HYPERSIALYLATED IMMUNOGLOBULIN
20230357813 · 2023-11-09
Inventors
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
C12Y204/99001
CHEMISTRY; METALLURGY
C12N9/1081
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
International classification
Abstract
Described herein are methods of preparing a hypersialylated human immunoglobulin G (hsIgG) preparations.
Claims
1. A method of preparing a hypersialylated human immunoglobulin G (hsIgG) preparation, the method comprising: (a) providing a composition comprising pooled human immunoglobulin G (IgG) wherein at least 5% or at least 10% wt/wt of protein in the composition is not IgG; (b) adding β1,4-Galactosyltransferase I (β4GalT) and uridine 5′-diphosphogalactose (UDP-Gal), together or sequentially, to the composition to create a reaction mixture, wherein the reaction mixture is in a buffer; (c) incubating the reaction mixture; (d) adding ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6Gal1) and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA), together or sequentially, to the reaction mixture; and (e) incubating the reaction mixture, thereby creating the hsIgG preparation.
2. A method of preparing hypersialylated (hsIgG), the method comprising: (a) providing a composition comprising pooled human IgG wherein at least 5% or at least 10% wt/wt of protein in the composition is not IgG in a buffer; (b) incubating the composition in a reaction mixture comprising β1,4-Galactosyltransferase I (β4GalT), UDP-Gal, ST6Gal1 and CMP-NANA, in a buffer, thereby creating the hsIgG preparation.
3. A method of preparing hypersialylated (hsIgG), the method comprising: (a) providing a composition comprising pooled human IgG wherein at least 5% or at least 10% wt/wt of protein in the composition is not IgG; (b) combining the composition with β4GalT and ST6Gal to create a reaction mixture, wherein the reaction mixture is in a buffer and contains UDP-Gal and CMP-NANA; and incubating the reaction mixture, thereby creating the hsIgG preparation.
4. The method of claim 1, wherein the buffer is selected from the group consisting of Bis(2-hydroxyethyl)amino0tris(hydroxymethyl)methane (BIS-TRIS), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), 1,4-Piperazinediethanesulfonic acid (PIPES), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), Triethanolamine (TEA), Piperazine-N—N′-bis(2-hydroxypropanesulfonic acid (POPSO), 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), and combinations thereof.
5. The method of claim 1, wherein providing a mixture of IgG antibodies includes (a) providing pooled plasma from at least 1000 human subjects; and (b) isolating a mixture of IgG antibodies from the pooled plasma.
6. (canceled)
7. The method of claim 1, wherein additional CMP-NANA is added to the reaction at a time after the first addition of CMP-NANA.
8-14. (canceled)
15. The method of claim 1, further comprising subjecting the hsIgG preparation to one or more purification steps.
16-36. (canceled)
37. The method of claim 1, wherein the reactions take place in BIS-TRIS at 10-500 mM pH 5.5-8.5.
38. The method of claim 1, wherein reaction mixtures comprise MnCl.sub.2 at 1-20 mM.
39. The method of claim 1, wherein the initial concentration of UDP-Gal in the reaction mixture comprising UDP-Gal is 20-500 UDP-Gal/g IgG antibody.
40. The method of claim 1, wherein initial concentration of CMP-NANA in the reaction mixture comprising CMP-NANA is 100-3000 μmol CMP-NANA/g IgG antibody.
41-44. (canceled)
45. The method of claim 1, wherein the hsIgG preparation is further treated to removed ST6Gal1 and β4GalT.
46. The method of claim 1, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in the hsIgG preparation have a sialic acid on both the α1,3 branch and the α1,6 branch.
47-58. (canceled)
Description
DESCRIPTION OF DRAWINGS
[0086]
[0087]
[0088]
[0089]
[0090]
[0091]
[0092]
[0093]
[0094]
[0095]
[0096]
[0097]
[0098]
[0099]
DETAILED DESCRIPTION
[0100] The present disclosure encompasses, in part, methods for preparing immunoglobulins (e.g., human IgG) having an Fc region having particular levels of branched glycans that are sialylated on both of the arms of the branched glycan (e.g., with a NeuAc-α 2,6-Gal terminal linkage). The levels can be measured on an individual Fc region (e.g., the number of branched glycans that are sialylated on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region), or on the overall composition of a preparation of polypeptides (e.g., the number or percentage of branched glycans that are sialylated on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region in a preparation of polypeptides). In some embodiments, the present disclosure concerns methods of treatment using hsIgG.
[0101] Immunoglobulins are glycosylated at conserved positions in the constant regions of their heavy chain. For example, IgG antibodies have a single N-linked glycosylation site at Asn297 of the C.sub.H2 domain. For human IgG, which is the type of antibody present in IVIG and hsIgG, the core oligosaccharide normally consists of GlcNAc2Man3GlcNAc and a core fucose, with differing numbers of outer residues. Variation among individual IgG's can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc). Various commercial preparations of IVIG are general less than 10% sialylated or even less than 5% sialylated on the Asn297 of the C.sub.H2 domain.
[0102] Naturally derived polypeptides that can be used to prepare hypersialylated IgG include, for example, IgG in human serum (e.g., human serum pooled from more than 1,000 donors), IgG derived from human serum, intravenous immunoglobulin (IVIg) and polypeptides derived from IVIg (e.g., polypeptides purified from IVIg (e.g., enriched for sialylated IgGs)) or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).
[0103] The level of sialylation can be measured on an individual Fc region (e.g., the number of branched glycans that are sialylated on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region), or on the overall composition of a preparation of glycoproteins (e.g., the number or percentage of branched glycans that are sialylated on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region in a preparation of glycoproteins).
[0104] Hypersialylated IgG is a composition comprising immunoglobulin in which at least 40% of the glycans in the Fc regions are disialylated, i.e., have a sialic acid on the α1,3 arm (e.g., with a NeuAc-α2,6-Gal terminal linkage) and on the α1,6 arm (e.g., with a NeuAc-α2,6-Gal terminal linkage).
[0105] In some embodiments, a sialylation level is an absolute level or range of (e.g., number of moles of or percent of) one or more glycans (e.g., branched glycans having a sialic acid on an α1,3 arm, and/or branched glycans having a sialic acid on an α1,6 arm, and/or branched glycans having a sialic acid on an α1,3 arm and on an α1,6 arm) in a glycoprotein preparation. In some embodiments, a predetermined or target level is a level or range of one or more glycans (e.g., branched glycans having a sialic acid on an α1,3 arm, and/or branched glycans having a sialic acid on an α1,6 arm, and/or branched glycans having a sialic acid on an α1,3 arm and on an α1,6 arm) in a glycoprotein preparation relative to total level of glycans in the glycoprotein preparation. In some embodiments, a predetermined or target level is a level or range of one or more glycans (e.g., branched glycans having a sialic acid on an α1,3 arm, and/or branched glycans having a sialic acid on an α1,6 arm, and/or branched glycans having a sialic acid on an α1,3 arm and on an α1,6 arm) in a glycoprotein preparation relative to total level of sialylated glycans in the glycoprotein preparation. In some embodiments, a predetermined or target level is expressed as a percent.
[0106] ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6 or ST6Gal1) (e.g., human ST6) is used in the methods described herein. Useful ST6 includes polypeptides whose amino acid sequence includes at least one characteristic sequence of and/or shows at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, or 70% identity with a protein involved in transfer of a sialic acid to a terminal galactose of a glycan through an α2,6 linkage (e.g., human ST6Gal1 or ST6), e.g., polypeptides that are at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, or 70% identity to any of SEQ ID NOs:1 and 4-9. A wide variety of ST6 sialyltransferase sequences are known in the art, such as those described herein; in some embodiments, an ST6 sialyltransferase shares at least one characteristic sequence of and/or shows the specified degree of overall sequence identity with one of the ST6 sialyltransferases set forth herein (each of which may be considered a “reference” ST6 sialyltransferase). In some embodiments, an ST6 sialyltransferase as described herein shares at least one biological activity with a reference ST6 sialyltransferase as set forth herein. In some such embodiment, the shared biological activity relates to transfer of a sialic acid to a glycan. In some embodiments, an ST6 sialyltransferase is a human sialyltransferase.
