CN117836313A - Chimeric IgG-Fc binding ligand polypeptides and their use for IgG affinity purification - Google Patents

Abstract

The present invention relates to chimeric IgG-Fc binding ligand polypeptides comprising protein fragments of SlyD, wherein the IF domain thereof is replaced by the affinity peptide Fc-III-4C or Fc-III-XC variants thereof, and related uses for IgG affinity purification.

Description

Chimeric IgG-Fc binding ligand polypeptides and their use for IgG affinity purification

The present invention relates to chimeric IgG-Fc binding ligand polypeptides comprising protein fragments of SlyD, wherein the IF domain thereof is replaced by the affinity peptide Fc-III-4C or Fc-III-XC variants thereof, and related uses for IgG affinity purification.

Background

With the success of monoclonal antibodies as therapeutic and diagnostic tools, the development of adequate and economical IgG purification procedures has become more necessary for both academic and industrial use. Apart from the different types of interaction and separation techniques, affinity strategies have been the most common and effective method to date.

With the increasing popularity of antibody applications, it is necessary to develop suitable mAb purification strategies, especially for industrial scale production. Compared with other biotechnological products, due to the higher administration dosage of monoclonal antibodies and the wide application field, hundreds of kilograms of raw material medicine substances of monoclonal antibodies are needed to be synthesized each year.

Streptococcal (Streptococcal) protein a is a natural IgG-Fc conjugate, which remains the gold standard for affinity ligands on a manufacturing scale. These affinity resins coupled to a suitable matrix (such as agarose beads) represent the first step in the purification of the chain to separate the antibodies from the crude protein mixture (such as serum, ascites fluid or cell supernatant) and thus make further downstream processes more economical.

The most common procedure used today for downstream purification of monoclonal antibodies comprises a first step, wherein production cell cultures are harvested via e.g. filtration to remove cells and cell debris and to produce a clear supernatant suitable for chromatography. The mabs present in the cell culture supernatant were then recovered in a single capture step by applying the filtered fluid to a protein a chromatography column. Process and product related impurities are removed by one or two refining steps, typically in combination with cation or anion exchange chromatography, hydrophobic interaction chromatography or mixed mode chromatography.

In addition, several steps are included in the process that bypass viral contamination, such as viral inactivation (e.g., low pH incubation) and viral filtration steps following protein a chromatography. The purified product is then transferred to a final formulation buffer via ultrafiltration or diafiltration.

WO 2004/076485A1 describes a method for purifying antibodies by means of protein A affinity chromatography.

Although protein a chromatography is very effective in removing process-related impurities and presents other significant features, such as high production yields and ease of handling, there are some drawbacks to the general requirements described above: first, protein a, as a product of bacterial origin, contaminates the final product by leaking from the column matrix and acts as an immunotoxin. Secondly, the cost is relatively high: on the one hand due to resin production, because protein a is obtained in recombinant form from escherichia coli (e.coli), and on the other hand due to column regeneration: protein a is not resistant to common cleaning and sanitizing agents (such as guanidine hydrochloride or urea), which represents not only a cost challenge, but also a disposal challenge. Another serious problem of concern is aggregate formation due to antibody elution conditions at low pH values, which may lead to loss of its biological activity (Arora, I., chromatographic Methods for the Purification of Monoclonal Antibodies and their Alternatives: A review. International Journal of Emerging Technology and Advanced Engineering,2013.3 (10): pages 475-481).

One of the major drawbacks of conventional IgG purification processes is elution at acidic pH values (up to pH 2.5-3.5). The high affinity of natural IgG-binding proteins (such as protein a) for their targets on the one hand causes high specificity and selectivity of the purification process, but on the other hand requires harsh conditions to disrupt the strong interactions between antibodies and ligands. The low pH may promote degradation of mAbs and may result in contamination of the final product due to leakage of column ligands

To overcome these drawbacks of protein a-based purification, great efforts are made to improve alternative Ab binding molecules, especially in view of binding properties as well as pH sensitivity. In addition to protein A, which is mainly used for Ab isolation due to its high affinity for IgG from different species, other proteins of bacterial origin have been studied, such as proteins G, Z and L (Choe, W., T.A. Durgannavar and S.J. Chung, fc-Binding Ligands of Immunoglobulin G: an Overview of High Affinity Proteins and peptides. Materials (Basel), 2016.9 (12)).

Affinity chromatography techniques using protein a and G resins remain the most widely used mAb capture method at manufacturing scale, as they represent well-proven techniques and are involved in many established platform processes.

SlyD (sensitive to cleavage D; product of slyD gene) is a chaperone protein and consists of two domains representing the following two functional units: the peptidyl prolyl cis/trans isomerase (PPIAse) activity in the FK506 binding protein (FKBP) domain and the 45 amino acid 'insertion flap' (IF) domain (Low, C. Et al, crystal structure determination and functional characterization of the metallochaperone SlyD from Thermus thermophilus. J Mol Biol,2010.398 (3): pages 375-90). Its excellent biophysical properties, such as excellent thermostability and proteolytic stability, due to its origin in the thermophilic organism Thermus thermophilus (Thermus thermophilus), makes TtSlyd a powerful tool for a wide range of applications. One of the great advantages of the TtSlyD scaffold is that the IF domain can be exchanged easily without affecting the physicochemical behaviour and tertiary structure of the FKBP core. Furthermore, its simple structure and its high solubility allow recombinant production in bacterial cells such as e.coli. However, due to its bacterial origin, therapeutic application of TtSlyD scaffolds is not feasible in its original composition, eliciting an immune response.

WO 2003/000878A2 relates to cloning and expression of heterologous proteins or polypeptides in bacteria such as E.coli. In particular, the present invention relates to expression means comprising FKBP-type peptidyl prolyl isomerase selected from the group consisting of FkpA, slyD and triggers, recombinant protein expression methods, recombinant polypeptides thus obtained and uses of such polypeptides.

WO 2014/071978A1 relates to a chimeric polypeptide protein scaffold for engineering a polypeptide domain displayed by a scaffold comprising one or more fragments from the FKBP family, displaying one or more polypeptides inserted in place of an insertion flap domain (IF domain), and its use in a method for screening and selecting a restricted peptide surrogate exhibiting binding activity for a polypeptide of a predetermined target molecule, in particular a MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRLIPGLEEALEGREEGEAFQAHVPAEKAY-X-GKDLDFQVEVVKVREATPEELLHGHA (SEQ ID NO: 2) sequence, wherein X is an amino acid sequence comprising a variable sequence to be displayed by a thermus thermophilus SlyD chimeric polypeptide.

It is an object of the present invention to provide an improved IgG purification procedure comprising a capture system that exhibits rapid complex formation between ligand and target analyte and allows dissociation under relatively mild conditions, and wherein the method also avoids the other drawbacks of the prior art. Other objects and advantages will become apparent to those skilled in the art upon a study of the present description of the invention.

In a first aspect of the invention the above object is solved by a chimeric IgG-Fc binding ligand polypeptide comprising a protein fragment of SlyD wherein its IF domain is replaced by the affinity peptide Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1) or an Fc-III-XC variant (X) ₁ DCAWHLGELVWCTX ₂ SEQ ID NO: 3) Replacement, wherein X ₁ Missing or independently selected from the group of: C. d, P, E and K, and X ₂ Independently selected from the group of: C. q, P and E. Preferably, the chimeric IgG-Fc binding ligand polypeptide according to the invention comprises SlyD from Thermus (Thermus) species, such as, for example, thermus thermophilus (Thermus thermophiles).

In a second aspect of the invention, the above object is solved by a bivalent binder molecule comprising two fused, preferably end-to-end fused, chimeric IgG-Fc binding ligand polypeptides according to the invention.

In a third aspect of the invention, the above object is solved by a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent conjugate molecule according to the invention coupled to a solid support, such as a solid matrix material, such as a bead and/or a column matrix.

In a fourth aspect of the invention, the above object is solved by a method for producing a chimeric IgG-Fc binding ligand polypeptide according to the invention, comprising recombinant expression of said ligand polypeptide in a suitable host cell, such as e.coli, or comprising chemical synthesis of said ligand polypeptide.

In a fifth aspect of the invention the above object is solved by a method for purifying an immunoglobulin, comprising contacting a solid support having a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent conjugate molecule according to the invention coupled thereto with said immunoglobulin and eluting said immunoglobulin from said chimeric IgG-Fc binding ligand polypeptide or said bivalent conjugate molecule, as appropriate, wherein said method preferably comprises Fast Protein Liquid Chromatography (FPLC).

