Protein L – Ypatent

See also Continuous Chromatography under Chromatography, Column arrangement.

Commercial Protein L Resins:

Capto L (GE Healthcare Life Sciences) Data file 29-0100-08 AB) combines a rigid, high flow agarose matrix with the immunoglobulin binding recombinant protein L ligand, which ahs strong affinity for the variable region of an antibody’s kappa light chain. It is thus suitable for capture of a wide range of antibody fragments such as Fabs, scFv and Dabs. The average ligand density is 10 mg/mL and particle size is 85 um.

Capto L (Data file 29-0100-08 AC GE, 2012-2014) discloses a recombinant Protein L with 4 binding domains that binds to the variable region of the kappa light chain of immunoglobulins and their fragments.

Tosoh Bioscience Toyopearl AF r-protein L-650F resin is based on polymethacryalte partciles. It offers a static binding capacity (SBC) fo 54 mg/mL for Fab fragments and remains stable at pH 2-12. Muller, “Intensificaiton of Fab-fragmetn Purification, Multicolumn chromatography using prepacked prtoein L columns”, BioProcess international, June 2023, 21(6)).

Introduction:

Many Gram-positive pathogenic bacteria have evolved cell-surface immunoglobulin-binding proteins, which may enable them to coat their cell surfaces with host protein and evade the immune response. The best characterized are staphylococcal protein A and streptococcal protein G, which interact with the immunoglobulin heavy chain, principally at the Fc CH2-CH3 interact, although SPG also interacts with FabCH1. Another protein, peptostreptococcal protein L (PPL) has been reported to be released form the bacterail cell wall by solubilization with proteolytic and muranolytic enzymes. It is expressed in about 10% of Peptostreptococcus magnus isolates and has been implicated in the virulence of these bacteria. (Murphy, Molecular Microbiology 12(6): 911-920, 1994).

Protein L is a cell surface protein expressed by Peptostreptoccocus magnus, which binds to the variable light chains of immunoglobulins without interfering with antigen binding. It can be used for purification of mammalian antibodies of all classes in contrast to the Ig-binding proteins protein A and protein G.

Both peptococcus and peptostreptococcus have been reported to produce Protein L, which binds to the Kappa light chain of human immunoglobulins. (Trowern, US 6,162,903).

Antibody fragmetns such as FAB, ScFvs and nanobodies have mereged as increasinly important therapeutic and diagnostic alternatives to full lenght mAbs. Whereas Mab downstream processing is well established and easy to scale based on protein A capture, the purificaiotn of antibody fragmetns is just on the verge of standardized prcoessing. The most promising candidate for effective capture of those containing a kappa light chain is protein L affintiy chromatography in the first step of downstream prcoessing. (Muller, “Intensificaiton of Fab-fragmetn Purification, Multicolumn chromatography using prepacked prtoein L columns”, BioProcess international, June 2023, 21(6)).

Unpredictability

Gore (WO 00/15803) disclsoes a Protein L mutant modified by an amino acid at one or more positions 39, 53 and 57 and/or by an amino acid instert between positions 59 and 60 such that the dissociation costant (dk) with respect to human immunogloubilin k-chain is 400 nM or more at pH 8. Gore further discloses that the rate of dissociation of the Y53F PpL k chain complex was affected by pH and dissociated faster at lower pH values. This was not the case for Y64W PpL, which dissociated faster at pH 9. Of note, Gore teaches that specific substitutions at positions 39, 53 and 57 and an insertion between positions 59 and 60 markedly affected the binding affinity. Some of the other subsitutions did not result in expression of any poolypeptide, potentially due to instability of the mutated polypeptide. (see Table 3).

Where Protein L binds:

Protein L binds to antibodies from a wide range of species including about 50% of human and 75% of mouse antibodies through the Vk region. The location of this unique binding site in the framework region of the light chain of antibodies allows protein L to bind an alternative subset of immunoglobuilins compared to protein A and G and also to bind the range of antibody fragment such as scFv, Fab and single domains, disulphide bonded Fv, a Fab fragment and a F9ab)2 fragment used in antibody engineering, if they have the correct K framework. Protein L has been found to bind to Vk of subgroups I, III and IV. (Enever 9WO/2005/033130).

Protein L binds human Fab via VL kappa 1, 3, and 4 but does not bind to VL kappa 2 and none of the VL domains of the lambda isotype. (Hermans US13/982970, published as US 2013/0337478)

Structure of Protein L:

Structure Depends on Peptostreptococcus Magnus: Strain:

Protein L (PpL) contains a plurality of VL-k-binding domains consisting of 70-80 residues. Yoshida Hyogo (US 15/660,365, published as US 2018/0016306). The number of FL-k-binding domains and the amino acid sequence of each domain are different depending on the kind of Protein L strain. For example, the number of Vl-k-binding domains in PpL of Peptostrepococcus magnus 312 strain is five, and the number of VL-k-binding domains in PpL of Peptostreptoccus mangus 3316 strain is four. Yoshida, Hyogo, (US 15/660, 373, published as US 2017/327535).