[0107] Beta-1,4-galactosyltransferase 1 (B4GalT), e.g., human B4GalT, is used in the methods described herein. Useful B4GalT can be a polypeptide whose amino acid sequence includes at least one characteristic sequence of and/or shows at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, or 70% identity with a protein involved in transfer of a transfers galactose from uridine 5′-diphosphosegalactose (UDP-Gal) to GlcNAc as a β-1,4 linkage. A wide variety of Beta-1,4-galactosyltransferase sequences are known in the art, such as those described herein; in some embodiments, an Beta-1,4-galactosyltransferase shares at least one characteristic sequence of and/or shows the specified degree of overall sequence identity with one of the Beta-1,4-galactosyltransferases set forth herein (each of which may be considered a “reference” Beta-1,4-galactosyltransferase). In some embodiments, an Beta-1,4-galactosyltransferase as described herein shares at least one biological activity with a reference Beta-1,4-galactosyltransferase as set forth herein. In some such embodiment, the shared biological activity relates to transfer of a galactose to a glycan. In some embodiments, an Beta-1,4-galactosyltransferase is a human galactosyltransferase.
[0108] Cohn II/III and/or Cohn I/II/III can be used as a source of IgG to prepare hsIgG. Various methods for preparing Cohn II/III and/or Cohn I/II/III are described in Cohn et al. (J. Amer. Chem. Soc. 68, 459-75 (1946)). For example, Cohn Fraction I is precipitated from pooled plasma and removed by adding ethanol at low temperatures (e.g., at 0.027 mole fraction to plasma at −3° C.). The pH of the supernatant (Cohn Fraction II/III/IV/V) is reduced by adding a buffer and salt (e.g., the pH of the supernatant is reduced to about 6.8 by addition of sodium acetate-acetic acid buffer with a molar ratio of salt to acid of 1.77) and precipitation at −5° C. with 0.091 mole fraction in ethanol yielding Cohn II/III.
[0109] Cohn II/III can be purchased from Sigma-Aldrich (St. Louis, MO) and various suppliers of plasma products.
[0110] In some embodiments, a first fractionation/precipitation can be performed to precipitate Cohn Fraction I leaving all other components soluble. A subsequent precipitation can yield Cohn II/III. In some embodiments, an initial fractionation can yield Cohn I/II/III.
Enzymes
Galactosylating Enzymes
[0111] Beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same are suitable for use in the methods described herein. B4Galt1 is one of seven beta-1,4-galactosyltransferase (beta4GalT) genes that each encode type II membrane-bound glycoproteins that appear to have exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a beta 1,4 linkage to similar acceptor sugars: GlcNAc, Glc, and Xyl. B4Galt1 adds galactose to N-acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains. B4GalT1 is also called GGTB2. Four alternative transcripts encoding four isoforms of B4GALT1 (NCBI Gene ID 2683) are described in Table 1.
TABLE-US-00001 TABLE 1 Human B4GALT1 isoforms Length Length Transcript (nt) Protein SEQ ID NO: (aa) Isoform NM_001497.4 4176 NP_001488.2 SEQ ID NO: 5 398 1 NM_001378495.1 3999 NP_001365424.1 SEQ ID NO: 6 385 2 NM_001378496.1 4053 NP_001365425.1 SEQ ID NO: 7 357 3 NM_001378497.1 1520 NP_001365426.1 SEQ ID NO: 8 225 4
TABLE-US-00002 TABLE 2 Topology of B4GALT1 isoform 1 (SEQ ID NO: 5) SEQ Feature AAs Description Length Sequence ID NO: Topologica 1-24 Cytoplasmic 9 MRLREPLLSGSAAMPGASLQR SEQ 1 domain ACR ID NO: 9 Trans- 25-44 Helical; 17 LLVAVCALHLGVTLVYYLAG SEQ membrane Signal- ID NO: anchor for 10 type II membrane protein Topological 45-398 Lumenal 380 RDLSRLPQLVGVSTPLQGGSN SEQ domain SAAAIGQSSGELRTGGARPPP ID NO: PLGASSQPRPGGDSSPVVDSG 11 PGPASNLTSVPVPHTTALSLP ACPEESPLLVGPMLIEFNMPV DLELVAKQNPNVKMGGRYAPR DCVSPHKVAIIIPFRNRQEHL KYWLYYLHPVLQRQQLDYGIY VINQAGDTIFNRAKLLNVGFQ EALKDYDYTCFVFSDVDLIPM NDHNAYRCFSQPRHISVAMDK FGFSLPYVQYFGGVSALSKQQ FLTINGFPNNYWGWGGEDDDI FNRLVFRGMSISRPNAVVGRC RMIRHSRDKKNEPNPQRFDRI AHTKETMLSDGLNSLTYQVLD VQRYPLYTQITVDIGTPS
TABLE-US-00003 TABLE 3 Binding sites of B4GALT1 isoform 1 (SEQ ID NO: 5) Position(s) Description Reference(s) 250 Metal binding; Manganese 310 Binding site; “Structural snapshots of beta-1,4- UDP-alpha-D- galactosyltransferase-I along the kinetic pathway.” galactose Ramakrishnan B., Ramasamy V., Qasba P. K. J. Mol. Biol. 357: 1619-1633(2006) 343 Metal binding; Manganese; via tele nitrogen 355 Binding site; N- “Oligosaccharide preferences of beta1,4- acetyl-D- galactosyltransferase-I: crystal structures of glucosamine Met340His mutant of human beta1,4- galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety.” Ramasamy V., Ramakrishnan B., Boeggeman E., Ratner D. M., Seeberger P. H., Qasba P. K. J. Mol. Biol. 353: 53-67(2005) “Deoxygenated disaccharide analogs as specific inhibitors of beta1-4-galactosyltransferase 1 and selectin-mediated tumor metastasis.” Brown J. R., Yang F., Sinha A., Ramakrishnan B., Tor Y., Qasba P. K., Esko J. D. J. Biol. Chem. 284: 4952-4959(2009)
TABLE-US-00004 TABLE 4 Post Translational Amino Acid Modifications of B4GALT1 isoform 1 (SEQ ID NO: 5) Feature key Position(s) Description Reference(s) Glycosylation 113 N-linked (GlcNAc . . .) asparagine Disulfide 130 .Math. 172 “Oligosaccharide preferences of beta1,4- bond galactosyltransferase-I: crystal structures of Disulfide 243 .Math. 262 Met340His mutant of human beta1,4- bond galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety.” Ramasamy V., Ramakrishnan B., Boeggeman E., Ratner D. M., Seeberger P. H., Qasba P. K. J. Mol. Biol. 353: 53-67(2005) “Structural snapshots of beta-1,4- galactosyltransferase-I along the kinetic pathway.” Ramakrishnan B., Ramasamy V., Qasba P. K. J. Mol. Biol. 357: 1619-1633(2006)
[0112] The soluble form of B4GalT1 derives from the membrane form by proteolytic processing. The cleavage site is at positions 77-78 of B4GALT1 isoform 1 (SEQ ID NO: 5).
[0113] In some embodiments, one or more of the amino acids of the B4GalT1 corresponding to amino acids 113, 130, 172, 243, 250, 262, 310, 343, or 355 of B4GALT1 isoform 1 (SEQ ID NO: 5) is conserved as compared to (SEQ ID NO: 5). In some embodiments, the enzyme is an enzymatically active portion of, e.g., B4GalT1. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 1 (SEQ ID NO: 5), or an ortholog, mutant, or variant of SEQ ID NO: 5. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 2 (SEQ ID NO: 6), or an ortholog, mutant, or variant of SEQ ID NO: 6. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 3 (SEQ ID NO: 7), or an ortholog, mutant, or variant of SEQ ID NO: 7. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 4 (SEQ ID NO: 8), or an ortholog, mutant, or variant of SEQ ID NO: 8.
[0114] In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 9. In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a transmembrane domain, e.g., SEQ ID NO: 10. In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 9 or a transmembrane domain, e.g., SEQ ID NO: 10.
[0115] In some embodiments, the enzymatically active portion of B4GalT1 comprises all or a portion of a luminal domain, e.g., SEQ ID NO: 11, or an ortholog, mutants, or variants thereof.
[0116] In some embodiments, the enzymatically active portion of B4GalT1 comprises amino acids 109-398 of SEQ ID NO: 5, or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of B4GalT1 consists of SEQ ID NO: 5, or an ortholog, mutant, or variant of SEQ ID NO: 5.
[0117] A suitable functional portion of an B4GalT1 can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 12.
[0118] Also suitable for use in the methods described herein is an amino acid sequence that comprises or consists of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 13.