In a sixth aspect of the invention, the above object is solved by the use of a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent conjugate molecule according to the invention for purifying immunoglobulins or for screening and selecting peptide conjugates against a predetermined target molecule.

In a main aspect of the invention, novel conjugates and corresponding immunoaffinity chromatography column materials suitable for IgG purification are developed. In a particularly preferred aspect, the new material is suitable for immunoglobulin affinity purification via Fast Protein Liquid Chromatography (FPLC). The present invention provides an efficient, less cumbersome and cost effective protein a substitute.

The present invention provides a chimeric IgG-Fc binding ligand polypeptide comprising a protein fragment of SlyD wherein its IF domain is replaced with an affinity peptide Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1).

The Fc binding ligands involved are based on the existing IgG-Fc affinity peptide, called Fc-III-4C, described by Gong et al in 2016 (Gong, Y. Et al Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein detection. Bioconjug Chem 2016.27 (7): pages 1569-73). By grafting a peptide onto the FKBP domain of the thermus thermophilus chaperone protein SlyD, an extremely stable and easy to produce affinity ligand, designated TtSlyD-Fc-III-4C, was surprisingly obtained. In addition, a chromatographic column was generated by covalently immobilizing the TtSlyD-Fc-III-4C chimera on NHS-activated agarose resin and tested successfully.

The crystal structure of domain B, where SpA binds to the IgG-Fc portion, was resolved in 1981 and revealed the site of interaction with the CH2 and CH3 domains of immunoglobulins. The B subunit contains two alpha helices and eleven residues participate in the binding process. The corresponding binding sites on the Fc fragment are also points of contact for other natural binding proteins (such as protein G and neonatal Fc receptor) and appear to be the preferred points of protein-protein interaction due to their physicochemical properties. DeLano and colleagues locked this specific interaction site in 2000 (DeLano, wi. Et al, convergent solutions to binding at a protein-protein interface.science,2000.287 (5456): pages 1279-83). By screening a cyclic peptide library via phage display, they identified a 13 residue sequence (DCAWHLGELVWCT, SEQ ID NO: 4) that binds to the common site of the IgG-Fc region, which was found to compete with the natural conjugate. Then, X-ray crystal structure analysis revealed the α -hairpin conformation of the selected Fc-III peptide. Although the structure is quite different from known binding motifs and is four times smaller, fc-III and protein a share many intermolecular interaction points at the surface of the Fc portion, and the binding affinity of Fc-III peptide is only about two times weaker.

In 2006, dias and colleagues have attempted to further stabilize the hairpin conformation by limiting the conformational freedom of the peptide (Dias, r.l. et al Protein ligand design: from phage display to synthetic protein epitope mimetics in human antibody Fc-binding peptides Chem Soc 2006.128 (8): pages 2726-32). They created two backbone cyclic mimetic peptides based on the original Fc-III. The resulting FcBP-1 and FcBP-2 peptides were generated by grafting Ala3-Trp11 or Asp1-Thr13 onto hairpin-induced D-Pro-L-Pro templates. FcBP-2 showed 80-fold higher affinity for the Fc domain compared to Fc-III. This is due to the skeletal cyclization and limitation of additional disulfide bridges. In contrast, fcBP-1 lacks a critical disulfide bridge and has weak interactions with the Fc domain. In 2012, gong and colleagues further tried to stabilize peptides in a double-ring structure (Gong, y. Et al, development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein detection. Bioconjug Chem,2016.27 (7): pages 1569-73).

In addition, to simplify expression and purification of the TtSlyD-FcIII-4C scaffold, derivatives of this molecule were designed and binding of chemically synthesized peptides to IgG (mAb) was tested in a high throughput microarray. The two cysteine residues forming the disulfide bridge in the stem of the emerging loop structure are either exchanged or completely removed.

Because of the difficulty in synthesizing the D-Pro-L-Pro backbone, two prolines are replaced at the N-and C-terminus by two cysteine residues, which are intended to form a second disulfide linkage. The newly generated peptide, called Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1), was analyzed via Surface Plasmon Resonance (SPR) and showed 30-fold higher binding affinity to human IgG compared to the original Fc-III. Furthermore, it shows strong interactions with multiple IgG from different species. The potential as ligand for antibody purification was demonstrated by selective capture of IgG from rabbit serum using agarose beads carrying immobilized affinity peptides.

Fc-III-4C peptide beads show even higher binding capacity and reusability than commercial protein a beads. The crystal structure of the complex of the Fc-III peptide and the IgG-Fc domain shows that the restricted peptide loop targets the same binding site as the native Fc conjugate. It interacts with similar amino acids found in the interface of commonly used Fc binding proteins.

Furthermore, complex stability of the Fc-III-4C binding peptide-IgG-Fc complex was altered by systematically engineering Fc-III-4C loop insertions.Trp11 appears to be one of the amino acids that plays a key role in complex formation, and the corresponding residue is exchanged with the other twelve amino acids, exhibiting different chemical properties. The resulting mutants were expressed, purified and tested via SPR analysis. The results obtained have been adopted and tested using the existing TtSlyD scaffold platform. They produced a chimeric IgG-Fc binding ligand polypeptide according to the invention comprising a protein fragment of SlyD in which its IF domain is replaced by its affinity peptide Fc-III-XC (X ₁ DCAWHLGELVWCTX ₂ SEQ ID NO: 3) Replacement, wherein X ₁ Missing or independently selected from the group of: C. d, P, E and K, and X ₂ Independently selected from the group of: C. q, P and E.

Peptide-based ligands are a promising new class of conjugates and have been successfully developed by different research communities. It is expected that protein a may be replaced with them, they may be produced at lower cost, and are not immunogenic compared to natural conjugates. Among this group of conjugates, in particular cyclic peptides have some promising features for use as affinity ligands. They exhibit higher enzymatic stability and conformational rigidity compared to their linear counterparts, leading to higher specificity and/or affinity for their targets and entropy advantage of binding. However, chemical synthesis of peptides is still necessary, and aggregation during synthesis may lead to low yields.

For chimeric IgG-Fc binding ligand polypeptides according to the invention, any suitable SlyD scaffold protein may be used, and SlyD from bacterial species, such as from e.coli, and SlyD orthologs from Yersinia pestis (Yersinia pestis), treponema pallidum (Treponema pallidum), pasteurella multocida (Pasteurella multocida) and Vibrio cholerae (Vibrio cholerae) are preferred. Further preferred is SlyD from a thermus species, such as, for example, thermus thermophilus.

More preferred is a chimeric IgG-Fc binding ligand polypeptide according to the present invention comprising the sequence

Further preferred is a chimeric IgG-Fc binding ligand polypeptide according to the invention, wherein its IF domain is replaced by an affinity peptide Fc-III-XC selected from the group consisting of:

DCAWHLGELVWCTX ₂ ，SEQ ID NO：6

CDCAWHLGELVWCTX ₂ ，SEQ ID NO：7

DDCAWHLGELVWCTX ₂ ，SEQ ID NO：8

PDCAWHLGELVWCTX ₂ ，SEQ ID NO：9

EDCAWHLGELVWCTX ₂ SEQ ID NO:10 sum of

KDCAWHLGELVWCTX ₂ SEQ ID NO:11, wherein X ₂ Independently selected from the group consisting of: C. q, P and E, and/or

Selected from the group consisting of:

X ₁ DCAWHLGELVWCTC，SEQ ID NO：12

X ₁ DCAWHLGELVWCTQ，SEQ ID NO：13

X ₁ DCAWHLGELVWCTP, SEQ ID NO:14 and

X ₁ DCAWHLGELVWCTE，SEQ ID NO：15，

wherein X is ₁ Missing or independently selected from the group consisting of: C. d, P, E and K.

Even further preferred is a chimeric IgG-Fc binding ligand polypeptide according to the invention, wherein its IF domain is replaced by an affinity peptide Fc-III-XC having the sequence: EDCAWHLGELVWCTE, SEQ ID NO:16 (TtSlyD-Fc-III-2C_Hit No. 4).

Still further preferred is a chimeric IgG-Fc binding ligand polypeptide according to the invention, which chimeric IgG-Fc binding ligand polypeptide further comprises a C-terminal amino acid tag, such as for example His ₈ And (5) a label. Due to the metal ion (Ni ²⁺ ) And interactions between 8 x-histidine side chains, proteins with affinity tags consisting of polyhistidine residues are advantageously captured (see examples).