Each of the VL-k-binding domains of PpL312 are referred to as B1, B2, B3, B4 and B5 domain in the order from the N-terminal and each of the VL-kbinding domains of PpL3316 are referred to as a C1, C2, C3, C4 domain in teh order from the N-terminal Yoshida Hyogo (US 15/660,365, published as US 2018/0016306),

The gene for protein L expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus has been cloned and sequence. Teh gene translates into a protein of 719 amino acid residues. Following a signal sequence of 18 amino acids and a NH2-temrinal region (“A) of 79 residues, the molecule contains five homlogogus “B” repeats of 72-76 amino acids each. Furtehr, toward the COOH terminus, two additional repeats (“C”) are found. These are not related to the “B” repeats, but are highly homologous to each other. After the C repeats (52 amino acids each), a hydrophilic, proline-rich putative cell wall-spanning region (W”) is found, followed at the COOH temrinal end by a hydrophobic membrane anchor (“M”). Fragmetns of the gene have been expressed, and the corresponding peptides analyzed for Ig binding activity. The B repeats are found to be responsible for the interaction with Ig light chains. (Kastern, J Biological Chemistry 267 (18): 12820-12825).

The two strains of P. magnus (312 and 3316) produce slightly different Protein L molecules. Strain 312 produces Protein L that contains five highly homologous Ig binding domains, wehreas strain 3316 produces a prtoein with four Ig-binding domains. (Housden, Biochemical Society Transactions, 31(3) June 2003).

312 Strain

Bjorck (US 5,965,390) discloses sequences of protein L including multiples of the domains B1-B5 which bind to light chains in Ig.

B5 domain of Strain 312:

Yoshida, Hyogo, (US 15/660, 373, published as US 2017/327535) discloses an affinity matrix based on the beta5 domain of Protein L dervied from Peptostreptococcus magnus 312 strain. Yoshida, Hyogo (US 15/660,373 published as US 2017/0327535; see also US Patnet Application No: 16/176,090, published as US 2019/0119362) also teaches the beta5 domain of Protein dervied from the 312 strain with an N-terminal deletion).

3316 Strain:

A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof have been expressed in E coli. The four C units were shown to be responible for binding to immunoglobulin and the four D units for binding to albumin. The protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. (Murphy, Molecular Microbiology 12(6): 911-920, 1994).

Protein L Mutants:

Substitions positions 39 53, 57; insertion between 59 and 60:

Mutations of Magnus 3316 Strain

–Substitution positions 7, 13, 22 and 29

Majima (15/770,150, published as US 2018//0305414) discloses a high binding capacity for an immunoglobulin kappa chain having excellent alkali stalisty by modifying an amino acid sequence of Protein L dervied from Peptostreptococcus magnus 3316 strain. In particular, Majima discloses substituting at least two or more sites from positions 7, 13, 22 and 29 with a basic amino acid other than lysine (e.g., R or H) or a hydroxyl group continaing amino acid (e.g., S, T or Y) in one of the four immunoglobulin-binding domains including in Protein L dervied from Peptostreptococcus magnus 3316 strain.

Mutations of 312 Strain:

–N-terminal deletion:

Yoshida (US 16/176090, published as US 2019/0119362) also teaches using the B5 domain for affinity chromatography purification of an antibody with a kappa chain variable region. In one preferred embodiment, the beta 5 domain of Protein L has an N terminal deletion.

–Substituions positions 15-18:

Yoshida Hyogo (US 15/660,365, published as US 2018/0016306) also discloses a substitution of one of the amino acid residues at positions 15-18 in one of the Vl-k-binding domains B1-B5 and C1-C4 of PpL which results shifting the pH to a neutral side for dissociation on an affinity resin of the VL-k.

–Mutation of B1 Domain

Enevery (“engineering high affinity superantigens by pahge display” J. Mol. Biol. 347, 107-120 (2005) discloses mutants of the N-temrinal B1 domain of P. magnus strain 312 with a 14 fold increased affinity for humVk1 and a greater than tenfold increased affinity for huVkIII.

Conditions

Binding:

Washing:

–Carpylate (caprylic acid)

Suda (WO 2014/141150) discloses a method of purifying an antibody by adsorbing the protein/antibody onto a Protein L resin and washing with an aliphatic carboxylate to remove contaminants prior to elution. In one embodiment the wash buffer further includes about 1-500 mM sodium acetiate.

Elution:

–acetic acid sodium acetate

Hyogo (US16183308, published as US 2019/0211082) discloses a method of purifying an antibody that contains a kappa-chain variable region using Protein L on a carrier where the antibody is eluted from the matrix using an acetate buffer. In some ebmodiments, the acetate buffer is at a pH value of 2.5-4.0 and the concentration of an acetate ion in the buffer is 10-500 mM.