Affinity resins for the purification of hepatitis b surface antigen

Abstract

Provided are affinity ligands that bind to Hepatitis B surface antigen (HBsAg), affinity resins comprising the HBsAg-binding ligands, and related compositions and methods for the isolation and / or purification of HBsAg.

Description

Docket No. 1580.00233WOAFFINITY RESINS FOR THE PURIFICATION OF HEPATITIS B SURFACE ANTIGENREFERENCE TO A SEQUENCE LISTING

[0001] The application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML sequence listing, created on October 27, 2025, is named 1580.00233 WO_Sequence_Listing and is 71,347 bytes in size.BACKGROUND

[0002] Protein-based vaccines have been used for the prophylaxis of bacterial and viral diseases in the United States for well over twenty-five years, the first vaccine including a viral protein having been approved in 1986. While protein-based vaccines are generally less immunogenic compared to vaccines comprised of either live-attenuated or inactivated whole organisms, their immunogenicity may be enhanced, for example, by mutation and / or by the use of adjuvants or other technologies including virus-like particles (VLPs) and protein micelles.

[0003] The polypeptide or protein component of a protein-based vaccine is typically manufactured using a recombinant eukaryotic expression systems, such as yeast, insect, or mammalian cells. It is important to obtain the recombinant polypeptide or protein at high purity for therapeutic use. Affinity purification allows for the efficient isolation of highly pure target molecules. However, the use of this technique is limited by the availability of suitable affinity agents which selectively bind to the target molecule. In the absence of a suitable affinity agent, purification typically involves inefficient, labor intensive, and expensive processes, such as a multi-column process. The present invention addresses the need for affinity ligands against molecules useful in protein-based vaccines, and related compositions and methods for purifying such molecules by affinity chromatography.BRIEF SUMMARY

[0004] Provided are affinity ligands that bind to Hepatitis B surface antigen (HBsAg) with high affinity and selectivity. Also provided are related compositions including affinity resins comprising the ligands described herein and methods for isolation of HBsAg by affinity chromatography utilizing the resins.

[0005] Accordingly, in one aspect, provided are affinity ligands including an amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 90%, or at least 93%,Docket No. 1580.00233WO or at least 96%, or at least 98% amino acid sequence identity thereto: VDAX17FDX18EX19X20X21X22AX23X24EIX25X26LPN NX27X28X29X30X31AFIX32SLX33DX34PS QSANLLAEAX35X36LNDAQAPK (SEQ ID NO: 58) where X17 is K or R, Xis is K or R, X19 is F, R or Y, X20 is I, W or Y, X21 is E H, L, Q or W, X22 is G or deleted, X23 is D, T, or V, X24 is F, H, Q, or W, X25 is A, F, Q or S, X26 is A, H, I , L, W or Q, X27 is A, L, V or W, X28 is H, I, V, W, or Y, X29 is E or Q, X30 is K, Q, R or V, X31 is I, S or W, X32 is A, F, I, N or W, X33 is H, L or W, X34 is D or N, X35 is K or R, X36 is K or R. The affinity ligand may also include a multimer of SEQ ID NO: 58. The affinity ligand may also include an amino acid sequence of any one of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71, or an amino acid sequence having at least 90%, at least 93%, at least 96%, or at least 98% amino acid sequence identity thereto, or a multimer of any of the foregoing. The affinity ligand may also include where the ligand includes or consists of an amino acid sequence of any one of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or an amino acid sequence having at least 90%, at least 93%, at least 96%, or at least 98% amino acid sequence identity thereto, or a multimer of any of the foregoing. The affinity ligand may also include where the multimer is a dimer, trimer, quatramer, pentamer, or hexamer.

[0006] In another aspect, provided is an affinity ligand having a structure represented by Li- D1-L2-DS-L -DT-L4 (Formula I) where Li, L2, L , and L4 are each independently absent or a linker, Di is a HBsAg binding domain, Ds is an optional structural domain, and DT is an optional C-terminal tag domain.

[0007] The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence having at least 86% identity to SEQ ID NO: 57: DLGKKLLE A ARAGQDDE VRILM ANGAD VNAKDX 1 X2GX3 TPLHL A AX4X5 GHLEI VE VLL KHGADVNAX6DX7X8GX9TPLHLAAX10X11GHLEIVEVLLKX12GADVNAX13DX14WGWTP LHLAAXisWGHLElVEVLLKYGADVNAXieDKFGKTAFDlSIDNGNEDLAElL, where Xi is S, or V, X2 is F or Y, X3 is A or G, X4 is W or Y, X5 is G or W, XG is I or Y, X7 is H or L, Xs is G or I, X9 is E or Y, X10 is P or R, X11 is G or T, X12 is H or Y, X13 is L or V, X14 is F or W, X15 is G or L, Xi6 is F or T, optionally wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.Docket No. 1580.00233WO

[0008] The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 86% identity to SEQ ID NO: 58: VDAX17FDX18EX19X20X21X22AX23X24EIX25X26LPNLNX27X28X29X30X31AFIX32SLX33DX34PS QSANLLAEAX35X36LNDAQAPK where X17 is K or R, Xis is K or R, X19 is F, R or Y, X20 is I, W or Y, X21 is E H, L, Q or W, X22 is G or deleted, X23 is D, T, or V, X24is F, H, Q, or W, X25 is A, F, Q or S, X26 is A, H, I , L, W or Q, X27 is A, L, V or W, X28 is H, I, V, W, or Y, X29 is E or Q, X30 is K, Q, R, or V, X31 is I, S or W, X32 is A, F, I, N, or W, X33 is H, L or W, X34 is D or N, X35 is K or R, X36 is K or R, optionally wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0009] The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having at least 86% identity to SEQ ID NO: 59: VDAX37X38X39X40X41X42EX43ARX44RIESLPNLTEEQRRAFIESLRDDPX45X46X47X4 X49LL WEAX50X51LNX52X53QAPK where X37 is K or R, X3s is H or L, X39 is D or H, X40 is E, H, V or Y, X41 is G, H, R, S, or W, X42 is H or L, X43 is W or Y, X44 is I, N or V, X45 is H or S, X46is H or Q, X47 is A or V, X4 is H or W, X49 is F, K or V, X50 is F or W, X51 is H or W, X52 is A, I or W, X53 is A or H, optionally wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0010] The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 57, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM. The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 57, and the ligand includes an amino acid sequence as defined by any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0011] The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 58, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM. The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 58, and the ligand includes an amino acid sequence as defined by any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQDocket No. 1580.00233WOID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0012] The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 59, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM. The affinity ligand may also include where Di is defined by the amino acid sequence of SEQ ID NO: 59, and the ligand includes an amino acid sequence as defined by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 50, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0013] The affinity ligand may also include where Di comprises an amino acid sequence as defined by any one of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71, or an amino acid sequence having at least 90%, at least 93%, at least 96%, or at least 98% amino acid sequence identity thereto, or a multimer of any of the foregoing. The affinity ligand may also include where the ligand includes or consists of an amino acid sequence as defined by any one of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity to any of the foregoing. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.Docket No. 1580.00233WG

[0014] In accordance with any of the foregoing embodiments where Di is defined by an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity to a reference sequence, e.g., SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 59, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM, any difference between the amino acid sequence of the variant Di and the reference sequence may consist of one or more conservative amino acid substitutions.

[0015] The affinity ligand may also include where Li is a polypeptide linker of from 1-15 amino acids in length. The affinity ligand may also include where at least one of L3 or L4 is present and includes one or more of a spacer molecule and a reactive thiol- or nitrogencontaining amino acid. The affinity ligand may also include where L3 comprises one or more of a spacer molecule and a reactive thiol- or nitrogen-containing amino acid, and DT and L4 is absent. The affinity ligand may also include where where L3 is absent or a spacer molecule, DT is present and L4 comprises one or more of a spacer molecule and a reactive thiol- or nitrogencontaining amino acid. The affinity ligand may also include where DT is a bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin-carboxy carrier protein (BCCP), polyhistidine (His-tag), polyaspartate (Asp-tag), polycysteine (Cys-tag), polyphenylalanine (Phe-tag), glutathione 5-transferase (GST), maltose binding protein (MBP), calmodulin binding peptide (CBP), intein-chitin binding domain (intein-CBD), a streptavidin / biotin-based tag, a tandem affinity purification (TAP) tag or TAP variant, a FLAG tag, human influenza hemagglutinin (HA), c-myc epitope, T7, or Glu-Glu, HSV epitope, or a green fluorescent protein (GFP) or GFP derivative. The affinity ligand may also include where the spacer molecule is selected from an amino acid, a polypeptide of from 1-50 amino acids, or a nonpolypeptide molecule. The affinity ligand may also include where the reactive thiol- or nitrogen-containing amino acid is cysteine or lysine.