The chimeric IgG-Fc binding ligand polypeptides according to the invention further have the advantage over protein a/G that the polypeptides exhibit high affinity binding to a broad IgG class, e.g. selected from the group consisting of: human, rabbit (see examples), mouse, rat, pig, goat, horse, and bovine IgG.

Protein ligand multimerization can result in even higher alkaline stability and improved binding capacity to the affinity column matrix due to the affinity effect. Thus, a bivalent conjugate of 29kDa in molecular weight was produced by head-to-tail ligation of TtSlyD-Fc-III-4C with the second scaffold protein, which exhibited the same Fc-III-4C peptide loop. In the SPR interaction assay, this dual conjugate showed even higher affinity for the Fc portion of IgG compared to the single scaffold (kd=5 nM, at 37 ℃ for rbIgG and at 25 ℃ for human IgG). Thus, another preferred aspect of the invention relates to a bivalent binder molecule comprising two chimeric IgG-Fc binding ligand polypeptides according to the invention fused, preferably end-to-end, to each other.

Yet another aspect of the invention relates to a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent conjugate molecule according to the invention coupled to a solid support, such as a solid matrix material, such as a bead, such as agarose beads, and/or a column matrix, preferably a NHS-activated matrix, such as agarose resin. Preferred are coupled ligand or conjugate molecules according to the invention, wherein the coupling is via the lysine side chain of SlyD with NHS esters contained in an agarose matrix.

Yet another aspect of the invention relates to the above-mentioned solid support material, such as beads, such as agarose beads, and/or a column matrix, preferably a NHS-activated matrix, such as agarose resin, coupled to a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent conjugate molecule according to the invention. The material has improved properties such as less leakage and increased reusability as described herein. Yet another aspect of the invention relates to an immunoaffinity chromatography column comprising a solid support material according to the invention, which is preferably suitable for IgG purification as also disclosed herein.

Yet another aspect of the invention relates to a method for producing a chimeric IgG-Fc binding ligand polypeptide according to the invention, comprising recombinant expression of the ligand polypeptide in a suitable host cell, such as e.coli, or comprising chemical synthesis of the ligand polypeptide. Corresponding methods are known in the art and are also disclosed herein. Preferred are methods according to the invention, which further comprise the step of coupling the chimeric IgG-Fc binding ligand polypeptide to a solid support, such as a solid matrix material, such as beads, and/or a column matrix, in particular a matrix as described above. The matrix may be packaged into a suitable column.

Yet another aspect of the invention relates to a method for isolating or purifying an immunoglobulin, comprising contacting a solid support having a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent conjugate molecule according to the invention coupled thereto with said immunoglobulin, and eluting said immunoglobulin from said chimeric IgG-Fc binding ligand polypeptide or said bivalent conjugate molecule, as appropriate. Preferred is a method according to the invention comprising Fast Protein Liquid Chromatography (FPLC). Exemplary methods are disclosed in this example and in the literature of affinity chromatography, such as Elliott L.Rodriguez et al (Affinity chromatography: A review of trends and developments over the past 50 years,Journal of Chromatography B, volume 1157, 2020, https:// doi.org/10.1016/j.jchromb.2020.122332, or Huse, klaus)&Hans-Joachim&Scholz, gerhard (2002) Purification of antibodies by affinity chromatography.journal of biochemical and biophysical methods.51.217-31.10.1016/S0165-022X (02) 00017-9). The method can be performed repeatedly using the matrix materials disclosed herein and can be scaled to industrial scale production.

Preferred is a method according to the invention, wherein the elution conditions for the immunoglobulin are milder compared to a solid support material comprising protein a coupled thereto. Particularly in the case of variants, a rapid association rate favors antibody capture, and a rapid dissociation rate allows for antibody separation under relatively mild pH conditions. For protein A, a low pH (e.g., acetic acid at a pH of about 3.3) has been used as a standard procedure for elution (Gulich, S., M.Uhlen and S.Hober, protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography.J Biotechnol,2000.76 (2-3): pages 233-44). The low pH may promote degradation of the mAb and may lead to contamination of the final product due to leakage of column ligands. The preferred pH elution range of the present invention is 3.4 to 4.5.

Also preferred is a method according to the invention comprising a chemical regeneration step for a solid support material comprising protein a coupled thereto using conditions that are harsher than those used for said solid support material. Successful regeneration involves removal of the bound analyte without isolating the ligand or limiting its activity. Protein a/G binds to the antibody relatively strongly and regeneration requires washing with e.g. glycine buffer (pH 2.7). The preferred pH regeneration range of the present invention is 2.5 to 2.0.

A further aspect of the invention relates to the use of a chimeric IgG-Fc binding ligand polypeptide according to the invention or a bivalent conjugate molecule according to the invention for purifying immunoglobulins or for screening and selecting peptide conjugates against a predetermined target molecule.

Chemically synthesized so-called Fc-III-4C peptides are limited by two disulfide bridges to facilitate hairpin conformation with binding capacity to accurately complement their targets. Cyclic 13-mer peptides identified based on phage display were studied, which were first described by De-Lano and colleagues in 2000 (DeLano, W.L. et al, convergent solutions to binding at a protein-protein interface. Science,2000.287 (5456): pages 1279-83), and were further optimized by Gong et al in terms of conformational stability and binding affinity (KD=2.45 nM for human IgG) in 2016 (Gong, Y. Et al, development of the Double Cyclic Peptide LiRand for Antibody Purification and Protein detection. Bioconjug Chem,2016.27 (7): pages 1569-73).

In contrast to the two disulfide bridges necessary in limited peptide synthesis, the scaffold conjugate requires only one bridge to function, which facilitates inexpensive recombinant production in E.coli in high yield. The chemical regeneration conditions of the affinity column may be more severe compared to protein a and the elution conditions of the antibody are milder compared to protein a.

In the present invention, the feasibility and efficiency of a novel immunoaffinity chromatography column suitable for IgG purification was demonstrated for the first time. It was shown that grafting of the Ig-Fc affinity peptide Fc-III-4C onto the TtSlyD scaffold protein was possible while retaining the specificity and affinity of the peptide.

The advantage of a single Fc-III-4C affinity peptide compared to protein a/G is that it shows high affinity for many IgG classes (human, rabbit, mouse, rat, pig, goat, horse, cow) and that Fc-III-4C immobilized on agarose beads as affinity ligand for mAb purification shows prolonged reusability compared to standard protein a beads. Furthermore, ttSlyD-Fc-III-4C scaffold proteins coupled to NHS-agarose have excellent chemical robustness (demonstrated by repeated denaturation and refolding of the protein) and are highly resistant to alkali treatment.

Affinity chromatography columns comprising TtSlyD-Fc-III-4C are an economical and effective alternative to alkali-sensitive protein A matrices. In addition, protein scaffolds provide high conformational stability and enhanced protease resistance of the displayed peptide loops, resulting in low ligand leakage and low contamination of the final product. Furthermore, by recombinant expression in E.coli, scaffold proteins can be produced in large quantities rapidly and cost effectively. About 600mg biomass and > 5mg final protein yield per 100mL bacterial cell culture were obtained for all expressed protein variants.

Furthermore, misfolding and oligomerization of proteins driven primarily by nonspecific intermolecular disulfide bridge formation are major problems. It was shown that synthesis can be improved by eliminating the two terminal cysteine residues of the restriction peptide loop. Variants TtSlyD-Fc-III-XC exhibiting only a single disulfide linkage are less prone to aggregation than wild-type scaffolds with four cysteines.

Furthermore, to overcome the problem of acidic elution conditions, the binding site of the TtSlyD-Fc-III ligand may be further engineered. The hotspot residue leucine 7 can be modified to obtain the desired association/dissociation characteristics.

