cells include, for example, Arabidopsis thaliana, rape seed, corn, wheat, rice, tobacco etc.) (Staub, et al. 2000 Nature Biotechnology 1(3): 333-338 and McGarvey, P. B., et al. 1995 Bio-Technology 13(13): 1484-1487; Bardor, M., et al. 1999 Trends in Plant Science 4(9): 376-380). Exemplary insect cells (for example, Spodoptera frugiperda Sf9, Sf21, Trichoplusia ni, etc. Exemplary bacteria cells include Escherichia coli. Various yeasts and fungi such as Pichia pastoris, Pichia methanolica, Hansenula polymorpha, and Saccharomyces cerevisiae can also be selected.

(14) Other suitable host cells are known to those skilled in the art.

(15) Methods to make and use host cells of the invention, and to make therapeutic glycoproteins in such host cells are known in the art. For example, methods are provided in Current Protocols in Cell Biology (2007, John Wiley and Sons, Inc., Print ISSN: 1934-2500); Current Protocols in Protein Science (2007, John Wiley and Sons, Inc., Print ISSN: 1934-3655); Wurm, Production of recombinant protein therapeutics in cultivated mammalian cells (2004) Nature Biotech. 22:1393-1398; Therapeutic Proteins: Methods and Protocols, Smales and James, eds. (2005, Humana Press, ISBN-10: 1588293904).

(16) Methods to Detect Gene Expression or Activity

(17) Nucleic acid based-detection methods encompass hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses (e.g., quantitative PCR), SAGE analyses, probe arrays or oligonucleotide arrays. Probes useful in such methods are routinely selected from known gene sequences. In some cases, cellular material from one species, (e.g., one rodent species such as Chinese Hamster) may be evaluated using probes identified from ortholog gene sequences (e.g., rat or mice-derived sequences) found in the art.

(18) Antibody-based techniques for detection of proteins include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, surface plasmon resonance. Other methods may include the detection of peptides or fragments thereof using mass spectrometric-based methods, including, but not limited to LC-MS, MS/MS, MS/MS/MS, MALDI-MS, multiple reaction monitoring (MRM).

(19) In one embodiment, detection methods described herein are part of determining a gene expression profile of the sample, wherein the profile includes a value representing the level of a gene's expression, among at least one other value for expression of at least one other gene. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to evaluate or screen CHO cells.

(20) In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of a gene in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, e.g., the cell type from which the sample was derived (e.g., a CHO cell strain), or a cell culture condition under which the cell that is the source of the sample was cultured. In one embodiment, the data record further includes values representing the level of expression of genes other than Ggta1 (e.g., other genes associated with glycan synthesis, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

(21) Methods of Modulating Gene Expression

(22) In certain methods of the invention, gene expression is modulated, e.g., increased or reduced. Such methods may include reducing (knocking-down) expression or abolishing (knocking out) expression of a subject gene. Methods of making and using antisense molecules to modulate biological activities are known in the art, see for example: Pan and Clawson, Antisense applications for biological control (2006) J. Cell Biochem. 98(1):14-35; Sioud and Iversen, Ribozymes, DNAzymes and small interfering RNAs as therapeutics (2005) Curr Drug Targets 6(6):647-53; Bhindi et al., Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies (2007) Am J. Pathol. 171(4):1079-88. Methods of making gene knockouts are known in the art, e.g., see Kuhn and Wurst (Eds.) Gene Knockout Protocols (Methods in Molecular Biology) Humana Pres (Mar. 27, 2009).

(23) In some embodiments, a cell can be selected which has been genetically engineered for permanent or regulated inactivation of a gene encoding a protein involved with the synthesis of a particular glycan as described herein. For example, genes encoding an enzyme such as the enzymes described herein can be inactivated. Permanent or regulated inactivation of gene expression can be achieved by targeting to a gene locus with a transfected plasmid DNA construct or a synthetic oligonucleotide. The plasmid construct or oligonucleotide can be designed to several forms. These include the following: 1) insertion of selectable marker genes or other sequences within an exon of the gene being inactivated; 2) insertion of exogenous sequences in regulatory regions of non-coding sequence; 3) deletion or replacement of regulatory and/or coding sequences; and, 4) alteration of a protein coding sequence by site specific mutagenesis.