[0016] In accordance with any of the foregoing aspects, the affinity ligand may be covalently attached to a solid support which may be in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith.

[0017] Also provided is an affinity chromatography resin comprising a solid support and a plurality of the affinity ligands described herein covalently attached to the solid support. The affinity chromatography resin may also include where the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith. The affinity chromatography resin may also include where the solidDocket No. 1580.00233WG support is in the form of discrete polymeric particles of a polysaccharide. The affinity chromatography resin may also include where the polysaccharide is selected from agar, agarose, dextran, starch, cellulose, pullulan, and stabilized variants or derivatives thereof.

[0018] Also provided is a method of isolating HBsAg from a mixture including steps of (i) contacting a liquid comprising HBsAg with an affinity chromatography resin functionalized with an affinity ligand as described herein; (ii) washing the resin with a wash buffer; and (iii) eluting the HBsAg from the resin with an elution buffer. The method may also include where the elution buffer comprises sodium thiocyanate, optionally from about 0.5 M to about 1.5 M sodium thiocyanate. The method may also include a cleaning in place step following elution of the HBsAg from the affinity resin. The cleaning in place step may comprise contacting the affinity resin with an alkaline solution comprising from 0.05-0.2 M NaOH for a period of time, optionally where the alkaline solution is 0.1 M NaOH and the period of time is at least 5 hours, at least 10 hours, or at least 20 hours. The method may also include where the liquid is a process intermediate of a yeast cell lysate selected from a polyethelyene glycol supernatant, a desalting / buffer exchange pool, and an ion exchange chromatography pool.

[0019] Also provided are polynucleotides encoding the affinity ligands as described herein. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows the breakthrough curve for an exemplary resin functionalized with an affinity ligand having an amino acid sequence as defined by SEQ ID NO: 2, in connection with a dynamic binding capacity assay.

[0021] FIG. 2 shows breakthrough curves for an exemplary resin functionalized with an affinity ligand having an amino acid sequence as defined by SEQ ID NO: 2 before and after successive 5-hour holds in 0.1 M NaOH.

[0022] FIG. 3 is an image of an exemplary electrophoresis gel of flowthrough fractions of a column packed with an exemplary resin functionalized with an affinity ligand as described herein. Identity of samples in each lane is given in Table 8.

[0023] FIG. 4 is an image of the electrophoresis gel used in an experiment to test the ability of representative resins prepared with affinity agents as described herein to purify HBsAg from certain yeast lysate intermediates. Identity of samples in each lane is given in Table 10.Docket No. 1580.00233WODETAILED DESCRIPTION

[0024] Provided are affinity ligands, which may also be referred to herein as “affinity agents” or in some instances simply as “ligands”, and related compositions, including affinity resins, and methods, including methods of isolating a target molecule utilizing the affinity ligands and resins described herein. The term "affinity ligand" refers to a molecule that binds with high affinity to a target molecule such that it is generally useful in methods of detection and / or purification of the target molecule. Exemplary target molecules may include antibodies, viral proteins including capsid proteins and surface antigens, nucleic acids, and other proteins. The affinity ligands described herein may be characterized as polypeptides or proteins. The terms polypeptide and protein are used interchangeably herein. In the context of the present invention, the target molecule is Hepatitis B surface antigen (HBsAg). In aspects, HBsAg may be displayed on a virus-like particle.

[0025] Provided are affinity ligands of Formula I:L1-D1-L2-DS-L3-DT-L4 (Formula I) whereinLi, L2, L3, and L4 are each independently absent or a linker;Di is a HBsAg binding domain;Ds is an optional structural domain; andDT is an optional C-terminal tag domain.

[0026] In aspects of Formula I, Li, L2, L3, or L4, if present, is a linker. In aspects, the linker is a peptide linker of from 1-15 amino acids in length, optionally from 1-10 amino acids in length or from 1-6 amino acids in length. In aspects, any one or more of Li, L2, L3, and / or L4, if present, is independently a poly(glycine) peptide, poly(alanine) peptide, poly((glycine)(alanine)) peptide, a poly((glycine)(serine)) peptide of from 1-5 amino acid residues in length. In other aspects, the linker may be as described in more detail infra.

[0027] In aspects of Formula I, L4is a C-terminal reactive thiol- or nitrogen-containing amino acid. In aspects, L4is a lysine or cysteine residue.Docket No. 1580.00233WO

[0028] In aspects of Formula I, Ds is a structural domain comprising or consisting of the amino acid sequence defined by SEQ ID NO: 60.

[0029] In aspects of Formula I, the tag domain DT may include one or more C-terminal tags, as described in more detail infra. In aspects, DT comprises at least one affinity tag to facilitate isolation during manufacture and / or to facilitate detection of the affinity ligand or its target molecule.

[0030] In aspects of Formula I, Di is defined by the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence having at least 86% identity to SEQ ID NO: 57: DLGKKLLEAARAGQDDEVRILMANGADVNAKDX1X2GX3TPLHLAAX4X5GHLEIVEVLL KHGADVNAX6DX7X8GX9TPLHLAAX10X11GHLEIVEVLLKX12GADVNAX13DX14WGWTP LHLAAXisWGHLEIVEVLLKYGADVNAXieDKFGKTAFDISIDNGNEDLAEIL, where Xi is S, or V, X2is F or Y, X3 is A or G, X4is W or Y, X5 is G or W, X6is I or Y, X7 is H or L, X8is G or I, X9 is E or Y, X10 is P or R, X11 is G or T, X12 is H or Y, X13 is L or V, X14 is F or W, X15 is G or L, Xi6 is F or T, optionally wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0031] In aspects, provided are affinity ligands of Formula I wherein Di is defined by the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 57, optionally wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0032] In aspects, provided is an affinity ligand of Formula I wherein Di is defined by the amino acid sequence of SEQ ID NO: 57, and the ligand comprises an amino acid sequence as defined by any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto, optionally wherein any difference between the amino acid sequence of Di and a reference sequence consists of one or more conservative amino acid substitutions, and further optionally wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0033] In aspects of Formula 1, Di is defined by the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 86% identity to SEQ ID NO: 58: VDAX17FDX18EX19X20X21X22AX23X24EIX25X26LPNLNX27X28X29X30X31AFIX32SLX33DX34PS QSANLLAEAX35X36LNDAQAPK where X17 is K or R, Xis is K or R, X19 is F, R or Y, X20 is I, W or Y, X21 is E H, L, Q or W, X22 is G or deleted, X23 is D, T, or V, X24 is F, H, Q, or W,Docket No. 1580.00233WOX25 is A, F, Q or S, X26 is A, H, I , L, W or Q, X27 is A, L, V or W, X2s is H, I, V, W, or Y, X29 is E or Q, X30 is K, Q, R, or V, X31 is I, S or W, X32 is A, F, I, N, or W, X33 is H, L or W, X34 is D or N, X35 is K or R, X36 is K or R.

[0034] In aspects, provided are affinity ligands of Formula I wherein Di is defined by the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 58.

[0035] In aspects, provided is an affinity ligand of Formula I wherein Di is defined by the amino acid sequence of SEQ ID NO: 58, and the ligand comprises an amino acid sequence as defined by any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto, optionally wherein any difference between the amino acid sequence of Di and a reference sequence consists of one or more conservative amino acid substitutions, and further optionally wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0036] In aspects, provided is an affinity ligand of Formula I wherein Di is defined by the amino acid sequence of SEQ ID NO: 58, and the ligand comprises or consists of an amino acid sequence as defined by any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto.

[0037] In aspects, provided is an affinity ligand of Formula I wherein Di is defined by the amino acid sequence of SEQ ID NO: 58, and the ligand comprises an amino acid sequence as defined by any one of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto.

[0038] In aspects of Formula I, Di is defined by the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having at least 86% identity to SEQ ID NO: 59: VDAX37X38X39X40X41X42EX43ARX44RIESLPNLTEEQRRAFIESLRDDPX45X46X47X48X49LLDocket No. 1580.00233WOWEAX50X51LNX52X53QAPK where X37 is K or R, X38is H or L, X39 is D or H, X4o is E, H, V or Y, X41 is G, H, R, S, or W, X42 is H or L, X43 is W or Y, X44is I, N or V, X45 is H or S, X46is H or Q, X47 is A or V, X4s is H or W, X49 is F, K or V, X50 is F or W, X51 is H or W, X52 is A, I or W, X53 is A or H.

[0039] In aspects, provided are affinity ligands of Formula I, wherein Di is defined by the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 59.

