Combinations of different chromatography modalities with particularly refined conditions significantly reduce acid charge variants in a preparation of monoclonal antibodies. The process for reducing acid charge variants utilizes a combination of anion exchange and hydrophobic interaction chromatography, followed by cation exchange chromatography polishing, whereby the levels of acidic or basic charge species of the monoclonal antibodies may be modulated to a desired level.

The present invention aims at providing a specific antibody that can simply and rapidly detect Mycoplasma pneumoniae which is a causative bacterium of mycoplasma pneumonia, with high sensitivity, and also an immunological detection method and a kit containing the same antibody. The present invention makes it possible to diagnose infection with Mycoplasma pneumoniae more rapidly and specifically than the conventional method, by producing an antibody recognizing a specific epitope of P30 protein of Mycoplasma pneumoniae and performing an immunological detection using the antibody. Also, the present invention enables easy and rapid detection of Mycoplasma pneumoniae and diagnosis of infection with the same at a hospital or the like without need of specialized instruments or skilled techniques.

The invention features methods of identifying and validating affinity reagents, such as antibodies. The methods of the invention generally involve screening an antibody library by, for example, phage display on bacteria (e.g., E. coli) to identify particular antibody clones capable of binding a desired target polypeptide. Clones identified in this way can then be validated using yeast 2-hybrid. In some instances, antibodies identified by their capacity to binding a partial antigen can be validated by their capacity to bind to the full-length antigen. Validated clones can be further screened by additional rounds of phage display and/or yeast 2-hybrid. Between each round, additional variants of particular antibody clones can be generated and screened to identify variants that demonstrate higher binding affinity to the target of interest.

The invention features methods of identifying and validating affinity reagents, such as antibodies. The methods of the invention generally involve screening an antibody library by, for example, phage display on bacteria (e.g., E. coli) to identify particular antibody clones capable of binding a desired target polypeptide. Clones identified in this way can then be validated using yeast 2-hybrid. In some instances, antibodies identified by their capacity to binding a partial antigen can be validated by their capacity to bind to the full-length antigen. Validated clones can be further screened by additional rounds of phage display and/or yeast 2-hybrid. Between each round, additional variants of particular antibody clones can be generated and screened to identify variants that demonstrate higher binding affinity to the target of interest.

Disclosed herein is a phage-displayed single-chain variable fragment (scFv) library, that comprised a plurality of phage-displayed scFvs characterized with (1) a specific CS combination; (2) a specific distribution of aromatic residues in each CDR; and (3) a specific sequence in each CDR. The present scFv library could be used to efficiently produce different antibodies with binding affinity to different antigens. Accordingly, the present disclosure provides a potential means to generate different antigen-specific antibodies promptly in accordance with the need in experimental researches and/or clinical applications.

The present invention concerns a method of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of: a) providing a storage liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; b) permeating the separation matrix with the storage liquid; and c) storing the storage liquid-permeated separation matrix for a storage time of at least days. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

VH domain, in which: (i) the amino acid residue at position 112 is one of K or Q; and/or (ii) the amino acid residue at position 89 is T; and/or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

VH domain, in which: (i) the amino acid residue at position 112 is one of K or Q; and/or (ii) the amino acid residue at position 89 is T; and/or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and in which said VH domain contains a C-terminal extension (X)n, in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

The present invention relates to variant Fc-containing molecules, such as antibodies and Fc-fusion molecules, having glycosylation characteristics favorable to large-scale production of therapeutic molecules containing such variant Fc.

The present invention relates to variant Fc-containing molecules, such as antibodies and Fc-fusion molecules, having glycosylation characteristics favorable to large-scale production of therapeutic molecules containing such variant Fc.