Sialylating Enzymes
[0119] ST6, e.g., ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same, are suitable for use in the methods described herein. Alpha-2,6-sialyltransferase 1 (ST6) is a Type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-NANA) to Gal as an α-2,6 linkage. ST6Gal1 is also called as ST6N or SIAT1. Four alternative transcripts encoding two isoforms of ST6GAL1 (NCBI Gene ID 6480) are described in Table 5.
TABLE-US-00005 TABLE 5 Human ST6GAL1 isoforms Length Length Transcript (nt) Protein SEQ ID NO: (aa) Isoform NM_173216.2 4604 NP_775323.1 SEQ ID NO: 14 406 a NM_173217.2 3947 NP_775324.1 SEQ ID NO: 15 175 b NM_003032.3 4303 NP_003023.1 SEQ ID NO: 14 406 a NM_001353916.2 4177 NP_001340845.1 SEQ ID NO: 14 406 a
TABLE-US-00006 TABLE 6 Topology of ST6Gall isoform a (SEQ ID NO: 14) SEQ ID Feature AAS Description Length Sequence NO: Topological 1-9 Cytoplasmic 9 MIHTNLKKK SEQ domain ID NO: 16 Trans- 10-26 Helical; 17 FSCCVLVFLLFAVICVW SEQ membrane Signal- ID anchor for NO: type II 17 membrane protein Topological 27-406 Lumenal 380 KEKKKGSYYDSFKLQTKEFQVLK SEQ domain SLGKLAMGSDSQSVSSSSTQDPH ID RGRQTLGSLRGLAKAKPEASFQV NO: WNKDSSSKNLIPRLQKIWKNYLS 18 MNKYKVSYKGPGPGIKFSAEALR CHLRDHVNVSMVEVTDFPFNTSE WEGYLPKESIRTKAGPWGRCAVV SSAGSLKSSQLGREIDDHDAVLR FNGAPTANFQQDVGTKTTIRLMN SQLVTTEKRFLKDSLYNEGILIV WDPSVYHSDIPKWYQNPDYNFFN NYKTYRKLHPNQPFYILKPQMPW ELWDILQEISPEEIQPNPPSSGM LGIIIMMTLCDQVDIYEFLPSKR KTDVCYYYQKFFDSACTMGAYHP LLYEKNLVKHLNQGTDEDIYLLG KATLPGFRTIHC
TABLE-US-00007 TABLE 7 Binding sites of ST6Gal1 isoform a (SEQ ID NO: 14) Position(s) Description Reference(s) 189 Substrate; via “The structure of human alpha-2,6-sialyltransferase amide nitrogen reveals the binding mode of complex glycans.” 212 Substrate Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., 233 Substrate Rudolph M. G. Acta Crystallogr. D 69: 1826- 353 Substrate; via 1838(2013) carbonyl oxygen 354 Substrate 365 Substrate 369 Substrate 370 Substrate “The structure of human alpha-2,6-sialyltransferase 376 Substrate reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69: 1826- 1838(2013)
TABLE-US-00008 TABLE 8 Post Translational Amino Acid Modifications of ST6Gal1 isoform a (SEQ ID NO: 14) Feature key Position(s) Description Reference(s) Disulfide 142 .Math. 406 “The structure of human alpha-2,6- bond sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69: 1826- 1838(2013) Glycosylation 149 N-linked “Glycoproteomics analysis of human (GlcNAc . . .) liver tissue by combination of multiple asparagine enzyme digestion and hydrazide chemistry.” Chen R., Jiang X., Sun D., Han G., Wang F., Ye M., Wang L., Zou H. J. Proteome Res. 8: 651-661(2009); and “The structure of human alpha-2,6- sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69: 1826- 1838(2013) Glycosylation 161 N-linked “Glycoproteomics analysis of human (GlcNAc . . .) liver tissue by combination of multiple asparagine enzyme digestion and hydrazide chemistry.” Chen R., Jiang X., Sun D., Han G., Wang F., Ye M., Wang L., Zou H. J. Proteome Res. 8: 651-661(2009) Disulfide 184 .Math. 335 “The structure of human alpha-2,6- bond sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69: 1826- 1838(2013) Disulfide 353 .Math. 364 “The structure of human alpha-2,6- bond sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69: 1826- 1838(2013) Modified 369 Phosphotyrosine “Quantitative phosphoproteomic residue analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions.” Mayya V., Lundgren D. H., Hwang S. I., Rezaul K., Wu L., Eng J. K., Rodionov V., Han D. K. Sci. Signal. 2: RA46-RA46(2009)
[0120] The soluble form of ST6Gal1 derives from the membrane form by proteolytic processing.
[0121] In some embodiments, one or more of the amino acids of the ST6Gal1 corresponding to amino acids 142, 149, 161, 184, 189, 212, 233, 335, 353, 354, 364, 365, 369, 370, 376, or 406 of ST6Gal1 isoform a (SEQ ID NO: 14) is conserved as compared to SEQ ID NO: 14.
[0122] Also provided herein is an enzymatically active portion of, e.g., ST6Gal1. In some embodiments, the enzyme is an enzymatically active portion of STG6Gal1 isoform a (SEQ ID NO: 14), or an ortholog, mutant, or variant of SEQ ID NO: 14. In some embodiments, the enzyme is an enzymatically active portion of STG6Gal1 isoform b (SEQ ID NO: 15), or an ortholog, mutant, or variant of SEQ ID NO: 15.
[0123] In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 16. In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a transmembrane domain, e.g., SEQ ID NO: 17. In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 16 or a transmembrane domain, e.g., SEQ ID NO: 17.
[0124] In some embodiments, the enzymatically active portion of ST6Gal1 comprises all or a portion of a luminal domain, e.g., SEQ ID NO: 18, or an ortholog, mutants, or variants thereof.
[0125] In some embodiments, the enzymatically active portion of ST6Gal1 comprises amino acids 87-406 of SEQ ID NO: 14 (SEQ ID NO: 19), or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of ST6Gal1 consists of SEQ ID NO: 19, or an ortholog, mutant, or variant of SEQ ID NO: 19.
[0126] A suitable functional portion of an ST6Gal1 can comprise or consist of an amino acid sequence that is at least 70%, (80%, 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 19.
[0127] In some embodiments, the ST6Gal1 comprises or consists of SEQ ID NO: 19, the portion of SEQ ID NO: 19 from amino acid 23 to 320, the portion of SEQ ID NO: 19 from amino acid 13 to 320, the portion of SEQ ID NO: 19 from amino acid 11 to 320, the portion of SEQ ID NO: 19 from amino acid 6 to 320, the portion of SEQ ID NO: 19 from amino acid 5 to 320, or the portion of SEQ ID NO: 19 from amino acid 4 to 320.
[0128] In some embodiments, the enzymatically active portion of ST6Gal1 comprises amino acids 109-406 of SEQ ID NO: 14 (SEQ ID NO: 20), or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of ST6Gal1 consists of SEQ ID NO: 20, or an ortholog, mutant, or variant of SEQ ID NO: 20.
[0129] A suitable functional portion of an ST6Gal1 can comprise or consist of an amino acid sequence that is at least 70%, (80%, 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 20.
[0130] Also suitable for use in the methods described herein is an amino acid sequence that comprises or consists of an amino acid sequence that is at least 70% (80%, 85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 21.
Variants
[0131] In some embodiments, the enzyme(s) described herein are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of an exemplary sequence (e.g., as provided herein), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein. In preferred embodiments, the variant retains desired activity of the parent, e.g., β-galactoside α-2,6-sialyltransferase activity or β-1,4-galactosyltransferase activity.
[0132] To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid “identity” is equivalent to nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
[0133] Percent identity between a subject polypeptide or nucleic acid sequence (i.e. a query) and a second polypeptide or nucleic acid sequence (i.e. target) is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); “BestFit” (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher Plus™, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed, pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for target proteins or nucleic acids, the length of comparison can be any length, up to and including full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of the present disclosure, percent identity is relative to the full length of the query sequence.
[0134] For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0135] In some embodiments, an enzyme that has a sequence that is not 100% identical to a sequence described herein differs by amino acid substitutions or deletions. In the case of substitution, the difference can be conservative or nonconservative substitution of one or more amino acid residues. Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of similar characteristics. Typical conservative substitutions are the following replacements: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine or vice versa; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
[0136] In some embodiments, a B4GalT or a ST6 sialyltransferase polypeptide includes a substituent group on one or more amino acid residues. Still other useful polypeptides are associated with (e.g., fused, linked, or coupled to) another moiety (e.g., a peptide or molecule). For example, a B4GalT or an ST6 sialyltransferase polypeptides can be fused, linked, or coupled to an amino acid sequence (e.g., a leader sequence, a secretory sequence, a proprotein sequence, a second polypeptide, or a sequence that facilitates purification, enrichment, or stabilization of the polypeptide).