[0040] In aspects, provided is an affinity ligand of Formula I, wherein Di is defined by the amino acid sequence of SEQ ID NO: 59, and the ligand comprises an amino acid sequence as defined by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ IDNO: 35, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 50, or an amino acid sequence having at least86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto, optionally wherein any difference between the amino acid sequence of Di and a reference sequence consists of one or more conservative amino acid substitutions, and further optionally wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0041] In accordance with any of the foregoing embodiments where Di is a variant defined by an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity to a reference sequence, e.g., SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, etc., wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM. any difference between the amino acid sequence of the variant Di and the reference sequence may consist of one or more conservative amino acid substitutions.

[0042] The affinity ligands described here advantageously are able to withstand being subjected to conditions of high pH, including a pH of 13 or higher, for prolonged periods of time, without a significant decrease in their binding capacity for HBsAg. The alkali stability of the proteins described here is particularly advantageous for chromatographic processes incorporating a cleaning in place procedure utilizing highly alkaline solutions, including for example 0.1 M NaOH solutions. For example, a resin functionalized with an affinity ligand as described herein may retain at least 80%, preferably at least 90%, of its binding capacityDocket No. 1580.00233WO following exposure to 0.1 M NaOH for at least 10 hours, or at least 20 hours, or at least 30 hours, where binding capacity is measured as a percentage of the dynamic binding capacity of the functionalized resin.

[0043] The affinity ligands described here also demonstrate high “dynamic binding capacity” or DBC, when coupled to a resin. DBC is defined as the mass loaded per unit of resin when there is 10% breakthrough in the column effluent. Typical units for DBC are given in milligrams per milliliter (mg / ml), e.g., milligrams target molecule per milliliter resin or equivalently, grams per liter (g / L). Column DBC must be maintained at a high level during the manufacturing process in order to maintain overall process efficiency in terms of cost and capacity. Accordingly, in preferred aspects a resin functionalized with an affinity ligand as described herein has a 10% breakthrough dynamic binding capacity for the target molecule of at least 3-6 g / L or higher, for example at a residence time of between 2-8 minutes. In aspects, a resin functionalized with an affinity ligand as described herein has a dynamic binding capacity (DBC) in the range of at least about 3 g / L, or about 5 g / L, or about 6 g / L, or about 7 g / L or higher for the target molecule. In aspects, a resin functionalized with affinity ligands as described herein has a DBC of from about 3-7 mg / ml, or from about 3-6 mg / ml for the target molecule.

[0044] Accordingly, provided are affinity ligands having chemical stability under alkali conditions such as commonly used for cleaning in place (CIP) procedures. Also provided are chromatography resins functionalized with the affinity ligands described here, which advantageously have a high dynamic binding capacity (DBC) for HBsAg proteins, and methods for their use in isolating HBsAg proteins, for example from host cell culture fluid or host cell lysate.Affinity ligands

[0045] Affinity ligands as described herein may be functionalized with one or more N- or C- terminal modifications to facilitate purification during manufacture and / or to facilitate its covalent attachment to a solid support to form an affinity chromatography resin as described herein. In this context, the term “functionalized” refers to a modification by covalent attachment. The one or more C-terminal modifications may include, for example, a linker molecule, a C-terminal reactive thiol- or nitrogen-containing amino acid, or a peptide, polypeptide, or protein tag.Docket No. 1580.00233WG

[0046] In the context of the present disclosure, the terms “spacer” and “linker” are used interchangeably. In aspects where an affinity ligand as described herein is functionalized with a linker, the linker may consist of a single amino acid residue, a polypeptide, or a nonpolypeptide molecule. In aspects, the single amino acid spacer may be an alanine, glycine^ serine, proline, aspartic acid, glutamic acid, or any natural or non-natural amino acid residue. In aspects, the single amino acid spacer is a glycine, alanine, or serine residue. In aspects, the polypeptide linker may consist of a polypeptide comprising a majority of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In aspects, the linker may consist of a polypeptide comprising a majority of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In aspects, the linker may consist of a polypeptide comprising a majority of amino acids selected from glycine, serine, and / or alanine. In some aspects, the linker is selected from a poly(glycine) peptide, a poly(alanine) peptide, a poly((glycine)(alanine)) peptide and a poly((glycine)(serine)) peptide. Polypeptide linkers may be from about 1 to 50 amino acids, from about 1 to 20 amino acids, from about 1 to 15 amino acids, from about 1 to 10 amino acids, from about 1 to 5 amino acids, from about 2 to 20 amino acids, from about 2 to 15 amino acids, from about 2 to 10 amino acids, or from about 2 to 5 amino acids in length. In aspects, the poly(glycine) peptide, poly(alanine) peptide, poly((glycine)(alanine)) peptide or a poly((glycine)(serine)) peptide linker consists of from about 2 to 10 amino acids, or from 2 to 6 amino acids, or from 2 to 4 amino acids.

[0047] In other aspects, the spacer or linker may be a non-polypeptide molecule, for example a substituted or unsubstituted C2-C30 alkyl spacer or a polyethylene glycol (PEG) spacer. In aspects, the C2-C30 alkyl spacer is -NH-(CH2)n-C(O)-, where n is from 2 to 30. In aspects, the alkyl spacer is substituted by a lower alkyl, e.g., Ci-Ce alkyl, acyl, halogen, -CN, -NH2, or phenyl. In aspects, a PEG spacer has a molecular weight of from about 100 to 5000 kDa, or from about 100 to 500 kDa.

[0048] In aspects, an affinity ligand as described herein may be functionalized with a peptide, polypeptide, or protein tag, for example a C-terminal affinity tag, to facilitate its isolation during manufacture and / or its detection. The term “tag” refers to a polypeptide covalently attached to another polypeptide or protein, generally to facilitate isolation and / or detection of the tagged polypeptide or protein. Such tags may be from about 4 to 500 amino acids in length. Tags allow for isolation, detection and / or localization of the tagged polypeptide orDocket No. 1580.00233WG protein using methods such as affinity purification or immunodetection with an appropriate antibody combined with detection e.g., by Western analyses, ELISA assays, or immunostaining. Exemplary tags that may be utilized include the bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin-carboxy carrier protein (BCCP), polyhistidine (His-tag), polyaspartate (Asp-tag), polycysteine (Cys-tag), polyphenylalanine (Phe-tag), glutathione S-transferase (GST), maltose binding protein (MBP), calmodulin binding peptide (CBP), intein-chitin binding domain (intein-CBD), a streptavidin / biotin-based tag such as streptavadin, streptavadin-binding peptide (SBP) or Strep-tag e.g., WSHPQFEK or AWAHPQPGG, a tandem affinity purification (TAP) tag, such as Protein A of Staphylococcus aureus and calmodulin-binding peptide (CBP), or other TAP variants, a short epitope tag such as FLAG (DYKDDDK), human influenza hemagglutinin (HA), c-myc epitope (CEQKLISEEDL), T7, or Glu-Glu (EYMPME or EFMPME), HSV epitope (QPELAPED), and small fluorescent proteins such as the green fluorescent protein (GFP) and derivatives thereof.

[0049] In aspects, an affinity ligand described here may include a C-terminal polyhistidine tag, also referred to as a “His-tag”. In aspects, an affinity ligand described here may include a combination of tags and linkers, for example a polypeptide tag, one or more N-terminal or C- terminal linkers and an additional C-terminal tag.

[0050] In aspects, provided is an affinity ligand comprising two or more units of a binding domain, for example a binding domain represented by SEQ ID NO: 57, SEQ ID NO: 58 or SEQ ID NO: 59. An affinity ligand comprising two or more units of a binding domain may be referred to as a “multimer” or by the number of units of the binding domain(s) present in the protein. For example an affinity ligand having two units of a binding domain may be referred to as a dimer. Similarly, an affinity ligand having three, four, five, or six units of a binding domain may be referred to as a trimer, a quatramer, a pentamer, or a hexamer, respectively. In some aspects, a multimer may comprise two or more units of the same binding domain. In some aspects, a multimer may comprise two or more units of different binding domains. In the context of a multimer, the two or more units of a binding domain are covalently attached to each other, either directly or via a linker molecule. Linker molecules are described in more detail infra, but generally a linker molecule is a single amino acid residue, a polypeptide, or a non-polypeptide molecule.

[0051] Also provided are affinity chromatography resins comprising a solid support functionalized with an affinity ligand as described herein. In this context, the termDocket No. 1580.00233WO“functionalized” refers to a modification of the solid support by covalent attachment of a plurality of affinity ligands.