In the context of the present invention, the Fc binding portion of the peptide is further altered by QC-PCR as described below, with the aim of selectively screening protein variants exhibiting the following characteristics: a rapid association rate that facilitates antibody capture, and a rapid dissociation rate that allows for antibody separation under relatively mild pH conditions. Surface plasmon resonance was used to determine the binding affinity of the resulting TtSlyD-Fc-III-4C variants to IgG. For this, the scaffold was immobilized on a Biacore chip, interacting with IgG in the flow cell as described below. Analyte (IgG) was applied at five different concentrations and each concentration was monitored in a single cycle, regenerating the chip surface after each run. Regenerative reconnaissance was performed using human IgG prior to kinetic measurements to check for optimal conditions for complete removal of analyte after each cycle. Regarding the results obtained in this experiment, it can be concluded that grafting of the Fc-III-4C peptide onto the TtSlyD domain did not negatively affect its affinity for IgG-Fc. Furthermore, protein variants exhibiting altered kinetics (such as faster association or dissociation rates) may not be detected by exchanging tryptophan residues at position 11. A possible explanation for this is that tryptophan contributes less to binding energy than originally expected. In future studies, emphasis may be shifted to another amino acid, showing a more significant contribution to binding energy. For example, leucine at position 7 in an Fc-III-4C peptide represents a typical hot spot at the protein-peptide interface. Furthermore, it has been shown in the experiments below that by exchanging the corresponding residues, the binding strength can be significantly influenced.

The four cysteine residues present in the Fc-III-4C peptide loop form a cyclic structure and define the rigidity and affinity of the conjugate. However, cysteine represents a major problem in protein expression and purification. Dias et al have demonstrated that complete loss of binding affinity occurs by removal of disulfide linkages in Fc-III peptides (Dias, R.L. et al Protein ligand design: from phage display to synthetic protein epitope mimetics in human antibody Fc-binding peptides Chem Soc,2006.128 (8): pages 2726-32). In the present invention, the TtSlyD scaffold retains the structural stability of the affinity peptide and may not require additional disulfide linkages. In subsequent experiments, cysteines were replaced with alternative amino acids to investigate whether these linkages are critical for loop stability and affinity of proteins for IgG-Fc.

In the context of the present invention, the feasibility and efficiency of a novel immunoaffinity chromatography column suitable for IgG purification was demonstrated for the first time. It was shown that grafting of the Ig-Fc affinity peptide Fc-III-4C onto the TtSlyD scaffold protein was possible while retaining at least the specificity and affinity of the peptide.

The advantage of a single Fc-III-4C affinity peptide compared to protein a/G is that it shows high affinity for many IgG classes (human, rabbit, mouse, rat, pig, goat, horse, cow) and that Fc-III-4C immobilized on agarose beads as affinity ligand for mAb purification shows prolonged reusability compared to standard protein a beads. Furthermore, ttSlyD-Fc-III-4C scaffold proteins coupled to NHS-agarose have excellent chemical robustness (demonstrated by repeated denaturation and refolding of the protein) and are highly resistant to alkali treatment. Thus, an affinity chromatography column comprising TtSlyD-Fc-III-4C is an economical and effective alternative to an alkali-sensitive protein A matrix. In addition, protein scaffolds provide high conformational stability and enhanced protease resistance of the displayed peptide loops, resulting in low ligand leakage and low contamination of the final product.

Furthermore, by recombinant expression in E.coli, scaffold proteins can be produced in large quantities rapidly and cost effectively. About 600mg biomass and a final protein yield of about 5mg per 100mL bacterial cell culture were obtained for all expressed protein variants. The amount generated is sufficient for the screening experiments performed in the present subject matter, but can be further optimized in terms of yield and solubility for potential mass production, for example, by lowering the culture temperature or the amount of inducing substances (Marisch, K. Et al, evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production. Microb Cell Fact,2013.12: page 58). Furthermore, misfolding and oligomerization of proteins driven primarily by nonspecific intermolecular disulfide bridge formation are major problems. It was shown that synthesis can be improved by eliminating the two terminal cysteine residues of the restriction peptide loop. Variants TtSlyD-Fc-III-XC exhibiting only a single disulfide linkage are less prone to aggregation than wild-type scaffolds with four cysteines.

Protein ligand multimerization can result in even higher alkaline stability and improved binding capacity to the affinity column matrix due to the affinity effect. Thus, a bivalent conjugate of 29kDa in molecular weight was produced by head-to-tail ligation of TtSlyD-Fc-III-4C with the second scaffold protein, which exhibited the same Fc-III-4C peptide loop. In the SPR interaction assay, this dual conjugate showed even higher affinity for the Fc portion of IgG compared to the single scaffold (kd=5 nM, at 37 ℃ for rbIgG and at 25 ℃ for human IgG). However, the production and purification of such dual conjugates is more complex in terms of protein aggregation and conjugation.

Furthermore, to overcome the problem of acidic elution conditions, the binding site of the TtSlyD-Fc-III ligand may be further engineered. The hotspot residue leucine 7 may be further modified to obtain the desired association/dissociation characteristics.

The invention will now be further described in the following examples with reference to the accompanying drawings, without being limited thereto. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety. In the drawing of the figure,

fig. 1 shows the general workflow of the present invention.

FIG. 2 shows an example of a coupling process of the conjugate of the invention to a substrate. The column used consisted of NHS ester linked to agarose HP via a six atom spacer. The activated ester reacts rapidly with the primary amino group containing ligand, forming a very stable amide linkage. AB = agarose beads

Examples

As a general outline, in the context of the present invention, a prokaryotic TtSlyD-Fc-III-4C expression construct was designed and TtSlyD-Fc-III-4C scaffold molecules and chromatographic columns were generated. IgG purification experiments were then performed. Subsequently, maturation of the Fc-III-4C binding site, replacement of the cyclic side cysteines, and kinetic screening of the engineered protein variants were performed. Suitable variants are selected.

Molecular cloning of E.coli expression constructs

Cloning of the TtSlyD-Fc-III-4C scaffold affinity peptide chimera was performed in a two-step reaction. First, restriction enzymes are usedAnd->The gene encoding the TtSlyD protein backbone and the 8 x-histidine tag was inserted into a prokaryotic expression vector. Subsequently, the sequence encoding the appropriate affinity peptide is passed through +.>And->Into the corresponding insertion site of the TtSlyD scaffold. For molecular grafting and generation of the TtSlyD-Fc-III-XC expression construct, a double stranded linear DNA fragment (so-called DNA strand) was designed (which contains the desired variation in the appropriate sites of the TtSlyD sequence) and cloned directly into the prokaryotic expression vector pQE 80-Kan.

The expression vectors pQE80-Kan-TtSlyD-Fc-III-4C and pQE80-Kan-TtSlyD-Fc-III-XC used in the present invention are derived from the pQE80-Kan vector and contain the following features: kanR: resistance to kanamycin antibiotics; colE1: an origin of replication; lacIq: a lac repressor; PT5: the T5 promoter (derived from e.coli phage T5); MCS: multiple cloning sites with restriction sites, especially for EcoRI and HindIII.

The expression system used in the present invention depends on the inducible T5-lac system [65]. In the absence of lactose, the lac repressor protein, lacIQ, encoded in the expression vector prevents the binding of bacterial RNA polymerase (RNAP) to the promoter of the lac operator. Isopropyl- β -D-thiogalactoside (IPTG) is a non-metabolizable structural analog of isolactose that binds to and inactivates laqIQ, which allows RNAP to transcribe the sequence downstream of the T5 promoter (PT 5). The resulting transcript may then be translated into a recombinant protein.

Quick Change PCR(QC-PCR)

Exchange of Trp11 was performed by the Quick change polymerase chain reaction (QCCCR, by Braman et al (Braman, G.P.C.G.A.), site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol Biol,1996.57: pages 31-44) on the expression construct pQE80-Kan-TtSlyDFc-III-4C according to QuikChange ^TM The instructions for the site-directed mutagenesis kit (Agilent) were carried out. Thus, the two mutagenic primers for each exchange are designed to be complementary to the target plasmid site. Both the forward and reverse primers contained the desired mutation and annealed to the same position on the opposite strand of the plasmid. During QC-PCR, the mutagenic primer was extended using a non-strand displacement clonotype Pfu DNA polymerase (Agilent) to generate a nicked circular strand. The non-mutant parent DNA template identified by its methylation site is digested by adding the restriction enzyme dpnl (NEB) to the final PCR reaction mixture. The nicked dsDNA is transformed into e.coli cells, where the nicks are repaired during replication. Primers were designed according to the Agilent kit instructions described above, were 25-45 bases in length (37 bases for forward primer and 41 bases for reverse primer), and had a melting temperature (Tm) of about 78 ℃. The desired mutation must be located in the middle of the primer, with the correct sequence of about 10-15 bases on both sides. All primers were synthesized by Metabion, planegg-Steinkirchen.