Glycans
[0137] In vivo, N-linked oligosaccharide chains are added to a protein, for example an immunoglobulin, in the lumen of the endoplasmic reticulum. Specifically, an initial oligosaccharide (typically 14-sugar) is added to the amino group on the side chain of an asparagine residue contained within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline. The structure of this initial oligosaccharide is common to most eukaryotes, and contains three glucose, nine mannose, and two N-acetylglucosamine residues. This initial oligosaccharide chain can be trimmed by specific glycosidase enzymes in the endoplasmic reticulum, resulting in a short, branched core oligosaccharide composed of two N-acetylglucosamine and three mannose residues. One of the branches is referred to in the art as the “α1,3 arm,” and the second branch is referred to as the “α1,6 arm,” as shown in
[0138] N-glycans can be subdivided into three distinct groups called “high mannose type,” “hybrid type,” and “complex type,” with a common pentasaccharide core (Man (α1,6)-(Man(α1,3))-Man(β1,4)-GlcNAc(β1,4)-GlcNAc(β1,N)-Asn) occurring in all three groups.
[0139] The more common Fc glycans present in IVIg include those shown in
[0140] After initial processing in the endoplasmic reticulum, the polypeptide is transported to the Golgi where further processing may take place. If the glycan is transferred to the Golgi before it is completely trimmed to the core pentasaccharide structure, it results in a “high-mannose glycan.”
[0141] Additionally or alternatively, one or more monosaccharides units of N-acetylglucosamine may be added to the core mannose subunits to form a “complex glycan.” Galactose may be added to the N-acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains that terminate with any of a sialic acid, a galactose or an N-acetylglucosamine residue. Additionally, a fucose residue may be added to an N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by specific glycosyl transferases.
[0142] “Hybrid glycans” comprise characteristics of both high-mannose and complex glycans. For example, one branch of a hybrid glycan may comprise primarily or exclusively mannose residues, while another branch may comprise N-acetylglucosamine, sialic acid, galactose, and/or fucose sugars.
[0143] Sialic acids are a family of 9-carbon monosaccharides with heterocyclic ring structures. They bear a negative charge via a carboxylic acid group attached to the ring as well as other chemical decorations including N-acetyl and N-glycolyl groups. The two main types of sialyl residues found in polypeptides produced in mammalian expression systems are N-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). These usually occur as terminal structures attached to galactose (Gal) residues at the non-reducing termini of both N- and O-linked glycans. The glycosidic linkage configurations for these sialyl groups can be either α2,3 or α2,6.
[0144] Fc regions are glycosylated at conserved, N-linked glycosylation sites. For example, each heavy chain of an IgG antibody has a single N-linked glycosylation site at Asn297 of the C.sub.H2 domain (see Jefferis, Nature Reviews 8:226-234 (2009)). IgA antibodies have N-linked glycosylation sites within the C.sub.H2 and C.sub.H3 domains, IgE antibodies have N-linked glycosylation sites within the C.sub.H3 domain, and IgM antibodies have N-linked glycosylation sites within the C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4 domains (see Arnold et al., J. Biol. Chem. 280:29080-29087 (2005); Mattu et al., J. Biol. Chem. 273:2260-2272 (1998); Nettleton et al., Int. Arch. Allergy Immunol. 107:328-329 (1995)).
[0145] Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions. For example, IgG has a single N-linked biantennary carbohydrate at Asn297 of the C.sub.H2 domain in each Fc polypeptide of the Fc region, which also contains the binding sites for C1q and FcγR (see Jefferis et al., Immunol. Rev. 163:59-76 (1998); and Wright et al., Trends Biotech 15:26-32 (1997)). For human IgG, the core oligosaccharide normally consists of GlcNAc2Man3GlcNAc, with differing numbers of outer residues. Variation among individual IgG can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc), and/or attachment of fucose.
Antibodies
[0146] Antibodies useful in the methods described herein include, for example, immunoglobulins, e.g., IgG antibodies. The antibodies, e.g., IgG antibodies, can be pooled. For example, IgG antibodies can be in pooled plasma. In some embodiments, the IgG antibodies comprise IgG antibodies isolated from at least 1000 donors. In some embodiments, at least 50%, 55%, 60%, 65% or 70% w/w of the IgG antibodies are IgG1 antibodies. In some embodiments, at least 90% of the donor subject has been exposed to a virus.
[0147] In some cases, the pooled IgG antibodies are produced using fractionation, e.g., cold ethanol fractionation (e.g., the Cohn-Oncley procedure (see Cohn et al., “Preparation and Properties of Serum and Plasma Proteins: IV. A System for the Separation into Fractions of the Protein and Lipoprotein Components of Biological Tissues and Fluids,” J Am Chem Soc 68:459-75 (1946))). See, e.g., Ofosu et al., “Plasma-Derived Biological Medicines Used to Promote Haemostasis,” Thromb Haemost 99:851-62 (2008); see also Cai et al., “Ensuring the Biological Safety of Plasma-Derived Therapeutic Proteins: Detection, Inactivation, and Removal of Pathogens,” BioDrugs 19(2):79-96 (2005). In some cases, fractionation comprises slowly thawing frozen plasma (e.g., at about 2° C.-4° C.) and centrifuging to remove cryoprecipitate prior to altering the solubility of the proteins remaining in the supernatant by manipulating, e.g., concentration of ethanol, temperature, pH, and/or ionic strength. In some cases, fractionation further comprises centrifugation, filtration (e.g., nanofiltration), and/or pathogen reduction treatments (see, e.g., Cai et al.).
[0148] In some cases, the pooled IgG antibodies pooled human plasma, pooled human serum, cryoprecipitate depleted plasma, pooled human serum or plasma that has been ethanol precipitated, human Cohn II/III plasma fraction, or human Cohn plasma I/II/III fraction. In some cases, the pooled IgG antibodies are human Cohn II/III plasma fraction, or human Cohn plasma I/II/III fraction.
[0149] In some cases, the human Cohn II/III plasma fraction is obtained by a process comprising: (a) cryoseparation; (b); alcohol precipitation; and (c) collecting the precipitate, thereby obtaining human Cohn I plasma fraction. In some cases, alcohol precipitation is ethanol precipitation, e.g., cold ethanol precipitation, e.g., ethanol precipitation from about −3° C. to about −5° C. In some cases, the alcohol precipitation comprises alcohol precipitation at or at about 8% (v/v) alcohol, e.g., at or at about 8% (v/v) ethanol. In some cases, alcohol precipitation is carried out at or at about pH 7.2. In some cases, the human Cohn II/III plasma fraction is obtained by a process comprising: (a) providing human Cohn I plasma fraction supernatant; (b) a second alcohol precipitation; and (c) collecting the precipitate, thereby obtaining human Cohn II/III plasma fraction. In some cases, the second alcohol precipitation comprises alcohol precipitation at or at about 20% (v/v) alcohol, e.g., at or at about 20% to or to about 25% (v/v) ethanol. In some cases, the second alcohol precipitation is carried out at or at about pH 6.9. In some cases, the human Cohn I/II/III fraction is obtained by combining human Cohn I plasma fraction and human Cohn II/III plasma fraction.
[0150] In some embodiments, the methods described herein include providing a mixture of IgG antibodies. In some embodiments, providing a mixture of IgG antibodies includes (a) providing pooled plasma from at least 1000 human subjects; and (b) isolating a mixture of IgG antibodies from the pooled plasma. In some embodiments, the mixture of IgG antibodies are isolated from intravenous immunoglobulin. In some embodiments, the mixture of IgG antibodies are intravenous immunoglobulin.
[0151] In some embodiments, isolating a mixture of IgG antibodies from the pooled plasma comprises precipitation (e.g., caprylate precipitation and/or polyethylene glycol (PEG) precipitation), chromatography (e.g., ion exchange chromatography, hydrophilic interaction chromatography, e.g., hydrophilic interaction liquid chromatography, and/or high performance liquid chromatography, e.g., C18 high performance liquid chromatography), filtration, delipidation, pathogen inactivation, e.g., viral inactivation (e.g., detergent treatment and/or caprylate precipitation), or a combination thereof.