[0052] The affinity ligands may be covalently attached to the solid support via a reactive thiol- or nitrogen-containing amino acid added at the C-terminus of the ligand. Alternatively or additionally, the affinity ligands may be covalently attached to the solid support via reactive epoxy or aldehyde groups of the support itself. Accordingly, in aspects, the polypeptides are covalently attached to the solid support directly, via a reactive thiol- or nitrogen-containing C- terminal amino acid of the polypeptide, or indirectly, via a polypeptide linker. In aspects, an affinity ligand as described herein may be modified to include a C-terminal reactive amino acid, e.g., a reactive thiol- or nitrogen-containing C-terminal amino acid, to facilitate covalent attachment of the polypeptide to the solid support. In aspects, the reactive amino acid is a lysine or cysteine. In aspects where the affinity ligand is covalently attached to the solid support via a C-terminal polypeptide linker, the C-terminal polypeptide linker may comprise from 2 to 50 amino acids, from 2 to 25 amino acids, from 2 to 15 amino acids, from 2 to 10 amino acids, or from 2 to 5 amino acids. In aspects, the C-terminal polypeptide linker comprises one or more reactive amino acids e.g., a reactive thiol- or nitrogen-containing amino acid, to facilitate covalent attachment of the linker to the solid support. In aspects, the one or more reactive amino acids includes one or more lysine and / or cysteine residues.

[0053] In aspects, the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith. The discrete polymeric particles may be made from a polysaccharide such as agar, agarose, dextran, starch, cellulose, pullulan, etc., and stabilized variants and derivatives thereof; or from a synthetic polymer such as polystyrene, polyvinylether, polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, etc. In aspects, the discrete particles may have a volume- weighted median diameter (d50,v or dv50) in the range of about 20 to 100 micrometers, or 40 to 90 micrometers, or 45 to 55 micrometers, or 80 to 90 micrometers. In aspects, the dv50 of the particles is 50 or 85 microns. In aspects, the particles are made from a material selected from agarose or a stabilized derivative thereof such as Praesto®Pure or Praesto® Jetted 50 or Praesto® Jetted 70, having a volume-weighted median diameter (d50,v) of about 50 or about 85 micrometers or having a d50v of from about 45 to 90 micrometers.

[0054] In aspects, provided is an affinity chromatography resin functionalized with at least about 1 to 20 g / Lresin of an affinity ligand as described herein. In aspects, an affinityDocket No. 1580.00233WO chromatography resin functionalized with the affinity ligands described here may be subjected to a typical cleaning in place or “CIP” process without significant decrease in ligand-binding capacity. Typical CIP processes include subjecting the affinity resin to conditions of alkaline pH, including for example 0. 1-0.5 M NaOH, acidic pH, or high concentration of a chaotropic agent such as 2-8 M urea or 2-6 M guanidine hydrochloride. In some aspects, an affinity chromatography resin functionalized with the affinity ligands described here may retain at least 80%, preferably at least 90%, of its ligand-binding capacity following exposure to 0. 1-0.5 M NaOH or 6 M guanidine hydrochloride for at least 10 hours, or at least 20 hours, or at least 30 hours, where ligand-binding capacity is measured as a percentage of the dynamic binding capacity (DBC) of the functionalized resin. In aspects, an affinity chromatography resin functionalized with the affinity ligands described here are provided as single-use columns having a ligand density of at least about 1 to 20 g / Lresin, for example about 1 to 5 g / Lresin or 5 to 15 g / Lresin or 10-20 g / resin or 15 to 20 g / Lresin. In aspects, a column comprising the affinity chromatography resin has a dynamic binding capacity (DBC) of at least about 0.1 to 20 g / Lresin or 1 to 10 g / Lresin for ligand. In aspects, a column comprising the affinity chromatography resin has a dynamic binding capacity (DBC) of at least about 2 g / Lresin, 8 g / Lresin, 12 g / Lresin, or 20 g / Lresin for the target molecule.

[0055] Suitable solid supports include, for example, agarose and stabilized derivatives of agarose (e.g. PraestoOPure, Praesto® Jetted 50, Praesto® Jetted 70, Sepharose 6B, Sepharose Fast Flow, etc.), cellulose or derivatives of cellulose, controlled pore glass, monolith (e.g. CIM® monoliths), silica, zirconium oxide (e.g. CM Zirconia or CPG®), titanium oxide, or synthetic polymers (e.g. polystyrene, polyvinylether, polyvinyl alcohol, monodisperse polyacrylate resin, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides, etc.) and hydrogels of various compositions. In certain aspects, the solid support comprises a polyhydroxy polymer, such as a polysaccharide.Examples of suitable polysaccharides include agar, agarose, dextran, starch, cellulose, pullulan, etc., and stabilized variants thereof. In some aspects, the solid support is made from agarose or a stabilized derivative thereof, such as Praesto®Pure, Praesto® Jetted 50, Praesto® Jetted 70 or polyvinyl divinyl benzene, silica, or control pore glass.

[0056] In other aspects the solid support may be a microchip, e.g., a silicon, silicon-glass, or gold microchip. In aspects, the solid support may be formed from nitrocellulose, paper, plastic,Docket No. 1580.00233WO nylon, metal, or any combination of the foregoing. In some aspects, the solid support may be a plate or dish, such as a microtiter plate or dish.

[0057] In aspects, an affinity chromatography resin functionalized with affinity ligands as described here may be subjected to a typical cleaning in place or “CIP” process without significant decrease in ligand-binding capacity. Typical CIP processes include subjecting the affinity resin to conditions of alkaline pH, including for example 0.1-0.5 M NaOH, acidic pH, or high concentration of a chaotropic agent such as 2-8 M urea or 2-6 M guanidine hydrochloride. In some aspects, an affinity chromatography resin functionalized with the affinity ligands described here may retain at least 80%, preferably at least 90%, of its ligandbinding capacity following exposure to 0.1-0.5 M NaOH or 6 M guanidine hydrochloride for at least 10 hours, or at least 20 hours, or at least 30 hours, where ligand-binding capacity is measured as a percentage of the dynamic binding capacity (DBC) of the functionalized resin. In aspects, an affinity chromatography resin functionalized with the ligand-binding polypeptides or chimeric proteins described here are provided as single-use columns.

[0058] Also provided are systems for purification of ligand-containing molecules in a manufacturing process. In aspects, the manufacturing system comprises at least one step of affinity chromatography utilizing an affinity chromatography resin functionalized with the affinity ligands described here.

[0059] The affinity ligands described herein may be produced synthetically e.g., by liquid or solid phase chemical synthetic methods, and / or other techniques such as polymerase chain reaction (PCR) based synthesis, concatemerization, seamless cloning, and recursive directional ligation (RDL). The affinity ligands may also be produced recombinantly via expression in a host cell from polynucleotides encoding the polypeptides. Such polynucleotides may also be produced synthetically or using recombinant techniques based on the amino acid sequences of the affinity ligands disclosed herein. It is understood that different nucleic acid sequences may be utilized to encode the same affinity ligand, for example where codon optimization is utilized to facilitate expression in a particular cell system. Methodologies and tools for constructing an optimized RNA sequence for protein expression are known in the art. See e.g., Strategies of codon optimization for high-level heterologous protein expression in microbial expression systems, Gene Reports 9:46-53 (2017); and “A new and updated resource for codon usage tables” Athey, J., Alexaki, A., Osipova, E. et al. A new and updated resource for codon usage tables. BMC Bioinformatics 18, 391 (2017).Docket No. 1580.00233WO

[0060] Polynucleotides encoding the polypeptides described herein may comprise control elements such as promoters, enhancers, ribosomal binding sites, transcription termination signals, and polyadenylation signals, or may be inserted into an appropriate expression vector containing one or more such control elements for expression of the polynucleotides in a host cell such as a bacterial cell, yeast cell, insect cell, plant cell or mammalian cell. Examples of prokaryotic host expression systems, viral expression systems, yeast expression systems, plant expression systems, and mammalian expression systems are known in the art. Insertion of of the polynucleotides into a suitable expression vector can be accomplished using in vitro recombinant DNA techniques, synthetic techniques, or in vivo recombination / genetic recombination techniques. Suitable expression vectors are commercially available and may include, for example, plasmid vectors, single and double-stranded phage vectors, or single and double-stranded RNA or DNA viral vectors. Phage and viral vectors may also be introduced into host cells in the form of packaged or encapsulated viral particles using known techniques for infection and transduction. Alternatively, cell-free translation systems may also be used to produce the polypeptides.

[0061] In an exemplary embodiment, affinity ligands are produced recombinantly in an appropriate expression system such as an E. Coli, Pichia Pastoris or in a mammalian cell system, using standard techniques. The recombinantly produced polypeptides are purified using multi-column chromatography. For example, histidine-tagged polypeptides may be purified using immobilized metal ion affinity chromatography (“IMAC”). The purity and identity of recombinant polypeptides may be assessed by a combination of gel electrophoresis, e.g., SDS- PAGE, reverse-phase ultra-high performance chromatography (“RP-UPLC”), quadrupole time- of-flight mass spectrometry, and size-exclusion chromatography (“SEC”).