The QC-PCR reaction mixtures were combined on ice in 0.2mL reaction tubes. The clonotype Pfu DNA polymerase was added shortly before the start of PCR amplification. The thermal cycling conditions are adjusted to accommodate the length of the DNA template and the type of mutation desired. For a single amino acid exchange in the 4.8kb plasmid, an extension time of 10 minutes and 16 PCR cycles were selected. A negative control without template DANN was prepared for each reaction. After temperature cycling, 2 μl dpnl was added directly to each amplification reaction to digest the parental plasmid DNA. The samples were incubated at 37℃for 1 hour, and then the restriction enzymes were subjected to a 20-minute heat-inactivation step at 80 ℃. The PCR samples were then analyzed on a 1% (w/v) agarose gel.

Excision of the desired band and purification of plasmid DNA and transformation as describedExpress competent E.coli (high efficiency) cells. A single colony of each construct was picked and DNA isolated. Sequence analysis was performed to verify the inserted mutations.

Small-scale protein test expression

Expression experiments were performed on small-scale tests on the various TtSlyD-Fc-III-4C proteins to assess the amount of protein produced and the solubility of the different variants. Expression is implemented in a 96-well deep hole module (DWB) Express competent escherichia coli (high potency), BL21 derivatives. An overnight starter culture was prepared by inoculating 1.2mL LB medium (50. Mu.g/mL kanamycin) from glycerol stock. Plates were incubated on an orbital shaker at 750rpm for 16 hours at 37 ℃. The next day, the expression culture (1.2 mL fresh Super Broth (SB) medium) was inoculated with 50. Mu.L of the preculture. After reaching the exponential growth phase (OD 600 of about 1-1.2), expression was induced by addition of IPTG to a final concentration of 0.5 mM. After five hours of incubation at 37 ℃, cells were harvested by centrifugation (4000 rcf,20 minutes). Samples (pre-induction preI and post-induction postI) were taken for subsequent SDS-PAGE analysis before inoculation and before cell harvest, respectively. The optical density of the bacterial culture was determined and the sample volume was adjusted. The cell suspension was centrifuged (8000 rcf,3 min) and the cells resuspended in 32.5. Mu.L 10mM Tris pH 8.0,4%SDS,2%2-mercaptoethanol to obtain a crude cell extract suitable for SDS-PAGE.

Mechanical cell disruption

The cell pellet was resuspended in 800. Mu.L of bead disruption buffer and transferred to a 2mL tube containing 150mg of 0.1-0.2mm glass beads. The solution was mixed with 50 μl of 1:1000 dilution defoamer 204 (Sigma-Aldrich) and the cells were mechanically disrupted using bead disrupter 24 (Omni International) according to the following procedure. mu.L of bacterial lysate was transferred to a 2mL tube and centrifuged (8000 rcf,3 min) to separate the soluble fraction from the insoluble fraction. The insoluble fractions were resuspended in 100. Mu.L of bead disruption buffer prior to application to SDS-PAGE. Protein expression levels and solubility were assessed via SDS-PAGE.

Medium-scale protein expression

To obtain sufficient biomass for subsequent protein purification, medium-scale expression cultures of the different TtSlyD scaffold variants were prepared. 5mL overnight cultures of each mutant (inoculated from glycerol stock) were used to seed 250mL cultures of SB medium containing 50. Mu.g/mL kanamycin. Cultures were incubated in a shaking incubator at 37℃and 250rpm until their OD600 reached 1-1.2. Expression was induced by addition of IPTG to a final concentration of 0.5 mM. After five more hours of incubation, the cells were harvested by centrifugation (6000 rcf,20 minutes). A 2mL sample of the final cell suspension was removed and the cells were mechanically disrupted to obtain a sample for subsequent SDS-PAGE analysis.

Chemical cell disruption

By adding 2mL of B-PER II PER mg of cells ^TM Bacterial protein extraction reagent (Thermo Fisher Scientific) to lyse cell pellet. Adding protease inhibitor phenylmethylsulfonyl fluoride (PMSF, PER mL of B-PER IIII) ^TM 3 μL 0.1M,Thermo Fisher Scientific) and DNaseI (Roche, PER mL B-PER IIII) ^TM A spatula tip, roche) to prevent protein degradation and reduce the viscosity of the lysate caused by unwanted DNA. The suspension was incubated on wet ice for 30 min and filled with 20mM Tris, 150mM NaCl (pH 7.5) (buffer A) to a final volume of 20mL. The lysate was centrifuged at 5000rcf for 15 minutes to separate the soluble fraction from the insoluble fraction. After resuspension of the insoluble fractions in 20mL of buffer a, samples were collected for SDS-PAGE. By Ni ²⁺ Affinity size exclusion chromatography further purified the supernatant containing the soluble protein.

Protein purification

Immobilized metal ion affinity chromatography (IMAC)

The scaffold proteins were initially purified via IMAC, first from Porth et al were formulated in 1975 (Porath, J. Et al, metal chelate affinity chromatography, a new approach to protein interaction. Nature,1975.258 (5536): pages 598-9). After cell lysis and centrifugation, the supernatant containing the soluble protein fraction was sterile filtered (0.2 μm) and applied directly to a nickel nitrotriacetate (Ni-NTA) gravity flow column (Pierce gravity flow column and filter apparatus loaded with Qiagen Superflow Ni-NTA agarose resin; column Volume (CV) of about 1 mL). Due to the metal ion (Ni ²⁺ ) And the 8 x-histidine side chains, capturing proteins with an affinity tag consisting of polyhistidine residues. Buffers and solutions used for purification are as follows.

Buffers and solutions for gravity flow purification. All buffers and solutions were sterile filtered (0.2 μm) and degassed prior to use. The pH was adjusted with HCl or NaOH.

Buffer/solution composition

Buffer A (sample buffer) 20mM Tris, 150mM NaCl (pH 7.5)

Buffer B1 (washing buffer) 20mM Tris, 150mM NaCl, 10 mM imidazole (pH 7.5)

Buffer B2 (washing buffer) 20mM Tris, 150mM NaCl, 50mM imidazole (pH 7.5)

Buffer C (elution buffer) 20mM Tris, 150mM NaCl, 250mM imidazole (pH 7.5)

Buffer D (regeneration buffer) 20mM Tris, 150mM NaCl, 500mM imidazole (pH 7.5)

Stripping solution 50mM EDTA, 1% SDS (pH 7.5)

Charging solution 100mM NiSO ₄

Storage solution 20% ethanol

Before use, the ddH is filtered and degassed ₂ The column was flushed with O. Equilibration was performed with 20CV of buffer A, followed by application of lysate supernatant. The non-specific and unbound proteins were removed by washing the column with 20CV of buffer B1 and 20CV of buffer B2. The 1mL fraction of protein was eluted with 11CV buffer C. The column was regenerated by washing with 10CV of buffer D. Respectively adding sample (flow through, FT), buffer B1 (wash out, WI) and bufferAfter liquid B2 (wash II, WII), 1mL of the column flow-through sample was collected. After three purification cycles, the column was stripped and charged as follows: first, 20CV ddH was added ₂ O, then 5CV of stripping solution containing chelating agent ethylenediamine tetraacetic acid (EDTA) was added to remove nickel ions. Then, 20CV ddH ₂ O was followed by 3CV 100mM NiSO ₄ To charge the column. By applying 20CV ddH ₂ O, then 20CV of storage solution was applied to prepare the column for storage.

Size Exclusion Chromatography (SEC)

After Ni-NTA chromatography, the protein solution was further purified via size exclusion chromatography. This technique allows separation according to molecular size and is applied in this work to remove oligomers and aggregates as well as low molecular components of the target protein. The system output is shown in a chromatogram showing the absorbance intensity in Absorbance Units (AU) over the retention volume or retention time (the volume/time required to elute the protein after injection). The signal intensity is proportional to the concentration of the eluting analyte. In the case of optimal separation, the multiple peaks or shifts on the chromatogram correspond to different components of the separated sample, and the molecular size can be directly specified by comparison with protein standards of known composition. The "peak integration" function of UNICORN 6.3 control software is used to identify and measure a variety of curve features, including peak area, retention time, and peak width. The required baseline is automatically calculated.