[0152] In some embodiments, the step of isolating a mixture of IgG antibodies from the pooled plasma comprises ethanol precipitation and/or caprylic acid (also called octanoic acid) precipitation.
[0153] In some embodiments, the step of isolating a mixture of IgG antibodies from the pooled plasma comprises PEG-4000 and/or PEG-6000 precipitation.
[0154] In some embodiments, the step of isolating a mixture of IgG antibodies from the pooled plasma comprises ethanol precipitation or caprylic acid precipitation followed by PEG-4000 and/or PEG-6000 precipitation, e.g., caprylic acid precipitation followed by PEG-4000 precipitation.
[0155] In some embodiments, the step of isolating a mixture of IgG antibodies from the pooled plasma comprises binding IgG antibodies to an ion exchange column and eluting the IgG antibodies from an ion exchange column.
[0156] In some embodiments, the step of isolating a mixture of IgG antibodies from the pooled plasma comprises adding one or more of: a delipidation agent, a high purity diatomite filter media, or a fumed silica to the mixture of IgG antibodies. In some embodiments, the delipidation agent, high purity diatomite filter media, or fumed silica is added to the mixture of IgG antibodies prior to buffer exchange.
[0157] In some cases, isolating a mixture of IgG antibodies, e.g., the Cohn I/II/III fraction or Cohn II/III fraction, comprises reducing the amount of non-IgG protein. In some cases, isolating a mixture of IgG antibodies, e.g., the Cohn I/II/III fraction or Cohn II/III fraction, comprises reducing the amount of non-protein components. In some cases, isolating a mixture of IgG antibodies, e.g., the Cohn I/II/III fraction or Cohn II/III fraction, comprises viral inactivation.
[0158] Purification techniques suitable for reducing the amount of non-IgG proteins and/or non-protein components are known and described in the art. See, e.g., Ofosu et al., “Plasma-Derived Biological Medicines Used to Promote Haemostasis,” Thromb Haemost 99:851-62 (2008).
[0159] Suitable viral inactivation methods are known and described in the art. See, e.g., Cai et al., “Ensuring the Biological Safety of Plasma-Derived Therapeutic Proteins: Detection, Inactivation, and Removal of Pathogens,” BioDrugs 19(2):79-96 (2005).
Enzymatic Galactosylation and Sialylation
[0160] Enzymatic galactosylation and sialylation reactions suitable for the methods described herein are described, for example, in PCT/US2021/033150.
[0161] The methods described herein can comprise a galactosylation step. An exemplary galactosylation reaction is depicted in
[0162] The methods described herein can comprise a sialylation step. An exemplary sialylation reaction is depicted in
[0163] In some embodiments, the galactosylation step and the sialylation step are carried out sequentially in the same reaction mixture, that is, the galactosylation reaction mixture becomes the sialylation reaction mixture upon addition of the sialylating enzyme and CMP-NANA or salt thereof. In some embodiments, the galactosylation reaction mixture is not filtered, fractionated, or purified prior to the sialylation step. In some embodiments, the galactosylation step and the sialylation step are carried out separately, e.g., pre-galactosylated antibod(ies) are provided, though they may have been processed (e.g., filtered, fractionated, or purified) and/or stored prior to the sialylation step.
[0164] Thus, the methods described herein can also comprise a sequential galactosylation and sialylation step. An exemplary galactosylation and sialylation reaction is depicted in
[0165] Also provided herein is a method for galactosylating and sialylating, e.g., hyper-sialylating antibod(ies), e.g., antibod(ies) described herein, by providing a composition comprising: antibod(ies), e.g., as described herein; a galactosylating enzyme, e.g., a galactosylating enzyme described herein, e.g., B4GalT or enzymatically active portion or variant thereof; UDP-gal or a salt thereof; a sialylating enzyme, e.g., a sialylating enzyme described herein, e.g., ST6Gal1 or enzymatically active portion or variant thereof; CMP-NANA or salt thereof; and d) incubating the composition under conditions effective for galactosylating and sialylating the antibod(ies), e.g., as described herein.
[0166] In some embodiments, the galactosylation reaction mixture and/or the sialylation reaction mixture comprises Bis (2-hydroxyethyl) aminotris (hydroxymethyl)methane (BIS-TRIS) buffer.
[0167] In some embodiments, the galactosylation reaction mixture and/or the sialylation reaction mixture comprises MnCl.sub.2.
[0168] In some embodiments, one or more component(s) of one or more of the reaction mixture(s) are supplemented during the incubation. That is, the reaction mixture may comprise an amount of the component at the beginning of the reaction (which may change during the course of the reaction), but also be supplemented with additional amounts of the component(s) during the reaction.
[0169] In some embodiments, the B4GalT comprises or consists of an amino acid sequence is at least 90% identical SEQ ID NO: 12 or SEQ ID NO: 13.
[0170] In some embodiments, the ST6Gal1 comprises or consists of an amino acid sequence that is at least 90% identical SEQ ID NO: 19 or SEQ ID NO: 21.
[0171] In some embodiments, at least or about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α1,3 branch and the α1,6 branch.
[0172] In some embodiments, about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α1,3 branch and the α1,6 branch.
[0173] In some embodiments, about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α 1,3 arm and the a 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.
[0174] In some embodiments, about or at least 80% of the branched Fc glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α1,3 branch and the α1,6 branch.
[0175] In some embodiments, about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α 1,3 arm and the a 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.
[0176] In some embodiments, about or at least 85% of the of the branched Fc glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α1,3 branch and the α1,6 branch.
[0177] In some embodiments, about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α 1,3 arm and the a 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.
[0178] In some embodiments, about or at least 90% of the of the branched Fc glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α1,3 branch and the α1,6 branch.
[0179] In some embodiments, about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the α 1,3 arm and the a 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.
[0180] In some cases, the hsIgG can be purified, e.g., as described in PCT/US2021/033156.
[0181] An exemplary galactosylation and sialylation reaction is shown in the table below.
Buffers
[0182] Suitable buffers for use in the compositions and methods described herein include, but are not limited to those shown in Table 9. In some cases, the buffer is present in the composition or reaction mixture at 10-500 mM, e.g., 10-100 mM, e.g., about 50 mM. In some cases, the buffer has a pH of 5.5-8.5, e.g., 6.5-7.5, e.g., about 7.0.
TABLE-US-00009 TABLE 9 Buffers Buffer Buffer Name Structure MOPS 3-(N- morpholino) propanesulfonic acid
Evaluation of Glycans
[0183] As described above, immunoglobulins, e.g., IgG antibodies, can be sialylated in vitro by carrying out a galactosylation step followed by a sialylation step. Beta-1,4-galactosyltransferase 1 (B4GalT) is a Type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5′-diphosphosegalactose (UDP-Gal) to GlcNAc as a β-1,4 linkage. Alpha-2,6-sialyltransferase 1 (also referred to ST6 or ST6Gal) is a Type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-NANA) to Gal as an α-2,6 linkage. Schematically, the reactions proceed as shown in
[0184] Glycans of polypeptides can be evaluated using any methods known in the art. For example, sialylation of glycan compositions (e.g., level of branched glycans that are sialylated on an α1,3 branch and/or an α1,6 branch) can be characterized using methods described in WO2014/179601.
[0185] In some embodiments of the hsIgG compositions prepared by the methods described herein, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans on the Fc domain have a sialic acid on both the α 1,3 arm and the a 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage. In addition, in some embodiments, at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain have a sialic acid on both the α 1,3 arm and the a 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage. Overall, in some embodiments, at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans have a sialic acid on both the α 1,3 arm and the a 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.
[0186] Glycans of glycoproteins can be evaluated using any methods known in the art. For example, sialylation of glycan compositions (e.g., level of branched glycans that are sialylated on an α1,3 arm and/or an α1,6 arm) can be characterized using methods described in, e.g., Barb, Biochemistry 48:9705-9707 (2009); Anumula, J. Immunol. Methods 382:167-176 (2012); Gilar et al., Analytical Biochem. 417:80-88 (2011); Wuhrer et al., J. Chromatogr. B. 849:115-128 (2007). In some embodiments, in addition to evaluation of sialylation of glycans, one or more parameters described in Table 9 are evaluated.