[0062] It should be noted that the N-terminal methionine of recombinantly produced polypeptides may be cleaved during expression resulting in polypeptides lacking the N-terminal methionine, or a mixture of polypeptides which contain or lack the N-terminal methionine. The presence or absence of the N-terminal methionine does not impact the function of the polypeptides or proteins described herein. Accordingly, the N-terminal methionine should be considered optional in the amino acid sequences of the affinity ligands described herein.Production of recombinant affinity ligands

[0063] Recombinant affinity ligands were expressed in E. Coli and / or Pichia Pastoris using standard techniques. Ligands were purified using multi-column chromatography. For his-taggedDocket No. 1580.00233WO ligands IMAC was used as the primary capture step. Biotinylated ligands were generated with the Avitag™ system (Avidity, Aurora, CO). Non-biotinylated ligands bearing the Avitag™ sequence were prepared by omitting exogenous biotin. The purity and identity of recombinant affinity ligands was assessed by a combination of SDS-PAGE, RP UPLC, quadrupole time-of- flight mass spectrometry and SEC. In many instances the ligand is isolated without the N- terminal methionine residue, which is presumed to be cleaved during expression. In many instances a mixture is obtained with only a proportion of the purified ligand containing the N- terminal methionine. As will be understood by the skilled artisan, the presence or absence of the N-terminal methionine does not alter the functionality of the affinity ligand. Accordingly, in the context of the amino acid sequences defining the affinity ligands of the present invention, the N- terminal methionine may be included but its presence is not required.Functional characterization of affinity ligands

[0064] The ability of the affinity ligands to bind to a target molecule is determined, for example, by measurement of binding affinity constants such as KA and / or KD using assays that may include one or more of competition analysis, equilibrium analysis, microcalorimetric analysis, and real-time interaction analysis based on surface plasmon resonance interaction (e.g., using a B1ACORE™ instrument). These methods are described, for example, in Neri et al. (1996) Tibtech 14:465-470 and Jansson et al. (1997) J Biol Chem 272:8189-8197. In aspects binding of the affinity ligands is characterized by one or more of sensogram, adsorption, SBC, and / or DBC analyses. In aspects, an affinity ligand described here exhibits a dissociation constant (KD ) for target molecule of about 1 * 10'4M to about 1 * 10'5M, about 1 * 10'5M to about 1 x 1 O’6M, about 1 * 1 O'6M to about I x lO'7M, about 1 x 1 O'7M to about I x lO'8M, about I x lO'8M to about l x IO’9M, about I x lO'9M to about I x lO'10M, about I x lO'10M to about I x lO’11M, or about I x lO'11M to about I x lO'12M.

[0065] The determination of KD may be performed under different conditions of pH and salt concentration using suitable buffers. For example, low pH solutions (<pH 5.5) can be made using citrate buffers, glycine-HCl buffers, or succinic acid buffers. High pH solutions can be made, for example, using Tris-HCl buffers, phosphate buffers, or sodium bicarbonate buffers.

[0066] The ability of the affinity ligands to bind target molecules may also be determined, for example, using biolayer interferometry (BLI) analysis. BLI is a technique that measures the thickness of a biological layer at the tip of a sensor though interferometry. Thickness is correlated to binding events such as protein :protein interactions. In an exemplary method, biotinylatedDocket No. 1580.00233WO ligands are immobilized on sensors and incubated with solutions containing the target molecule at various concentrations. The binding curves are fitted with a 1 : 1 binding model using appropriate software, e.g., ForteBio software, to determine the KD values. In some aspects, an affinity ligand described here binds target HBsAg protein with a biolayer interferometry (BLI) response of less than or equal to 4 nm, 3 nm, 2 nm, 1 nm, or 0.5 nm.

[0067] Also provided are methods of isolating HBsAg with the affinity ligands described here and affinity chromatography resins functionalized with same.

[0068] In aspects, provided is a method for isolating HBsAg from a mixture.

[0069] In aspects, provided is a method for isolating HBsAg where the method includes steps of (i) contacting a liquid comprising HBsAg with an affinity chromatography resin as described here; (ii) washing the affinity resin with a wash buffer; and (iii) eluting the HBsAg from the affinity resin with an elution buffer. The method may also include a cleaning in place (CIP) step following elution of the HBsAg from the affinity resin. In aspects, the CIP process includes contacting the affinity resin with an alkaline solution comprising from about 0.1-0.5 M NaOH or 6 M guanidine hydrochloride for a period of at least 10 hours, optionally at least 20 hours or at least 30 hours.EXAMPLESProduction of recombinant affinity ligands

[0070] Recombinant affinity ligands were expressed in E. Coli and / or Pichia Pastoris using standard techniques. Ligands were purified using multi-column chromatography. For his- tagged ligands IMAC was used as the primary capture step. Biotinylated ligands were generated with the Avitag™ system (Avidity, Aurora, CO). Non-biotinylated ligands bearing the Avitag™ sequence were prepared by omitting exogenous biotin. The purity and identity of recombinant affinity ligands was assessed by a combination of SDS-PAGE, RP UPLC, quadrupole time-of-flight mass spectrometry and SEC. In many instances the ligand is isolated without the N-terminal methionine residue, which is presumed to be cleaved during expression. In many instances a mixture is obtained with only a proportion of the purified ligand containing the N-terminal methionine. As will be understood by the skilled artisan, the presence or absence of the N-terminal methionine does not alter the functionality of the affinity ligand. Accordingly, in the context of the amino acid sequences defining the affinity ligands of the present invention, the N-terminal methionine may be included but its presence is not required.Docket No. 1580.00233WOBinding selectivity of affinity ligands

[0071] Characterization of ligand binding to HBsAg was determined using biolayer interferometry (BLI; ForteBio, Menlo park, CA). Biotinylated ligands were immobilized on sensors and incubated with solutions containing HBsAg at various concentrations. The binding curves were fitted with a 1 : 1 binding model using the ForteBio software to determine the affinity and the resulting data is shown in Table 1 below. The data show that all affinity ligands bind to HBsAg with high affinity. In most cases, the binding affinity was less than 1 nM.

[0072] Table 1: Binding affinity (KD) for representative affinity ligands of the indicated SEQID NOs, also indicating the sequence of the binding domain for representative ligands.*The ligands demonstrated an avid binding mode due to multivalent presentation of the epitope on the antigen surface, which resulted in a KD lower than the limit of detection for ForteBio Octet.Docket No. 1580.00233WOAlkaline Stability

[0073] In addition, the sodium hydroxide stability of the affinity ligands was evaluated as follows. Ligands were incubated in either 0.1 M NaOH for 24 hours followed by neutralization. The binding of the NaOH treated ligands was measured as described in above and compared to untreated ligand. The binding retained was calculated according to the following formula: % binding retained = (measured response after NaOH treatment) (measured response of untreated) x 100. The results are shown in Table 1 above. The data indicate that many of the affinity ligands tested exhibit high alkaline stability under tested conditions.Production and characterization of affinity resins

[0074] Affinity resins comprising affinity ligands as described herein were produced as follows. Affinity resins were prepared by conjugating ligands to agarose beads. The following is a non-limiting example of affinity ligand conjugation to a commercially available base bead: Praesto® Jetted 50 beads (Purolite, King of Prussia, PA) were activated with disuccinimidyl carbonate and coupled with excess ethylenediamine. After washing, bromoacetate was conjugated to the aminated beads using EDC activation. After washing, ligands were conjugated to the beads at room temperature. Epoxy activated beads were either obtained from the vendor or prepared using standard methods well known in the art. Ligands were coupled to epoxy activated beads at 37-40 °C. Targeted ligand densities were varied from 5-30 g / L. Following washing, the beads were deactivated with excess thioglycerol. The actual ligand density for all resins was measured using a subtractive RP-HPLC method according to the following formula:Actual Ligand Density = (Measured [ligand] in feed - Measured [ligand] in effluent).Measurement of binding on resin

[0075] Binding capacity toward HBsAg of affinity resins functionalized with the ligands described herein was determined as follows. Bulk drug substance containing purified HBsAg was applied to 96-well filter plates containing affinity resins prepared from ligands having amino acid sequences as defined by SEQ ID Nos. 15-30. Plates were incubated on a plateDocket No. 1580.00233WO shaker running at 1100 RPM for the various times during the equilibration, load, wash, elution, strip, and cleaning in place (CIP) steps, as indicated in Table 2.

[0076] Table 2: Process for measuring SBC and alkaline stability (base CIP).