(24) In the case of insertion of a selectable marker gene into coding sequence, it is possible to create an in-frame fusion of an endogenous exon of the gene with the exon engineered to contain, for example, a selectable marker gene. In this way following successful targeting, the endogenous gene expresses a fusion mRNA (nucleic acid sequence plus selectable marker sequence). Moreover, the fusion mRNA would be unable to produce a functional translation product.

(25) In the case of insertion of DNA sequences into regulatory regions, the transcription of a gene can be silenced by disrupting the endogenous promoter region or any other regions in the 5 untranslated region (5 UTR) that is needed for transcription. Such regions include, for example, translational control regions and splice donors of introns. Secondly, a new regulatory sequence can be inserted upstream of the gene that would render the gene subject to the control of extracellular factors. It would thus be possible to down-regulate or extinguish gene expression as desired for glycoprotein production. Moreover, a sequence which includes a selectable marker and a promoter can be used to disrupt expression of the endogenous sequence. Finally, all or part of the endogenous gene could be deleted by appropriate design of targeting substrates.

(26) Other methods of affecting gene expression, e.g., increasing or reducing gene expression, may involve addition of agents, e.g., addition of specified agents to culture media, e.g., as described herein.

(27) Biology of High Mannose Structures

(28) The presence of mannose containing glycans on proteins is known to have an effect on the interaction of these proteins with several receptors and binding partners through which the function, distribution, and stability of these mannose-containing proteins are influenced. Such binding partners includes Fc receptors, FcRn, mannose binding lectins (MBL), C1q, mannose receptor, DC and L-sign, and receptors on specific cells (see, e.g., Li et al., (2009) Curr Opin Biotechnol. 20, 678-684). Thus, methods described herein are useful to evaluate or modulate targeting of glycoproteins to specific tissues (e.g., bone marrow, mammary epithelia, intestinal epithelia), to specific cell types (e.g., dendritic cells, macrophages) or specific compartments (e.g., lysosome). The methods described herein are also useful to evaluate or modulate biological activity through receptor binding (e.g., Fc receptors), serum half lives/clearance (e.g., through binding to the mannose receptor, FcRn) and adsorption.

(29) The methods described herein are also useful to evaluate or modulate other biological activities, including: antibody deposition and aggregation. The glycan structure on the Fc portion of an antibody change the 3 dimensional structure of the antibody. Alterations in antibody structure are known to have the potential to lead to antibody aggregation and deposition. The methods used herein would be useful to generate antibodies with targeted levels of high mannose so as to decrease antibody sera deposition, complex formation and aggregation. Similarly, these structural changes can be utilized to increase or decrease immunogenicity of an antibody.

(30) In some embodiments, the antibody molecules may also contain glycosylation on the Fab portion of the molecule. In these instances, in addition to those biology described above, the presence of a high mannose structure may also alter the affinity for the epitopes, or the ability for the antibody to form cross linked complexes on the cell surface. The methods described herein would be useful to generate antibodies with altered levels of high mannose structures so as to dial in desired affinity, as well as achieve a desired level of receptor cross linking so as to decrease off target effects and increase the therapeutic window. In addition design of high mannose containing peptides and polypeptides may inhibit protein degradation through ubiquitin ligase mediated pathway.

(31) Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

(32) This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

EXAMPLES

Example 1: High Throughput Analysis of High Mannose Protein Glycoforms

(33) One of the major challenges in characterizing low abundance glycoforms in a mixture is the presence of higher abundance glycoforms as well as the diverse array of glycoforms in the mixture. The analysis of low abundance glycoforms typically involves the release of the glycans from the protein either by enzymatic (PNGAse-F or Endo-H) or chemical treatment (i.e. hydrazinolysis). The released glycans are then purified and subsequently analyzed without further derivatization or after the labeling with different chromophores/fluorophores. However, the laborious sample preparation steps involved in this process as well as the high amounts of material needed for these analyses hampers the ability to use these types of methods in a high throughput setting.

(34) The method described here is useful to analyze high mannose containing glycoforms, and in particular high mannose glycoforms present in low abundance (e.g., <10%, <5%, <4%, <3%, <2%, <1%, <0.05%).