[0187] In some instances, glycan structure and composition as described herein are analyzed, for example, by one or more, enzymatic, chromatographic, mass spectrometry (MS), chromatographic followed by MS, electrophoretic methods, electrophoretic methods followed by MS, nuclear magnetic resonance (NMR) methods, and combinations thereof. Exemplary enzymatic methods include contacting a glycoprotein preparation with one or more enzymes under conditions and for a time sufficient to release one or more glycan(s) (e.g., one or more exposed glycan(s)). In some instances, the one or more enzymes include(s) PNGase F. Exemplary chromatographic methods include, but are not limited to, Strong Anion Exchange chromatography using Pulsed Amperometric Detection (SAX-PAD), liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof. Exemplary mass spectrometry (MS) include, but are not limited to, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), and combinations thereof. Exemplary electrophoretic methods include, but are not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, and combinations thereof. Exemplary nuclear magnetic resonance (NMR) include, but are not limited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear Overhauser effect spectroscopy NMR (ROESY-NMR), nuclear Overhauser effect spectroscopy (NOESY-NMR), and combinations thereof.
[0188] In some instances, techniques described herein may be combined with one or more other technologies for the detection, analysis, and or isolation of glycans or glycoproteins. For example, in certain instances, glycans are analyzed in accordance with the present disclosure using one or more available methods (to give but a few examples, see Anumula, Anal. Biochem., 350(1):1, 2006; Klein et al., Anal. Biochem., 179:162, 1989; and/or Townsend, R.R. Carbohydrate Analysis” High Performance Liquid Chromatography and Capillary Electrophoresis., Ed. Z. El Rassi, pp 181-209, 1995; WO2008/128216; WO2008/128220; WO2008/128218; WO2008/130926; WO2008/128225; WO2008/130924; WO2008/128221; WO2008/128228; WO2008/128227; WO2008/128230; WO2008/128219; WO2008/128222; WO2010/071817; WO2010/071824; WO2010/085251; WO2011/069056; and WO2011/127322). For example, in some instances, glycans are characterized using one or more of chromatographic methods, electrophoretic methods, nuclear magnetic resonance methods, and combinations thereof. In some instances, methods for evaluating one or more target protein specific parameters, e.g., in a glycoprotein preparation, e.g., one or more of the parameters disclosed herein, can be performed by one or more of following methods.
[0189] In some instances, methods for evaluating one or more target protein specific parameters, e.g., in a glycoprotein preparation, e.g., one or more of the parameters disclosed herein, can be performed by one or more of following methods.
TABLE-US-00010 TABLE 10 Exemplary methods of evaluating parameters: Method(s) Relevant Literature Parameter C18 UPLC Mass Chen and Flynn, Anal. Glycan(s) Spec.* Biochem., 370: 147-161 (e.g., N-linked glycan, (2007) exposed N-linked glycan, Chen and Flynn, J. Am. glycan detection, glycan Soc. Mass Spectrom., identification, and 20: 1821-1833 (2009) characterization; site specific glycation; glycoform detection (e.g., parameters 1-7); percent glycosylation; and/or aglycosyl) Peptide LC-MS Dick et al., Biotechnol. C-terminal lysine (reducing/non- Bioeng., 100: 1132-1143 reducing) (2008) Yan et al., J. Chrom. A., 1164: 153-161 (2007) Chelius et al., Anal. Chem., 78: 2370-2376 (2006) Miller et al., J. Pharm. Sci., 100: 2543-2550 (2011) LC-MS (reducing/non- Dick et al., Biotechnol. C-terminal lysine reducing/alkylated) Bioeng., 100: 1132-1143 (2008) Goetze et al., Glycobiol., 21: 949-959 (2011) Weak cation exchange Dick et al., Biotechnol. C-terminal lysine (WCX) Bioeng., 100: 1132-1143 chromatography (2008) LC-MS (reducing/non- Dick et al., Biotechnol. N-terminal pyroglu reducing/alkylated) Bioeng., 100: 1132-1143 (2008) Goetze et al., Glycobiol., 21: 949-959 (2011) PeptideLC-MS Yan et al., J. Chrom. A., N-terminal pyroglu (reducing/non- 1164: 153-161 (2007) reducing) Chelius et al., Anal. Chem., 78: 2370-2376 (2006) Miller et al., J. Pharm. Sci., 100: 2543-2550 (2011) Peptide LC-MS Yan et al., J. Chrom. A., Methionine oxidation (reducing/non- 1164: 153-161 (2007); reducing) Xie et al., mAbs, 2: 379- 394 (2010) Peptide LC-MS Miller et al., J. Pharm. Site specific glycation (reducing/non- Sci., 100: 2543-2550 reducing) (2011) Peptide LC-MS Wang et al., Anal. Chem., Free cysteine (reducing/non- 83: 3133-3140 (2011); reducing) Chumsae et al., Anal. Chem., 81: 6449-6457 (2009) Bioanalyzer Forrer et al., Anal. Glycan (e.g., N-linked (reducing/non- Biochem., 334: 81-88 glycan, exposed N-linked reducing)* (2004) glycan) (including, for example, glycan detection, identification, and characterization; site specific glycation; glycoform detection; percent glycosylation; and/or aglycosyl) LC-MS (reducing/non- Dick et al., Biotechnol. Glycan (e.g., N-linked reducing/alkylated)* Bioeng., 100: 1132-1143 glycan, exposed N-linked *Methods include (2008) glycan) removal (e.g., Goetze et al., Glycobiol., (including, for example, enzymatic, chemical, 21: 949-959 (2011) glycan detection, and physical) of Xie et al., mAbs, 2: 379- identification, and glycans 394 (2010) characterization; site specific glycation; glycoform detection; percent glycosylation; and/or aglycosyl) Bioanalyzer Forrer et al., Anal. Light chain: Heavy chain (reducing/non- Biochem., 334: 81-88 reducing) (2004) Peptide LC-MS Yan et al., J. Chrom. A., Non-glycosylation-related (reducing/non- 1164: 153-161 (2007) peptide modifications reducing) Chelius et al., Anal. (including, for example, Chem., 78: 2370-2376 sequence analysis and (2006) identification of sequence Miller et al., J. Pharm. variants; oxidation; Sci., 100: 2543-2550 succinimide; aspartic acid; (2011) and/or site-specific aspartic acid) Weak cation exchange Dick et al., Biotechnol. Isoforms (including, for (WCX) Bioeng., 100: 1132-1143 example, charge variants chromatography (2008) (acidic variants and basic variants); and/or deamidated variants) Anion-exchange Ahn et al., J. Chrom. B, Sialylated glycan chromatography 878: 403-408 (2010) Anion-exchange Ahn et al., J. Chrom. B, Sulfated glycan chromatography 878: 403-408 (2010) 1,2-diamino-4,5- Hokke et al., FEBS Lett., Sialic acid methylenedioxybenzene 275: 9-14 (1990) (DMB) labeling method LC-MS Johnson et al., Anal. C-terminal amidation Biochem., 360: 75-83 (2007) LC-MS Johnson et al., Anal. N-terminal fragmentation Biochem., 360: 75-83 (2007) Circular dichroism Harn et al., Current Secondary structure spectroscopy Trends in Monoclonal (including, for example, Antibody Development alpha helix content and/or and Manufacturing, S. J. beta sheet content) Shire et al., eds, 229-246 (2010) Intrinsic and/or ANS Harn et al., Current Tertiary structure dye fluorescence Trends in Monoclonal (including, for example, Antibody Development extent of protein folding) and Manufacturing, S. J. Shire et al., eds, 229-246 (2010) Hydrogen-deuterium Houde et al., Anal. Tertiary structure and exchange-MS Chem., 81: 2644-2651 dynamics (including, for (2009) example, accessibility f amide protons to solvent water) Size-exclusion Carpenter et al., J. Pharm. Extent of aggregation chromatography Sci., 99: 2200-2208 (2010) Analytical Pekar and Sukumar, Anal. ultracentrifugation Biochem., 367: 225-237 (2007)
[0190] The literature recited above are hereby incorporated by reference in their entirety or, in the alternative, to the extent that they pertain to one or more of the methods for determining a parameter described herein.
Pharmaceutical Compositions and Administration
[0191] Hypersialylated IgG can be incorporated into a pharmaceutical composition. For example, the pharmaceutical composition can be formulated by suitably combining the hsIgG with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. The amount of active ingredient included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided.
[0192] The hsIgG can be formulated for intravenous administration.
[0193] The sterile composition for injection can be formulated in accordance with conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80™, HCO-50 and the like.
[0194] Nonlimiting examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampule.