[0077] The flow through and elution samples were collected by centrifugation of the filter plate and analyzed by UV280 nm in a plate reader. The difference in mass of HBsAg between the load solution and the flow through sample divided by the resin volume is reported as the static binding capacity (SBC) of the resin. Next, the alkaline stability of the resins was tested. The process outlined in Table 2 was repeated after incubating the affinity resins in 0.1 M sodium hydroxide (NaOH) while shaking during a 16-hour hold. The static binding capacity measured after the 16-hour hold in sodium hydroxide relative to the initial static binding capacity is reported as the percentage of retained binding. The data are listed in Table 3 below for exemplary affinity resins produced from the affinity ligands corresponding to SEQ ID Nos. 15-30.

[0078] Table 3: Static binding capacity (SBC) of representative resins functionalized with affinity ligands of the indicated amino acid sequence and percentage retained binding capacity following incubation of the resin for 16 hours in 0.1 M NaOH. Also shown are the binding domain sequences for representative ligandsDocket No. 1580.00233WO

[0079] As illustrated by the DBC results in Table 3, exemplary resins prepared with representative affinity ligands as described herein demonstrated high binding capacity toward HBsAg and many of the resins also exhibited very high alkaline stability.

[0080] The dynamic binding capacity of resins prepared with exemplary affinity ligands was evaluated by flowing target molecule at a concentration of 0.4 g / L over a 0.3 x 5 cm (0.353 mL) columns packed with resin functionalized with affinity ligands having the amino acid sequences shown in Table 5. The column was operated according to Table 4,

[0081] Table 4: Column operating parameters for DBC assay

[0082] The materials were analyzed by measuring absorbance at 280 nm in a plate reader and calculating HBsAg concentration using a standard curve. The aggregate mass of the target molecule challenge at the point when the effluent measures 10% of the load solution is reported as the dynamic binding capacity of the resin. Data for an exemplary resin produced from the affinity ligand corresponding to SEQ ID NO: 2 is shown in FIG. 1. Dynamic binding capacity measured for additional exemplary affinity resins is reported in Table 5.Docket No. 1580.00233WO

[0083] Table 5: Dynamic Binding Capacity (DBC) of representative resins functionalized with affinity ligands of the indicated amino acid sequence.Enhanced alkaline stability

[0084] Representative resins were tested for enhanced alkaline stability using a protocol that represents repeated cleaning in place (CIP) with an alkaline solution. FIG. 2 shows breakthrough curves for an exemplary resin before and after successive 5-hour holds in 0.1 M NaOH. The dynamic binding capacity relative to the initial measured dynamic binding capacity (t=0) for each time point (5, 10, 15 hours) is reported as the retained binding capacity in Table 6. The data show that the resins demonstrate robust tolerance to repeated exposure 0.1 M NaOH under conditions simulating repeated sodium hydroxide based CIP protocols. This indicates that the resins retain sufficient binding capacity to HBsAg to enable their use in sodium hydroxide based CIP protocols during industrial manufacture of HBsAg.

[0085] Table 6: Retained binding capacity of representative resins following repeat exposure to 0.1 M NaOH (5 hours each)Docket No. 1580.00233WOUse of affinity ligands in purification of ligand from host cell culture fluid

[0086] This example demonstrates use of resins comprising affinity ligands described herein for affinity purification of HBsAg from an ion-exchange pool intermediate. Packed columns were used to purify HBsAg at a titer of 1.7 g / L from the ion-exchange pool. The resin was prepared from ligand corresponding to SEQ ID NO: 2 and SEQ ID NO: 28. The resin was packed into a 0.3 x 5.0 cm (0.353 mL) column and the column was operated as described in Table 7.

[0087] Table 7: Specifications for representative purification column runs

[0088] The eluate fractions were collected and analyzed by HBsAg ELISA and polyacrylamide gel electrophoresis (SDS-PAGE). Purity of the affinity resin elution pools are assessed by comparison to the HBsAg bulk drug substance banding pattern on the SDS-PAGE gel. The yields were measured to be 64% and 78% for resins functionalized with ligands represented by SEQ ID NO: 2 and SEQ ID NO: 28, respectively. A representative gel is illustrated in FIG. 3. Samples in each lane of the gel are given in Table 8.

[0089] Table 8: Samples in each lane of gel pictured in FIG. 3Docket No. 1580.00233WGUse of affinity agents in purification of HBsAg from yeast lysate intermediates

[0090] This example demonstrates use of resins comprising affinity ligands as described herein for affinity purification of HBsAg from several downstream process intermediates, namely polyethelyene glycol (PEG) supernatant, buffer-exchanged Aerosil desorbate, ion exchange chromatography (IEC) pool, and bulk drug substance. Packed columns were used to purify HBsAg from the four intermediates. Resins were functionalized with ligands represented by SEQ ID NO: 2, SEQ ID NO: 17, and SEQ ID NO: 23. The resins were packed into 0.3 x 5.0 cm (0.353 mL) columns and were operated as described in Table 9.

[0091] Table 9: Column operating parameters for purification of HBsAg from yeast lysate intermediatesDocket No. 1580.00233WG

[0092] The eluate fractions were collected and analyzed by HBsAg ELISA, polyacrylamide gel electrophoresis (SDS-PAGE), and Quant-It PicoGreen dsDNA assay. Purity of the affinity resin elution pools was assessed by comparison to the HBsAg bulk drug substance banding pattern on the SDS-PAGE gel shown in FIG. 4. Table 10 below identifies the sample in each lane of the gel.

[0093] Table 10: Samples in each lane of gel pictured in FIG. 4.

[0094] The affinity resins corresponding to ligand SEQ ID NO: 2, SEQ ID NO: 17, and SEQ ID NO: 23 achieved a 2.6, 1.2, and 2.2 log reduction in host cell DNA, respectively, from the PEG supernatant intermediate and yields of 133% (where 1 M sodium thiocyanate (NaSCN) was added to the elution buffer), 10%, and 76%, respectively.Additional Embodiments

[0095] Embodiment 1. An affinity ligand having a structure of Formula I, L1-D1-L2-DS-L3- DT-L4(Formula I) wherein Li, L2, L3, and L4 are each independently absent or a linker; Di is a HBsAg binding domain; Ds is an optional structural domain; and DT is an optional C-terminal tag domain.Docket No. 1580.00233WO

[0096] Embodiment 2. The affinity ligand of Embodiment 1, wherein Di is defined by the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 86% identity to SEQ ID NO: 61 : DLGKKLLEAARAGQDDEVRILMANGADVNAKDX1X2GX3TPLHLAAX4X5GHLEIVEVLL KHGADVNAX6DX7X8GX9TPLHLAAX10X11GHLEIVEVLLKX12GADVNAX13DX14WGWTP LHLAAXisWGHLEIVEVLLKYGADVNAXieDKFGKTAFDISIDNGNEDLAEIL, where Xi is S, or V, X2 is F or Y, X3 is A or G, X4 is W or Y, Xs is G or W, Xe is I or Y, X7 is H or L, Xs is G or I, X9 is E or Y, X10 is P or R, X11 is G or T, X12 is H or Y, X13 is L or V, X14 is F or W, Xis is G or L, Xi6 is F or T.

[0097] Embodiment 3. The affinity ligand of Embodiment 2, wherein Di is defined by the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 57.

[0098] Embodiment 4. The affinity ligand of Embodiment 2, wherein Di is defined by the amino acid sequence of SEQ ID NO: 61, and the ligand comprises an amino acid sequence as defined by any one of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto.

[0099] Embodiment 5. The affinity ligand of Embodiment 3 or 4, wherein any difference between the amino acid sequence of Di and SEQ ID NO: 61, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48 consists of one or more conservative amino acid substitutions.

[0100] Embodiment 6. The affinity ligand of any one of Embodiments 1 to 5, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0101] Embodiment 7. The affinity ligand of Embodiment 1, wherein Di is defined by the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 86% identity to SEQ ID NO:58: VDAX17FDX18EX19X20X21X22AX23X24EIX25X26LPNLNX27X28X29X30X31AFIX32SLX33DX 34PSQSANLLAEAX35X36LNDAQAPK where X17 is K or R, Xis is K or R, X19 is F, R or Y, X20 is I, W or Y, X21 is E H, L, Q or W, X22 is G or deleted, X23 is D, T, or V, X24 is F, H, Q, or W, X25 is A, F, Q or S, X26 is A, H, I , L, W or Q, X27 is A, L, V or W, X28 is H, I, V, W, or Y,Docket No. 1580.00233WOX29 is E or Q, X30 is K, Q, R, or V, X31 is I, S or W, X32 is A, F, I, N, or W, X33 is H, L or W, X34 is D or N, X35 is K or R, X36 is K or R.