Prior to application, the protein sample was concentrated to a final volume of about 5mL and used The Avant chromatography system was manually loaded via capillary loop (5 mL) onto GE Healthcare HiLoad/600 superdex 75pg column 7. Before use, 1.5CV ddH was used ₂ O removes the storage solution. The column was equilibrated and the 2mL fractions of protein were eluted with 1.5CV running buffer in 96 well DWB at a flow rate of 1 mL/min. The run was performed at Room Temperature (RT) and monitored at a wavelength of 280 nm. Then, using ddH ₂ O and storage solution rebalance the column. Protein purity was confirmed by SDS-PAGE and calibrated according to GE HealthcareThe Molecular Weight (MW) is determined from the quasi-curve. The desired peak fractions were combined and concentrated. Aliquots of sufficient volume were flash frozen in liquid nitrogen and stored at-80 ℃.

Buffers and solutions for size exclusion chromatography. All buffers and solutions were sterile filtered (0.2 μm) and degassed prior to use. The pH was adjusted with HCl.

Buffer/solution composition

Running buffer 20mM Tris, 150mM NaCl (pH 7.5)

Storage solution 20% ethanol

Buffer exchange and protein concentration

Prior to SEC, protein samples eluted from the Ni-NTA gravity flow column were buffer exchanged and concentrated to remove residual imidazole present in the elution buffer. Buffer exchange was achieved by dialysis. Transferring the protein solution to a molecular weight cut-off (MWCO) of 3500Da 3 dialysis membrane (Spectrum Laboratories) and incubated overnight at 4℃in 5L running buffer with continuous stirring. Application of dialyzed proteins to 5000Da MWCOThe column was concentrated by centrifugation (Sartorius) and centrifuged to obtain a final volume of about 5 mL. After SEC, via 5000Da MWCO +.>A20-centrifuge concentration column (Sartorius) concentrates the protein pool.

Generation of immunoaffinity chromatography columns

Within the scope of the present invention, novel immunoaffinity columns for antibody purification have been developed. The TtSlyD-Fc-III-4C scaffold affinity peptide chimeras previously described were used as capture molecules/ligands for IgG and permanently coupled to the chromatography column matrix.

Briefly, for immunoaffinity chromatography, a crude solution containing the desired antibody (e.g., cell culture supernatant) is applied to a column. Capturing the antibody by means of an affinity ligand contained in the column matrix; contaminants are removed by vigorous washing and the immunoglobulins are eluted by adding appropriate buffers.

The immobilization of the Fc specific ligand was performed by covalent coupling of the primary amino group to N-hydroxysuccinimide (NHS) activated highly crosslinked agarose beads contained in a HiTrap NHS activated HP column (1mL,GE Healthcare). The coupling procedure is performed according to the manufacturer's protocol. The required buffers are shown below. The ligand was dissolved in standard coupling buffer and concentrated to 1mL, resulting in a final concentration of 0.5-10 mg/mL. The column was washed with ice-cold 1mM HCl to remove the isopropanol present in the manufacturer's storage buffer. 1mL of ligand solution was injected manually and the column incubated at 25℃for 30 minutes. Then the column and the chromatographic system are combined Avant, ge healthcare) and wash out the ligand solution with 3CV of standard coupling buffer, thereby collecting column flow-through. To inactivate any excess active groups not coupled to the ligand and wash out non-specifically bound ligand, 6CV buffer A, 6CV buffer B and the other 6CV buffer A are injected. The eluate was collected and the column incubated for 30 minutes at room temperature. Thereafter, 6CV of buffer B, 6CV of buffer A and 6CV of buffer B were injected, and the eluate was collected as well. Finally, the pH was adjusted by applying 10CV of binding buffer. If the column is not used immediately, it is washed with 5CV of storage buffer and stored at 8 ℃.

Buffer for use in the generation of immunoaffinity chromatography columns. all buffers were sterile filtered (0.2 μm) and degassed prior to use. The pH was adjusted with HCl or NaOH.

Buffer composition

Standard coupling buffer 0.2M NaHCO ₃ ，0.5M NaCl(pH 8.3)

Buffer A0.5M ethanolamine-HCl, 0.5M NaCl (pH 8.3)

Buffer B0.1M NaOAc,0.5M NaCl (pH 4)

Storage buffer 0.05M Na ₂ HPO ₄ ，0.1％NaN ₃ (pH 7)

Binding buffer 20mM Tris,150mM NaCl (pH 7.5). The binding buffer corresponds to the SEC running buffer. Purified scaffold proteins were dissolved in this buffer for long term storage.

To assess the maximum coupling capacity of the column matrix, increasing ligand concentrations from 1.5-3mg/mL were applied. The flow-through containing excess ligand was analyzed by SDS-PAGE and the coupling efficiency was assessed as follows: the PD-10 stripper column (GE Healthcare) was equilibrated with 25mL 0.1M NaH2PO4, 150mM NaCl (pH 7) (equilibration buffer 8) and then 0.5mL of eluate was added. Elution was performed using an equilibration buffer in the following two-step procedure: first 2mL of equilibration buffer was added to remove salts and other low molecular weight components, then 1.5mL equilibration buffer was added to elute the desired high molecular weight protein. Using NanoDrop ^TM The absorbance of the eluted fraction was measured by OneC micro UV-Vis spectrophotometer (Thermo Fisher Scientific). The coupling efficiency was calculated. See fig. 2.

Evaluation of column parameters

TtSlyD-Fc-III-4C immunoaffinity chromatography columns were generated and used to evaluate the required parameters such as affinity, chemical stability and reusability using commercial high purity rabbit immunoglobulin G (rbIgG) supplied by Sigma-Aldrich. UsingThe Avant chromatography system was run for testing. The affinity column was equilibrated with 20CV of equilibration buffer prior to application of rbIgG. rbIgG was diluted to a final concentration of 1 mg/mL. 1mL of sample was injected manually using a 1mL syringe and the column was incubated at 8℃for 30 minutes to allow binding to the ligand. Unbound antibody was removed by adding 10CV of wash buffer. In 96 well DWB, igG from the 0.2mL fraction was eluted using 5CV of elution buffer. To neutralize the eluate, 40 μl of 1M arginine solution was added per well. The column in situ wash (CIP) procedure was performed by first washing with 10CV of regeneration buffer, followed by a 15CV of 100mM NaOH rinse. If the column is not immediately reused, it is equilibrated in storage buffer and stored at 8 ℃.

Buffers and solutions used for rbIgG purification are as follows. All buffers and solutions were sterile filtered (0.2 μm) and degassed prior to use. Adjusting pH with HCl or NaOH

Buffer/solution composition

Balanced buffer 50mM Tris-HCl,0.05%20(pH 7.8)

Washing buffer 50mM Tris-HCl (pH 6.0)

Elution buffer NH ₄ Ac-AcOH(pH 3.4)

Regeneration buffer NH ₄ Ac-AcOH(pH 2.2)

CIP solution 100mM NaOH

Storage buffer 0.05M Na ₂ HPO ₄ ，0.1％NaN ₃ (pH 7)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Protein fractions were analyzed by SDS-PAGE under reducing and non-reducing, reducing or non-reducing conditions. Mixing the sample with a proper amount ofLDS sample buffer (4X) (Thermo Fisher Scientific) and +.>The reducing agent (10 x) (Thermo Fisher Scientific) (for reducing gels) or water (for non-reducing gels) was mixed to obtain a total volume of 50 μl and incubated in a thermocycler at 95 ℃ for 10 minutes. At->Gel runs were performed on Bis-Tris 4-12% gel (Thermo Fisher Scientific) using 15. Mu.L/10. Mu.g sample per lane and 5. Mu.L Novex ^TM Sharp pre-stains protein ladder (Thermo Fisher Scientific) to estimate the molecular weight of protein bands. Electrophoresis was performed at a constant voltage of 200V. InstantBuue for gel ^TM Protein stain (Expedeon) staining and staining in ddH ₂ Decolorized in O overnight. The image is in Chemidoc Mp ^TM Photographed in a device (Bio-Rad).

Spectrophotometric determination of protein concentration

Using NanoDrop ^TM The OneC micro UV-Vis spectrophotometer (Thermo Fisher Scientific) determines the concentration of purified protein after SEC by spectrophotometry. An appropriate dilution buffer was used as a blank. Measurement was performed using a universal reference setup based on 0.1% (1 mg/mL) protein solution, yielding an absorbance of 1.0A at 280nm (where the optical path length is 1 em). The protein concentration was calculated according to the beer-lambert law, followed by consideration of the specific protein absorbance at 280 nm. The corresponding Absorbance Units (AU) were calculated by Vector NTI (Thermo Fisher Scientific) software.