[0195] A suitable means of administration can be selected based on the age and condition of the patient. A suitable dose of hsIgG prepared by the methods described herein can be about the same or less than (e.g., 20%, 35%, 40%, 50%, 60%, 70% or 80% less) the suitable or approved dose of commercially available IVIg preparations. The dose and method of administration varies depending on the weight, age, condition, and the like of the patient, and can be suitably selected as needed by those skilled in the art.
EXAMPLES
[0196] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1: Hypersialylation of IgG from Cohn fraction
[0197] IgG in which more than 50% of the overall branched glycans are disialylated can be prepared as follows.
[0198] Briefly, a mixture of IgG antibodies (e.g., from Cohn II/III or Cohn I/II/III) is exposed to an enzymatic reaction using β1,4 galactosyltransferase 1 (B4GalT) and α2,6-sialyltransferase (ST6Gal1) enzymes. The B4GalT does not need to be removed from the reaction before addition of ST6Gal1 and no partial or complete purification of the product is needed between the enzymatic reactions. Therefore the reaction may be sequential or concurrent.
[0199] The galactosyltransferase enzyme selectively adds galactose residues to pre-existing asparagine-linked glycans. The resulting galactosylated glycans serve as substrates for the sialic acid transferase enzyme which selectively adds sialic acid residues to cap the asparagine-linked glycan structures attached to the protein. Thus, for disialylation the overall sialylation reaction employed two sugar nucleotides (uridine 5′-diphosphogalactose (UDP-Gal) and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA)). The latter can be replenished periodically to increase disialylated product relative to monosialylated product. The reaction includes the co-factor manganese (II) chloride.
[0200] A representative example of the IgG-Fc glycan profile for such a reaction starting with IVIg and the reaction product is shown in
[0201] The glycan data is shown per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg the sum of all the nonsialylated glycans is more than 80% and the sum of all sialylated glycans is <20% (top graph). For the reaction product, the sum for all nonsialylated glycans is <20% and the sum for all sialylated glycans is more than 80% (bottom graph). Nomenclature for different glycans listed in the glycoprofile use the Oxford notation for N linked glycans.
[0202] The starting material for the following examples was a lyophilized solid of Cohn II,III sourced from Sigma Aldrich (G2388; 78% protein by weight, primarily γ-globulin and β-globulin). A portion of the material was not completely soluble in the reaction buffer of choice (BIS-TRIS) nor at the concentration of choice (>100 mg/mL). Insoluble materials were generally gel-like rather than solid precipitates, possibly due to the presence of lipoproteins.
[0203] Five different preparations of the Cohn II/III material were generated (Preparations A-E) and used to prepare hsIgG. Concentrations were estimated by A280 using the extinction coefficient for IgG (1.37). It should be noted that the IgG concentration was assessed by absorption at A280, but this signal likely reflects the presence of non-IgG protein as well as non-protein material. Thus, the actual IgG concentration may be lower than indicated.
[0204] Preparation A: Cohn fraction II,III dissolved in 50 mM pH 6.9 BIS-TRIS buffer to a measured (A280) concentration of 109 mg/mL. Undissolved material was allowed to settle.
[0205] Preparation B: Cohn fraction II,III dissolved in 50 mM pH 6.9 BIS-TRIS buffer to a measured (A280) concentration of 97 mg/mL. Undissolved material was allowed to settle.
[0206] Preparation C: Cohn fraction II,III (640 mg) dissolved in 30 mL 50 mM pH 6.9 BIS-TRIS buffer (21.3 mg/ml), sterile filtered (0.2 μm) to remove undissolved material and then then buffer exchanged into 50 mM pH 6.9 BIS-TRIS buffer using a G25 desalting column. The resulting material was allowed to stand, the liquid was decanted away from the settled solids, and the liquid was concentrated to 50.6 mg/mL using 10 kDa MWCO Vivaspin Turbo 15 devices.
[0207] Preparation D: Cohn fraction II,III dissolved in 50 mM pH 6.9 BIS-TRIS buffer, buffer exchanged into 50 mM pH 6.9 BIS-TRIS buffer using a 10 kDa G2 dialysis cassette. Undissolved material was allowed to settle and the supernatant was decanted off. Concentration in the supernatant was 37 mg/mL.
[0208] Preparation E: Cohn fraction II,III material (622 mg) dissolved in 9 mL 50 mM pH 6.9 BIS-TRIS buffer (691 mg/ml) and buffer exchanged into 50 mM pH 6.9 BIS-TRIS buffer using a 10 kDa G2 dialysis cassette over 54 h with one change in dialysis buffer. The material was centrifuged to settle undissolved material and the supernatant was decanted off. Concentration in supernatant was 38 mg/mL.
[0209] Galactosylation using human B4GalT enzyme, UDP-Gal sugar nucleotide donor, and manganese(II) chloride (MnCl.sub.2) in BIS-TRIS pH 6.9 buffer was done first for 18-22h at 37° C. This was followed by sialylation with addition of a deletion mutant of human ST6 sialyltransferase that includes amino acids 109-406 of SEQ ID NO: 14 (SEQ ID NO: 20) and half of the total amount of the CMP-NANA sugar donor. The second half of the CMP-NANA was added at −9 h post sialylation start. Total sialylation time was 30-32 h. The reaction scale was 20 mg protein. The amounts of reagents were varied across the reactions.
[0210] The extent of Fc galactosylation and sialylation was determined by glycopeptide analysis as follows. A 50 μg sample was denatured, reduced, and alkylated. The resulting material was subjected to trypsin digestion and analysis by LCMS. The following glycans on the Fc glycopeptide were quantified.
TABLE-US-00011 Glycoform G0F G1F G2F A1F A2F G1F + NeuAc G0F + bisecting_GlcNAc G1F + bisecting_GlcNAc G2F + bisecting_GlcNAc A1F + bisecting_GlcNAc A2F + bisecting_GlcNAc G0 G1 G2 A1 A2 G1 + NeuAc
[0211] Total sialylation was defined as the sum of the LCMS peak areas for the glycans which were fully galactosylated and sialylated (A2, A2F, and A2F+bisecting GlcNAc) (
[0212] A first set of reactions was run on Cohn II/III fraction material directly dissolved in BIS-TRIS buffer using the conditions in Table 11.
TABLE-US-00012 TABLE 11 Conditions for first set of reactions B4GalT ST6 UDP-Gal CMP-NANA (mU/mg (mU/mg (nmol/mg (nmol/mg MnCl2 Reaction Material protein) protein) protein) protein) (mM) JS1155-2 B (37 mg/mL) 9.3 19 37.5 220 2.4 JS1155-1 B (97 mg/mL) 9.3 19 37.5 220 5.3 JS1160-1 B (97 mg/mL) 14.8 31.1 38 220 7.4 JS1160-8 B (97 mg/mL) 15.2 30.9 95 445 7.4 JS1160-5 B (97 mg/mL) 22.5 46.9 37.5 220 7.4 JS1160-11 B (97 mg/mL) 22.5 46.9 95 445 7.4 JS1149-3 A (109 mg/mL) 93 201 380 2200 58 JS1149-4 B (97 mg/mL) 93 201 380 2200 9.3 JS1149-5 B (37 mg/mL) 93 201 380 2200 9.2
[0213] A second set of reactions was run on Cohn II/III fraction material buffer exchanged into BIS-TRIS buffer using the conditions in Table 12.
TABLE-US-00013 TABLE 12 Conditions for second set of reactions B4GalT ST6 UDP-Gal CMP-NANA (mU/mg (mU/mg (nmol/mg (nmol/ MnCl2 Reaction Material protein) protein) protein) protein) (mM) JS1155-3 D (37 mg/mL) 9.3 19 37.5 220 2.3 JS1160-2 C (37 mg/mL) 14.8 31.1 38 220 7.4 JS1160-3 E (38 mg/mL) 14.8 31.1 38 220 7.5 JS1160-4 D (37 mg/mL) 14.8 31.1 38 220 7.4 JS1160-9 C (37 mg/mL) 15.2 30.9 95 445 7.4 JS1160-10 E (38 mg/mL) 15.2 30.9 95 445 7.5 JS1160-6 C (37 mg/mL) 22.5 46.9 37.5 220 7.4 JS1160-7 E (38 mg/mL) 22.5 46.9 37.5 220 7.5 JS1160-13 E (38 mg/mL) 22.5 46.9 95 445 7.5 JS1160-12 C (37 mg/mL) 22.5 93.8 95 700 7.4 JS1149-1 D (37 mg/mL) 37 81 152 880 15.7 JS1149-6 D (37 mg/mL) 93 201 380 2200 9.0 JS1149-2 D (37 mg/mL) 93 201 380 2200 21
[0214] The starting material had essentially no species defined as fully sialylated (A2F, A2, A2F+bisecting GlcNAc). However, under several of the reaction conditions, greater than 90% full sialylation (sialylated on both arms of branched glycan) could be achieved. Reactions run on material directly dissolved in BIS-TRIS achieved significantly lower levels of fully sialylated IgG than when using buffer exchanged material even when the reactions using directly dissolved material were conducted at a higher protein concentration. This might be because the sodium chloride in the starting material has a negative impact on the sialylation reaction. Interestingly, the NaCl did not seem to significantly impact the sialylation of non-IgG proteins.