(35) Starting with a glycoprotein-containing sample such as an antibody sample (e.g., from either media, or a protein preparation) optionally perform a buffer exchange to buffer compatible with enzymatic digest of step #2 and/or mass spectrometry analysis of step #3. This step is optional depending on the sample formulation or the chromatographic method of step 3. Treat the glycoprotein sample with an enzyme that cleaves complex fucosylated glycans from the glycoprotein in the sample (e.g., Endoglycosydase F3 (http://glycotools.qa-bio.com/s.nl/it.A/id.96/.f)). This step unexpectedly reveals low abundance glycoforms such as high mannose species, in the remaining sample. In the next step, optionally reduce and alkylate the sample and/or perform a buffer exchange to buffer compatible with MS. In a next step, analyze the enzymatically treated sample by a reverse phase LC-MS or targeted reverse phase-LC-MS analysis to identify high mannose-containing glycoforms. Other types of column chemistries can also be employed. Knowing the theoretical masses of the high mannose-containing glycoforms, a targeted MS experiment can be established to only monitor selected set of m/z signatures corresponding to these species. The analysis can be repeated with multiple samples in a comparative analysis setting.

(36) The method described above provides an excellent balance between throughput, resolution and sensitivity making it particularly suitable for the analysis of low abundance glycoforms containing high mannose in a high throughput setting.

Example 2: Effect of MGAT1 Levels on Man5 Structures

(37) Wild type CHO (WT) and Lec1 (MGAT1 null) cell lines were stably transformed to express an IgG model fusion protein. The recombinant product was harvested from the cells, and N-glycans analyzed by Amide LC/MS. LC data from WT (top) and MGAT1 deficient (bottom) CHO cells with representative glycans identified with relative percentage is shown in FIG. 2. As can be seen in FIG. 2, the glycoprotein produced from the Lec1 mutant lacks Man5 structures.

(38) However, it has been found that complete inhibition of MGAT1 is not necessary to produce this effect. FIG. 3 a set of plots reflecting glycan levels modeled to reflect varying levels of MGAT1. Each plot refers to the level of the indicated glycan (% of starting) based on the level of MGAT1 expression (% of starting). These illustrate that an elevation of high mannose structures does not require complete abolition ore depletion of the MGAT1 transferase.

Example 3: Identification of Non-Linear Correlations Related to High Mannose Content

(39) This example illustrates the identification of unexpected, non-linear correlations between glycosylation regulators and high mannose content.

(40) Stably transformed clones expressing an IgG fusion model protein were generated from each of the cell lines CHOK1, CHOS, DG44 and Dhfr-. The IgG product was isolated from each clone and the glycans characterized by 2D LC/MS. The level of Man5 was determined as a percentage of total glycans. Concurrently, mRNA was extracted from each clone and the levels of various enzymes involved in disparate aspects of glycobuilding were characterized. Such enzymes include glycosyltransferases, transporters, metabolic enzymes, and others involved in the biosynthesis of glycans. These data were subjected to linear analysis to identify relations between particular biosynthetic steps and Man5 content. FIG. 4A shows the level of gene expression of UGP-2 as it unexpectedly shows a linear correlation to Man5 content on a glycoprotein produced by the cell. While not bound by theory, FIG. 4B illustrates how this gene can, in retrospect, be correlated to metabolites involved in Man5 biosynthesis.

Example 4: Man5 Levels Affected by High Concentrations of Divalent Cations

(41) This example illustrates the inhibitory affect of elevated levels of divalent cations on glycoenzyme activity.

(42) CHO cells expressing an IgG fusion model protein were cultured in the presence of increasing levels of MnCl.sub.2. Product was harvested from these cells after 5 days, purified, and subjected to N-glycan analysis by normal phase HPLC. The levels (% of total glyans) of Man5 were quantified and are shown in FIG. 5. As can be seen, as the levels of Mn in the media are elevated there is a concommittant increase in high mannose content. While not bound by theory, this may be driven primarily by Mn cofactor activity on transferases.

(43) Extensions and Alternatives

(44) All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. While the methods have been described in conjunction with various embodiments and examples, it is not intended that the methods be limited to such embodiments or examples. On the contrary, the methods encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.