Surface Plasmon Resonance (SPR)

The affinity and binding kinetics of the generated TtSlyD-Fc-III-4C mutants were evaluated by SPR using a Biacore biosensor system. The Biacore system enables real-time monitoring of intermolecular interactions between ligands immobilized on the gold surface of the sensor chip and analytes free in solution and passing through the ligands (Healthcare, g., biacore ^TM Assay Handbook 29-0194-00 AA). The interaction of the binding partners generates an SPR signal (response), which is measured in Resonance Units (RU) and shown as a graph over time (sensorgram).

With respect to this process, briefly, the analyte is injected onto the chip to interact with the immobilized ligand. Variations in the concentration of molecules on the chip surface can result in variations in refractive index, monitored in Response Units (RU). The continuous buffer flow triggers dissociation of the analyte, which is then completely removed (chip regeneration) and a new analysis cycle is started. The obtained SPR data is fitted to a mathematical model to derive kinetic parameters such as association rate constant ka (M-1 s-1) and dissociation rate constant kd (s-1).

Interaction kinetics were checked by monitoring the different analyte concentrations over time. Kinetic parameters such as association (ka) and dissociation rate constants (kd) are evaluated against a mathematical model. One model used is the "Langmuir interaction model" (O' Shannessy, D.J. et al, determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods. Animal Biochem,1993.212 (2): pages 457-68), assuming a 1:1 interaction, wherein one ligand molecule interacts with one analyte molecule. The kinetic model of "divalent analyte binding" assumes a divalent analyte in which one analyte molecule may bind to one or both ligand molecules. Kinetic analysis experiments performed in the present invention were performed with multi-cycle kinetics, testing each analyte concentration in a single cycle and regenerating the chip surface after each cycle. Successful regeneration means that the bound analyte is removed without isolating the ligand or limiting its activity. Therefore, regenerative reconnaissance is performed prior to kinetic measurements.

Regenerative reconnaissance

Regeneration scouts were performed using the Biacore 3000 system provided by GE Healthcare and CM5 chips. The chip showed carboxymethylated dextran covalently attached to the gold surface. The ligand was immobilized on the chip surface via amine coupling, in an attempt to achieve ligand densities around 3000 RU. The analyte (human IgG,300 nM) was diluted in sample buffer. Regeneration was tested according to the following protocol. In the first cycle, 300nM human IgG was injected. The bound antibody was replaced by adding system buffer and two 1 minute pulses of 10mM glycine pH 2. This cycle was repeated six times and then two additional cycles were performed using 10mM glycine (pH 1.75 and pH 1.5), respectively.

Operation procedure of regeneration reconnaissance

Kinetic screening

Kinetic measurements were performed using the Biacore 3000 and Biacore 8K systems with C1 chips (GE Healthcare). The chip assembly is similar to the C5 chip except that dextran threads are absent. The scaffold is immobilized via amine coupling, i.e. the protein is covalently linked to carboxymethyl groups present on the chip surface. The chip surface was rinsed with 270nM analyte prior to measurement to saturate (modulate) the free binding sites remaining on the chip surface and regenerated with glycine buffer (pH 1.75). Briefly, scaffold proteins were immobilized on Biacore C1 chips via amine coupling. Different concentrations of IgG were injected and the chip was regenerated after each cycle.

Immobilization of ligand

The desired ligand was diluted to a final concentration of 5. Mu.g/. Mu.L in sample buffer. The chip surface was cleaned by two one minute injections of wash buffer and finally filled with system buffer. The reactive groups were activated by applying 40. Mu.L NHS/EDC (50% mixture) at a flow rate of 20. Mu.L/min. The ligand solution was injected at a flow rate of 50 μl/min to achieve RU response suitable for subsequent performance. (RU values of no more than around 100 to avoid steric crowding effects at high analyte concentrations). Excess NHS activated ester was quenched by exposing the chip to 100. Mu.L of 1M ethanolamine hydrochloride (EA-HCl) solution (pH 8.5) at a flow rate of 20. Mu.L/min.

Kinetic screening

The analyte is gradually diluted in the sample buffer to achieve the desired concentration. The specified concentrations of IgG (human or rabbit IgG, sigma-Aldrich) were injected onto the chip at 60. Mu.L/min or 40. Mu.L/min and 25℃or 37℃respectively. At the end of each analysis period, the chip surface was regenerated using two 1 minute pulses of 10mM glycine pH 1.75.

Results

In the present invention, peptide transplantation is accomplished by generating a DNA vector construct encoding the FKBP domain of TtSlyD protein and Fc-III-4C peptide. The appropriate sequences were inserted into the prokaryotic expression vector pQE80-Kan, allowing DNA amplification and expression in E.coli. IgG affinity columns were generated by coupling recombinantly produced TtSlyD-Fc-III-4C to a commercially available NHS agarose matrix. Principle verification purification experiments were performed using high purity IgG solutions. The chemical stability and reusability of the column matrix was further assessed by exposure to extreme alkaline conditions and repeated column runs. Maturation of the Fc binding moiety is accomplished via Quick change PCR (QC-PCR) for the purpose of promoting milder elution conditions. Tryptophan 11, which plays a key role in the interaction of peptides with the Fc portion of IgG, was replaced with twelve amino acids exhibiting different biochemical properties. Expression levels and solubilities of the different variants were pre-tested in 96-well plates.

The selected mutants were expressed in medium-scale form (250 mL) and purified via immobilized metal ion affinity chromatography (IMAC) and Size Exclusion Chromatography (SEC) of the TtSlyD-FcIII-4C scaffold. To simplify expression and purification of potential column ligands, a library of chemically synthesized Fc-III-4C peptides lacking bridge-forming cysteines was created and screened for high affinity variants in the HT-NimbleGen microarray. Ten optimal binders were identified and transplanted onto TtSlyD scaffold, expressed and purified. The interaction of the resulting protein variants with IgG was assessed by kinetic screening using Surface Plasmon Resonance (SPR) techniques.

Grafting of Fc-III-4C peptide onto FKBP domain of Thermus thermophilus Slyd protein was achieved by molecular cloning. Sequence analysis revealed the correct sequence of the final pQE80Kan-TtSlyD-Fc-III-4C expression construct.

TtSlyd scaffolds carrying the original unchanged Fc-III-4C insert were expressed on a 250mL scale and then purified via immobilized metal ion affinity (IMAC) and preparative Size Exclusion Chromatography (SEC). In a proof of principle experiment, proteins were coupled to the column matrix to assess their potential as affinity ligands for IgG-FPLC purification.

Ligand-matrix combination was performed by covalently coupling primary amine groups present in the scaffold protein to NHS-activated highly cross-linked agarose beads contained in HiTrap NHS-activated HP column (1mL,GE Healthcare). Primary amine groups are found in lysine residues and the N-terminus of proteins. Lysine residues tend to act as conjugation sites because they typically have exposed positions. The TtSlyD FKBP domain has five lysine residues that may react with NHS ester groups. A coupling efficiency of 93% was achieved.

The IgG-Fc affinity scaffold was attached to the column via random amine coupling to create a heterogeneous matrix composition. The affinity peptide ring is not necessarily opposite the analyte solution. The binding capacity of the final column may thus be affected or even compromised. One possible strategy is Thiol-directed immobilization by introducing a single cysteine residue in the C-terminal part of the scaffold protein, followed by coupling to a Thiol-containing solid matrix (Ljungquist, C.et al, thio-directed immobilization of recombinant IgG-binding acceptors. Eur J Biochem,1989.186 (3): pages 557-61). Furthermore, potential effects of ligand density must be considered.

In principle verification experiments. The IgG binding ability of one of the generated NHS-sepharose-TtSlyd-Fc-III-4C columns was tested. Purification of rabbit IgG via NHS-Sepharose-TtSlyd-Fc-III-4C column did not show ligand leakage. Results are consistent with those described by Gong et al (Gong, y. Et al, development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein detection. Bioconjug Chem,2016.27 (7): pages 1569-73), which have shown that the efficiency of enrichment of rabbit IgG on Fc-III-4C-sepharose beads is comparable to that of protein a beads at a selected pH, which successfully demonstrates the competitive IgG binding capacity of scaffold affinity chimeras compared to commercially available affinity matrices.