Example 2: Cohn precipitate is about 65% IgG
[0215] Cohn fraction II/III sourced from Sigma-Aldrich was dialyzed into BIS-TRIS across a 10KDa MWCO filter and/or was additionally buffer exchanged into BIS-TRIS using a desalting column (G25).
[0216] LC-MS was used to quantify the relative abundance of proteins in the starting material, in Privigen®, a commercially available IVIG preparation, and in both the soluble fraction and the insoluble fractions of Cohn II/III after dialysis based on molecular weight corrected normalized peptide spectral matches (nPSMs).
[0217]
[0218] Based on normalized peptide spectral matches, Cohn II/III was about 65% IgG and 35% non-IgG, which is consistent with reported values. Privigen® was greater than 99% IgG. Thus, it can be appreciated that the material used in Example 1 to prepare hsIgG included a significant portion of protein that was other than IgG.
[0219]
Example 3: Delipidation
[0220] Processing of Cohn II/III paste using no precipitation step but with delipidation prior to buffer exchange into BIS-TRIS was carried out as follows:
[0221] Cohn II/III paste was resuspended in water at 4° C. and the pH adjusted to 4.81 using 0.5 M acetic acid. Stirring was continued overnight at 4° C. The pH was adjusted to 7.00 using 2.4 M BIS-TRIS base buffer. After centrifugation the supernatant was filtered using 0.45 urn and then 0.22 urn filters and placed at 4° C. Lipid removal agent (LRA, Advanced Minerals Corp) and Celpure® P-100 (Advanced Minerals Corp) were added, the suspension stirred 1.5 h at ambient temperature, stirred 1.5 h at 4° C., and then 0.22 urn filtered. The conductivity was adjusted to 9.2 mS/cm using 5 M NaCl solution. Fumed silica 350-420 m.sup.2/g (Thermo Fisher) and Celpure® P-100 were added, the solution stirred 1 h at ambient temperature, 0.22 urn filtered, and then 0.1 urn filtered.
[0222] The solution was concentrated and buffer exchange by TFF (Pellicon 3 membrane, EMD Millipore) using six diavolumes 50 mM BIS-TRIS 7.20 buffer. After elution from the TFF system the material was further concentrated to 100 mg/mL (using an extinction coefficient of 1.37) in Vivaspin Turbo 15 devices (Sartorius). Designate material “F”.
[0223]
[0224] Cohn II/III material F obtained above was subjected to the galactosylation reaction.
TABLE-US-00014 Fully Fully Fully mU nmol galactosylated galactosylated galactosylated B4GalT/mg UDP-Gal/mg MnCl.sub.2 glycans IgG1 glycans IgG2 glycans IVIg IVIg (mM) (%) (%) IgG3/4 (%) JS1221-1 11 48 5.0 100 99 100
[0225] Reaction JS1212-1 material was then sialylated.
TABLE-US-00015 ST6 nmol CMP- Disialylated Disialylated Disialylated activity NANA/mg glycans glycans glycans (mU/mg) IVIg total IgG1 (%) IgG2 (%) IgG3/4 (%) JS1221-3 18 150 82 63 100 JS1221-4 36 300 94 83 100 JS1221-5 54 450 97 90 100
[0226] Galactosylation (JS1212-1 stoichiometry) and sialylation (JS1212-4 stoichiometry) reactions were performed in which the two enzyme reactions were run concurrently or sequentially, as described below.
[0227] Sequential reaction:
[0228] Processed Cohn II/III (8.0 mL, 800 mg) in BIS-TRIS was mixed with 38 umol UDP-Gal, 9.0 U B4GalT, 5.0 mM manganese(II) chloride, and incubated at 37° C. 24 h. 120 umol CMP-NANA and 28.8 U ST6 were added and incubation continued. At 9 h an additional 120 umol CMP-NANA was added. Reaction was stopped at 31 h.
[0229] In the concurrent reaction all the reagents amount given above were mixed at T=0. At 9 h an additional 120 umol CMP-NANA was added. Reaction was stopped at 31 h total reaction time.
[0230] Both gave the same high levels of galactosylation and sialylation. Reaction on this material proceeded with less enzyme than Example 1. This is due in part to the high protein concentration achieved with low salt content.
TABLE-US-00016 Disialylated Disialylated Disialylated Reaction glycans glycans glycans Type IgG1 (%) IgG2 (%) IgG3/4 (%) JS1221-15 Sequential 95 85 100 JS1221-16 Concurrent 94 82 100
TABLE-US-00017 SEQUENCES (IgG1) SEQ ID NO: 1 EEQYNSTYR (IgG2/3) SEQ ID NO: 2 EEQFNSTFR (IgG3/4) SEQ ID NO: 3 EEQYNSTFR (IgG3/4) SEQ ID NO: 4 EEQFNSTYR (NP_001488.2 B4GALT1 [organism = Homo sapiens] [GeneID = 2683] [isoform = 1]) SEQ ID NO: 5 TPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNL TSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKONPNVKMGGRYAPRD CVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLORQQLDYGIYVINQAGDTIFNRAKLL NVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQY FGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIR HSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS (NP_001365424.1 B4GALT1 [organism = Homo sapiens] [GeneID = 2683] [isoform = 2]) SEQ ID NO: 6 GQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSL PACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPF RNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDY TCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFL TINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQR FDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS (NP_001365425.1 B4GALT1 [organism = Homo sapiens] [GeneID = 2683] [isoform = 3]) SEQ ID NO: 7 MRLREPLLSGSAAMPGASLORACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVS TPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNL TSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRD CVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLL NVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFRLVFRGM SISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQR YPLYTQITVDIGTPS (NP_001365426.1 B4GALT1 [GeneID = 2683] [isoform = 4]) [organism = Homo sapiens] SEQ ID NO: 8 MRLREPLLSGSAAMPGASLORACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVS TPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNL TSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRD CVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQYEKIRRLLW SEQ ID NO: 9 MRLREPLLSGSAAMPGASLORACR SEQ ID NO: 10 LLVAVCALHLGVTLVYYLAG SEQ ID NO: 11 RDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSS PVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQN PNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVIN QAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVA MDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSIS RPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPL YTQITVDIGTPS (B4GalT) SEQ ID NO: 12 GPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGG RYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIF NRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFS LPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVG RCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVD IGTPS (B4GalT) SEQ ID NO: 13 gssplldmGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQ NPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVI NQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISV AMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSI SRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYP LYTQITVDIGTPSprdhhhhhhh (NP_001340845.1 (NP_003023.1, NP_775323.1) ST6GAL1 [organism = Homo sapiens] [GeneID = 6480] [isoform = a]) SEQ ID NO: 14 MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMG SDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNLIPRLQKIWK NYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPK ESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTT IRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYR KLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYE FLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLP GFRTIHC (NP_775324.1 ST6GAL1 [organism = Homo sapiens] [GeneID = 6480] [isoform = b]) SEQ ID NO: 15 MNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLH PNQPFYILKPOMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLP SKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFR TIHC SEQ ID NO: 16 MIHTNLKKK SEQ ID NO: 17 FSCCVLVFLLFAVICVW SEQ ID NO: 18 KEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRG LAKAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEAL RCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQ LGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGIL IVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEI SPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGA YHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC (ST6Gal1) SEQ ID NO: 19 AKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCH LRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGR EIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVW DPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPE EIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHP LLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC (ST6Gal1) SEQ ID NO: 20 LOKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEW EGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQD VGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFN NYKTYRKLHPNQPFYILKPOMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCD QVDIYE (ST6Gal1) SEQ ID NO: 21 gssplldmlehhhhhhhhmAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKV SYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPW GRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVT TEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYI LKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDV CYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC
OTHER EMBODIMENTS
[0231] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.