[0102] Embodiment 8. The affinity ligand of Embodiment 7, wherein Di is defined by the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 62.

[0103] Embodiment 9. The affinity ligand of Embodiment 7, wherein Di is defined by the amino acid sequence of SEQ ID NO: 62, and the ligand comprises an amino acid sequence as defined by any one of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto.

[0104] Embodiment 10. The affinity ligand of Embodiment 8 or 9, wherein any difference between the amino acid sequence of Di and SEQ ID NO: 62, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60 consists of one or more conservative amino acid substitutions.

[0105] Embodiment 11. The affinity ligand of any one of Embodiments 7 to 10, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.

[0106] Embodiment 12. The affinity ligand of claim 1, wherein Di is defined by the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 86% identity to SEQ ID NO: 63 : VDAX37X38X39X40X41X42EX43ARX44RIESLPNLTEEQRRAFIESLRDDPX45X46X47X48X49LL WEAX50X51LNX52X53QAPK where X37 is K or R, X3 is H or L, X39 is D or H, X4o is E, H, V or Y, X41 is G, H, R, S, or W, X42 is H or L, X43 is W or Y, X44is 1, N or V, X45 is H or S, X46is H or Q, X47 is A or V, X48 is H or W, X49 is F, K or V, X50 is F or W, X51 is H or W, X52 is A, I or W, X53 is A or H.Docket No. 1580.00233WO

[0107] Embodiment 13. The affinity ligand of Embodiment 12, wherein Di is defined by the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity to SEQ ID NO: 63.

[0108] Embodiment 14. The affinity ligand of Embodiment 12, wherein Di is defined by the amino acid sequence of SEQ ID NO: 63, and the ligand comprises an amino acid sequence as defined by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 54, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity thereto.

[0109] Embodiment 15. The affinity ligand of Embodiment 13 or 14, wherein any difference between the amino acid sequence of Di and SEQ ID NO: 63, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 54 consists of one or more conservative amino acid substitutions.

[0110] Embodiment 16. The affinity ligand of any one of Embodiments 12 to 15, wherein the affinity ligand binds to HBsAg with a KD of less than 100 nM.[OHl] Embodiment 17. The affinity ligand of any one of Embodiments 1 to 16, wherein the affinity ligand is covalently attached to a solid support.

[0112] Embodiment 18. An affinity chromatography resin comprising a solid support and a plurality of the affinity ligands of any one of Embodiments 1 to 16, or multimer thereof, covalently attached to the solid support.

[0113] Embodiment 19. The affinity chromatography resin of Embodiment 18, wherein the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith.

[0114] Embodiment 20. The affinity chromatography resin of Embodiment 19, wherein the solid support is in the form of discrete polymeric particles of a polysaccharide.Docket No. 1580.00233WO

[0115] Embodiment 21. The affinity chromatography resin of Embodiment 20, wherein the polysaccharide is selected from agar, agarose, dextran, starch, cellulose, pullulan, and stabilized variants or derivatives thereof.

[0116] Embodiment 22. A method of isolating HBsAg from a mixture comprising steps of (i) contacting a liquid comprising HBsAg with an affinity chromatography resin according to any one of Embodiments 18 to 21; (ii) washing the resin with a wash buffer; and (iii) eluting the HBsAg from the resin with an elution buffer.

[0117] Embodiment 23. The method of Embodiment 22, wherein the elution buffer comprises sodium thiocyanate, optionally from about 0.5 M to about 1.5 M sodium thiocyanate.

[0118] Embodiment 24. The method of Embodiment 22, wherein the method comprises a cleaning in place step following elution of the HBsAg from the affinity resin.

[0119] Embodiment 25. The method of Embodiment 24, wherein the cleaning in place step comprises contacting the affinity resin with an alkaline solution comprising from 0.05-0.2 M NaOH for a period of time, optionally wherein the alkaline solution is 0.1 M NaOH and the period of time is at least 5 hours, at least 10 hours, or at least 20 hours.

[0120] Embodiment 26. The method of any one of Embodiments 22 to 25, wherein the liquid comprising HBsAg is a process intermediate of a yeast cell lysate selected from a polyethelyene glycol supernatant, a desalting / buffer exchange pool, and an ion exchange chromatography pool.

[0121] Embodiment 27. A polynucleotide encoding the affinity ligand of any one of Embodiments 1 to 16.

[0122] While the invention herein disclosed has been described by means of specific embodiments and applications thereof, modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims.

[0123] It will be appreciated that the present invention is set forth in various levels of detail in this application. In certain instances, details not necessary for one of ordinary skill in the art to understand the invention, or that render other details difficult to perceive may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.Docket No. 1580.00233WO

[0124] Various features of a process system may be used independently of, or in combination, with each other. It will be appreciated that a system as disclosed herein may be embodied in different forms and should not be construed as limited to the illustrated embodiments of the figures.

[0125] It should be understood that, as described herein, an “embodiment” (such as illustrated in the accompanying Figures) may refer to an illustrative representation of an environment or article or component in which a disclosed concept or feature may be provided or embodied, or to the representation of a manner in which just the concept or feature may be provided or embodied. However such illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. In addition, it will be appreciated that while the Figures may show one or more embodiments of concepts or features together in a single embodiment of an environment, article, or component incorporating such concepts or features, such concepts or features are to be understood (unless otherwise specified) as independent of and separate from one another and are shown together for the sake of convenience and without intent to limit to being present or used together. For instance, features illustrated or described as part of one embodiment can be used separately, or with one or more other features to yield a still further embodiment. Thus, it is intended that the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0126] In the foregoing description and the following claims, the following will be appreciated. The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0127] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” means that the value may vary by + / - 10%.Docket No. 1580.00233WO

[0128] In the claims, the term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. In addition, singular references do not exclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

[0129] The term “dissociation constant” or “KD” refers to the dissociation equilibrium constant of the particular interaction between a first protein and a second protein. In the context of the present invention, the term KD is particularly used to describe the binding affinity between an affinity ligand as described herein and a target molecule. An affinity ligand is considered to bind to a target where it has a dissociation constant KD of less than about 15 uM or less than about 10 uM. In some aspects, an affinity ligands described herein may have a KD of 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, or 1 nM or less for a target. KD may be determined, for example by Surface Plasmon Resonance (SPR) using a commercially available kit such as the Biacore™ assay sold by Cytiva Life Sciences.

[0130] The terms “protein” and “polypeptide” refer to any linear molecular chain of two or more amino acids linked by peptide bonds and does not refer to a specific length of the product. Thus, “peptides”, “protein”, “amino acid chain,” or any other term used to refer to a chain of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-translational modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, proteolytic cleavage, modification by non-naturally occurring amino acids and similar modifications which are well-known in the art.

[0131] The terms “alkaline stable” or “alkaline stability” may be used interchangeably herein and refer to the ability of the affinity ligand of the invention to withstand alkaline conditions without significantly losing the ability to bind to its target molecule. The skilled person in thisDocket No. 1580.00233WO field can easily test alkaline stability by incubating an affinity ligand with, for example, sodium hydroxide solutions, e.g., as described in the Examples, and subsequent testing of the binding capacity or binding activity to target molecule by routine experiments known to those skilled in the art, for example, by chromatographic approaches. The alkaline stability may be determined by coupling the affinity ligand of the invention to a surface plasmon resonance (SPR) sensor chip, and assaying the binding capacity or binding activity for target molecule before and after exposure to an alkaline solution. The alkaline treatment can be performed, for instance, in 0.5 M NaOH for an extended period of time, e.g., at least 5 hours, at least 8 hours, at least 12 hours, at least 16 hours, or at least 20 hours.

[0132] The term “percent (%) identity”, in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Percent identity may be determined using a computer algorithms such as the Basic Local Alignment Search Tool (“BLAST”) or a related tool, including other BLAST-based tools available at the US National Library of Medicine, National Center for Biotechnology Information website. The BLAST and related algorithms have been described, for example, in Altschul et al., 1990, J. Mol. Biol. 215:3, 403-410; and Altschul et al., 1997, Nucleic Acids Res. 25: 17, 3389-402.

[0133] The term “substitution” in the context of an amino acid substitution refers to an exchange of an amino acid at a particular position in a polypeptide sequence with a different amino acid. Similarly, the term “deletion” in the context of an amino acid deletion refers to the removal of an amino acid at a particular position in a polypeptide sequence; and the term “insertion” in the context of amino acid insertion refers to the addition of an amino acid to the polypeptide sequence.