To further assess the chemical stability of the ligand and the reusability of the column matrix, the purification process was automated and the scope of the protocol was narrowed to run multiple cycles consecutively. The yield of eluted antibody was almost identical for all runs as confirmed by spectrophotometric determination of protein concentration and monitoring peak area. Even after 30 cycles, a reproducible chromatogram without significant peak shifts or broadening can be recorded.

To test for improvement of IgG elution conditions by engineering Fc-III-4C binding sites, the Fc binding moiety was altered by QC-PCR in order to selectively screen protein variants exhibiting the following characteristics: a rapid association rate that facilitates antibody capture, and a rapid dissociation rate that allows for antibody separation under relatively mild pH conditions.

Tryptophan, one of the major driver of binding and strongest interaction molecules between Fc-III-4C and IgG-Fc portions, was exchanged with twelve amino acids (glycine, serine, alanine, arginine, lysine, glutamic acid, lysine, threonine, asparagine, glutamine, tyrosine, or histidine). As far as reducing the binding strength, the introduction of histidine residues has been successfully applied to engineered affinity ligands (Watanabe, H. Et al Optimizing pH response of affinity between protein G and IgG Fc: how electrostatic modulations affect protein-protein interactions. J Biol Chem,2009.284 (18): pages 12373-83). Thus, electrostatic repulsion between the ligand and the antibody promotes dissociation of the antibody under relatively mild acidic conditions. Sequence analysis confirmed the successful insertion of all the desired mutations.

For further purification attempts, expression of all protein variants was performed in 250mL form as described above. After harvesting the cells, the cell pellet was subjected to chemical disruption, centrifugation, sterile filtration, and the soluble fraction was applied to a Ni-NTA gravity flow column. The desired protein was eluted by dissolving the binding of the polyhistidine tag present in the scaffold protein to a nickel column immobilized at a concentration of 250mM imidazole. The monomer fractions were pooled and used for further analysis. The ratio of the protein monomers obtained with respect to the amount of the initially loaded protein ranges from 12% (W to a mutation) to 97% (wild type). The results show that tryptophan at position 11 is critical to the stability of the monomeric protein. The purpose of the alternating Fc binding sites is to generate protein variants that are characterized by sufficiently high affinity and at the same time exhibit rapid association/dissociation to enable protein elution under mild pH conditions for subsequent IgG purification processes. However, all variants tested revealed affinities and association/dissociation rates within the same range.

In other experiments, cysteines were replaced with alternative amino acids to investigate whether these linkages were critical for loop stability and affinity of the protein for IgG-Fc.

The cysteine residues of the Fc-III (DCAWHLGELVWCT, seq ID NO: 17) and Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1) peptides were randomized and the resulting cyclic peptide library was tested for binding to mAbs in a high throughput NimbleGen microarray. For Fc-III peptides only one high affinity variant, the wild type peptide, can be identified.

For Fc-III-4C 299 variants were identified with IgG-Fc affinity superior to the wild-type peptide. However, in all these high affinity variants, only two internal cysteines were found to remain intact. Variants lacking internal cysteines all belong to low intensity variants, indicating that internal cysteines are absolutely necessary. In contrast, terminal disulfide bridges are not necessary to maintain the affinity of the peptide for IgG, and may even improve binding strength in some cases.

Several positions can be identified in which amino acid exchanges cause changes in intensity and binding affinity. The most notable position is the leucine residue at position 7 in Fc-III-4C. The corresponding substitution of glutamine significantly improved the affinity. Ten high affinity variants were grafted onto TtSlyD FKBP domains and further studied for purification profile and IgG-Fc affinity. Variants lacking an external cysteine are referred to as Fc-III-XC. Only the first 50 variants exhibiting the highest affinity against CD44 antibody were analyzed. At the 3' end, a clear trend towards acidic amino acids (aspartic acid and glutamic acid) as well as polar amino acids (asparagine) can be identified. However, the most abundant cysteine substitution at the C-terminus of the peptide is proline. Proline has a low conformational freedom and thus may stabilize the ring structure, resulting in a higher affinity. The occurrence of 5' -substitution was quite random, and no obvious trend was observed. In most cases, cysteines are simply deleted or exchanged with the same amino acids named above. Sequencing data revealed the correct sequence for all the desired constructs.

As expected, the TtSlyD-Fc-III-XC mutants were less prone to aggregation than variants exhibiting four cysteine residues. The cysteine-deficient protein variants were tested for molecular interactions and affinities for both rabbit IgG and human IgG at different temperatures (25 ℃ and 37 ℃).

KD values in the range of 8-43nM were obtained in the interaction analysis with hIgG. For rbIgG, KD values range between 15 and 52 nM. According to Gong et al, KD values of chemically synthesized Fc-III-4C peptides for human beings2.45nM, 5.67nM for rabbit IgG, representing higher affinity for Fc than native protein A conjugate. TtSlyD-Fc-III-2C_Hit No.4 was identified as the most promising candidate for purification of IgG via FPLC. It exhibits a rapid association rate (t/2 diss=36 minutes) and a relatively high binding affinity (K _A ＝1.09×10 ⁸ 1/M) (for hIgG, measured at 25 ℃). Covalent attachment of the affinity protein to a matrix (e.g., agarose resin) will reveal its full potential as a chromatographic ligand.

Claims (15)

1. A chimeric IgG-Fc binding ligand polypeptide comprising a protein fragment of SlyD wherein the IF domain thereof is replaced by the affinity peptide Fc-III-4C (CDCAWHLGELVWCTC, SEQ ID NO: 1) or an Fc-III-XC variant (X ₁ DCAWHLGELVWCTX ₂ SEQ ID NO: 3) Replacement, wherein X ₁ Missing or independently selected from the group of: C. d, P, E and K, and X ₂ Independently selected from the group of: C. q, P and E.

2. The chimeric IgG-Fc binding ligand polypeptide according to claim 1, wherein said SlyD is from a thermus species, such as, for example, thermus thermophilus.

3. The chimeric IgG-Fc binding ligand polypeptide according to claim 1 or 2, comprising the sequence:

4. the chimeric IgG-Fc binding ligand polypeptide of any one of claims 1 to 3, further comprising a C-terminal amino acid tag, such as, for example, his ₈ And (5) a label.

5. The chimeric IgG-Fc binding ligand polypeptide according to any one of claims 1 to 4, wherein said polypeptide exhibits high affinity binding to an IgG class selected from the group consisting of: human, rabbit, mouse, rat, pig, goat, horse and cow.

6. A bivalent conjugate molecule comprising two fused, preferably end-to-end fused, chimeric IgG-Fc binding ligand polypeptides according to any one of claims 1 to 5.

7. The chimeric IgG-Fc binding ligand polypeptide according to any one of claims 1 to 5 or the bivalent conjugate molecule according to claim 6, coupled to a solid support, such as a solid matrix material, such as beads and/or a column matrix.

8. The coupled ligand or conjugate molecule according to claim 7, wherein the coupling is via the lysine side chain of SlyD with a NHS ester contained in an agarose matrix.

9. A method for producing a chimeric IgG-Fc binding ligand polypeptide according to any one of claims 1 to 5, comprising recombinant expression of the ligand polypeptide in a suitable host cell such as e.coli, or comprising chemical synthesis of the ligand polypeptide.

10. The method of claim 9, further comprising the step of coupling the chimeric IgG-Fc binding ligand polypeptide to a solid support, such as a solid matrix material, such as beads and/or a column matrix.

11. A method for purifying an immunoglobulin, the method comprising contacting a solid support having the chimeric IgG-Fc binding ligand polypeptide of any one of claims 1 to 5 or the bivalent conjugate molecule of claim 6 coupled thereto with the immunoglobulin, and eluting the immunoglobulin from the chimeric IgG-Fc binding ligand polypeptide or the bivalent conjugate molecule as appropriate.

12. The method of claim 11, comprising Fast Protein Liquid Chromatography (FPLC).

13. The method of claim 11 or 12, wherein the elution conditions for the immunoglobulin are milder compared to a solid carrier material comprising protein a coupled thereto.

14. The method according to any one of claims 11 to 13, comprising a chemical regeneration step for a solid support material comprising protein a coupled thereto using conditions that are harsher than the solid support material.

15. Use of the chimeric IgG-Fc binding ligand polypeptide according to any one of claims 1 to 5 or the bivalent conjugate molecule according to claim 6 for purifying immunoglobulins or for screening and selecting peptide conjugates against a predetermined target molecule.