[0134] The term “chromatography” refers to separation technologies which employ a mobile phase and a stationary phase to separate one type of molecules from other molecules (e.g., host cell nucleic acids or proteins) in a sample. A liquid mobile phase contains a mixture of molecules and transports these across or through a stationary phase (such as a solid resin, support, or matrix, which terms are used interchangeably). Due to the differential interaction of the different molecules in the mobile phase with the stationary phase, molecules in the mobile phase can be separated. The term “affinity chromatography” refers to a specific type ofDocket No. 1580.00233WO chromatography in which a ligand having a specific affinity for a target molecule is coupled to the stationary phase. The ligand interacts with the target molecule in the mobile phase thereby separating it from the mobile phase. Generally, the target molecule is eluted from the stationary phase in a separate step. The terms “solid support” or “solid matrix” or “resin” are used interchangeably to refer to the stationary phase.

[0135] A “conservative” amino acid substitution is one in which one amino acid residue is replaced with another having similar side chain chemistry and / or size. Families of amino acid residues having similar side chain chemistry have been defined in the art. For example, those having basic side chains, e.g., lysine (K), arginine (R), histidine (H); acidic side chains, e.g., aspartic acid (D), glutamic acid (E); uncharged polar side chains, e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C); nonpolar side chains, e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W); beta-branched side chains, e.g., threonine (T), valine (V), isoleucine (I); and aromatic side chains, e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H).Informal Sequence ListingDocket No. 1580.00233WODocket No. 1580.00233WODocket No. 1580.00233WODocket No. 1580.00233WODocket No. 1580.00233WO

Claims

Docket No. 1580.00233WOCLAIMSWhat is claimed is:

1. An affinity ligand comprising an amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 90%, or at least 93%, or at least 96%, or at least 98% amino acid sequence identity thereto:VDAX17FDX18EX19X20X21X22AX23X24EIX25X26LPNLNX27X28X29X30X31AFIX32SLX33 DX34PSQSANLLAEAX35X36LNDAQAPK (SEQ ID NO: 58) wherein X17 is K or R, Xis is K or R, X19 is F, R or Y, X20 is I, W or Y, X21 is E H, L, Q or W, X22 is G or deleted, X23 is D, T, or V, X24 is F, H, Q, or W, X25 is A, F, Q or S, X26 is A, H, I , L, W or Q, X27 is A, L, V or W, X28 is H, I, V, W, or Y, X29 is E or Q, X30 is K, Q, R, or V, X31 is I, S or W, X32 is A, F, I, N, or W, X33 is H, L or W, X34 is D or N, X35 is K or R, X36is K or R.

2. The affinity ligand of claim 1, comprising a multimer of SEQ ID NO: 58.

3. The affinity ligand of claim 2, wherein the multimer is a dimer, trimer, quatramer, pentamer, or hexamer.

4. The affinity ligand of any one of claims 1 to 3, wherein the ligand comprises an amino acid sequence of any one of SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71, or an amino acid sequence having at least 90%, at least 93%, at least 96%, or at least 98% amino acid sequence identity thereto, or a multimer of any of the foregoing.

5. The affinity ligand of any one of claims 1 to 4, wherein the ligand comprises or consists of an amino acid sequence of any one of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, or an amino acid sequence having at least 90%, at least 93%, at least 96%, or at least 98% amino acid sequence identity thereto, or a multimer of any of the foregoing.

6. An affinity ligand having a structure of Formula I,L1-D1-L2-DS-L3-DT-L4 (Formula I)Docket No. 1580.00233WO whereinLi, L2, L3, and L4 are each independently absent or a linker;Di is a HBsAg binding domain comprising SEQ ID NO: 58, or an amino acid sequence having at least 90%, or at least 93%, or at least 96%, or at least 98% amino acid sequence identity thereto, or a multimer thereof;Ds is an optional structural domain; andDT is an optional C-terminal tag domain.

7. The affinity ligand of claim 6, wherein Di comprises an amino acid sequence as defined by any one of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71, or an amino acid sequence having at least 90%, at least 93%, at least 96%, or at least 98% amino acid sequence identity thereto, or a multimer of any of the foregoing.

8. The affinity ligand of claim 6, wherein the ligand comprises or consists of an amino acid sequence as defined by any one of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56, or an amino acid sequence having at least 86% identity, at least 90% identity, at least 95% identity, or at least 98% identity to any of the foregoing.

9. The affinity ligand of any one of claims 6 to 8, wherein Li is a polypeptide linker of from 1- 15 amino acids in length.

10. The affinity ligand of claim 6 or 9, wherein at least one of L3 or L4 is present and comprises one or more of a spacer molecule and a reactive thiol- or nitrogen-containing amino acid.

11. The affinity ligand of claim 6 or 9, wherein L3 comprises one or more of a spacer molecule and a reactive thiol- or nitrogen-containing amino acid, and DT and L4 is absent.

12. The affinity ligand of claim 6 or 9, wherein wherein L3 is absent or a spacer molecule, DT is present and L4 comprises one or more of a spacer molecule and a reactive thiol- or nitrogencontaining amino acid.Docket No. 1580.00233WG13. The affinity ligand of claim 12, wherein DT is a bacteriophage T7 epitope (T7-tag), bacteriophage V5 epitope (V5-tag), biotin-carboxy carrier protein (BCCP), polyhistidine (His- tag), polyaspartate (Asp-tag), polycysteine (Cys-tag), polyphenylalanine (Phe-tag), glutathione 5-transferase (GST), maltose binding protein (MBP), calmodulin binding peptide (CBP), intein- chitin binding domain (intein-CBD), a streptavidin / biotin-based tag, a tandem affinity purification (TAP) tag or TAP variant, a FLAG tag, human influenza hemagglutinin (HA), c- myc epitope, T7, or Glu-Glu, HSV epitope, or a green fluorescent protein (GFP) or GFP derivative.

14. The affinity ligand of any one of claims 10 to 13, wherein the spacer molecule is selected from an amino acid, a polypeptide of from 1-50 amino acids, or a non-polypeptide molecule.

15. The affinity ligand of any one of claims 10 to 14, wherein the reactive thiol- or nitrogencontaining amino acid is cysteine or lysine.

16. The affinity ligand of claim 6 or 8, wherein the affinity ligand is covalently attached to a solid support, optionally wherein the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith.

17. An affinity chromatography resin comprising a solid support and a plurality of the affinity ligands of any one of claims 1 to 16, or multimers thereof, covalently attached to the solid support.

18. The affinity chromatography resin of claim 17, wherein the solid support is in the form of discrete polymeric particles, ceramic or glass beads, a woven or non-woven membrane, or a polymeric monolith.

19. The affinity chromatography resin of claim 18, wherein the discrete polymeric particles are particles of a polysaccharide or synthetic polymer, optionally wherein the polysaccharide is agarose or cellulose, or a stabilized variant or derivative thereof.

20. The affinity chromatography resin of claim 19, wherein the polysaccharide is selected from agar, agarose, dextran, starch, cellulose, pullulan, and stabilized variants or derivatives thereof.

21. The affinity chromatography resin of any one of claims 17 to 20, wherein the solid support comprises the affinity ligand at a density of from about 1-20 g / Lresin.Docket No. 1580.00233WO22. The affinity chromatography resin of any one of claims 17 to 21, wherein the resin has a dynamic binding capacity of at least 1-20 g / L for HBsAg.

23. A method of isolating HBsAg from a mixture comprising steps of (i) contacting a liquid comprising HBsAg with an affinity chromatography resin according to any one of claims 17 to 22; (ii) washing the resin with a wash buffer; and (iii) eluting the HBsAg from the resin with an elution buffer.

24. The method of claim 23, wherein the elution buffer comprises sodium thiocyanate, optionally from about 0.5 M to about 1.5 M sodium thiocyanate.

25. The method of claim 23, wherein the method comprises a cleaning in place step following elution of the HBsAg from the affinity resin.

26. The method of claim 25, wherein the cleaning in place step comprises contacting the affinity resin with an alkaline solution comprising from 0.05-0.5 M NaOH for a period of time, optionally wherein the alkaline solution is 0.1 M NaOH and the period of time is at least 5 hours, at least 10 hours, or at least 20 hours.

27. The method of any one of claims 23 to 26, wherein the liquid comprising HBsAg is a process intermediate of a yeast cell lysate selected from a polyethelyene glycol supernatant, a desalting / buffer exchange pool, and an ion exchange chromatography pool.

28. A polynucleotide encoding an affinity ligand comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 or SEQ ID NO: 56, or an amino acid sequence having at least 90%, at least 93%, at least 96%>, or at least 98% amino acid sequence identity thereto, or a multimer of any of the foregoing, optionally wherein the ligand contains a C-terminal cysteine.

29. A recombinant cell or viral particle comprising the polynucleotide of claim 28.

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