Direct determination of antibody chain pairing

A method of determining chain pairing of an antibody fragment, comprising:

• ionizing the antibody fragment into precursor ions;
• determining a mass of the antibody fragment from a mass spectrum of the precursor ions;
• selecting one or more precursor ions for MS/MS analysis;
• dissociating by a first dissociation method, a light chain of the antibody fragment from an Fd portion of the antibody fragment to generate a first type of product ions of the light chain and a first mass spectrum of the light chain, and a first type of product ions of the Fd portion and a first mass spectrum of the Fd portion, wherein the Fd portion is derived from a heavy chain;
• dissociating by a second dissociation method, the light chain of the antibody fragment from the Fd portion of the antibody fragment to generate a second type of product ions of the LC chain and a second mass spectrum of the LC chain, and a second type of product ions of the Fd portion and a second mass spectrum of the Fd portion, wherein the first dissociation method cleaves a backbone of the Fd portion at a different location than the second dissociation method;
• comparing the first mass spectrum of the light chain to the second mass spectrum of the light chain to identify a first group of the first type of product ions that differ in mass from a second group of the second type of product ions by a constant value;
• comparing the first mass spectrum of the Fd portion to the second mass spectrum of the Fd portion to identify a first group of the first type of product ions that differ in mass from a second group of the second type of product ions by a constant value;
• determining, from the first and/or second mass spectrum of the light chain, an amino acid sequence of the light chain based on a mass difference between consecutive product ions of the first group and/or consecutive product ions of the second group;
• determining an amino acid sequence of the light chain based on a mass difference between consecutive product ions of the first group and/or consecutive product ions of the second group;
• determining, from the first and/or second mass spectrum of the Fd portion, an amino acid sequence of the Fd portion based on a mass difference between consecutive product ions of the first group and/or consecutive product ions of the second group;
• determining an amino acid sequence of the Fd portion based on a mass difference between consecutive product ions of the first group and/or consecutive product ions of the second group;
• comparing the determined sequence of the light chain and Fd portion with a database comprising amino acid sequences of light chain sequences and heavy chain sequences;
• and
• determining heavy chain and light chain pairing of the antibody fragment.

A method of determining chain pairing in an intact antibody or antibody fragment, comprising:

• ionizing the intact antibody or antibody fragment into precursor ions;
• determining a mass of the intact antibody or a mass of the antibody fragment from a mass spectroscopy spectrum of the precursor ions;
• selecting one or more precursor ions for MS/MS analysis;
• dissociating at least a light chain from the intact antibody by cleaving one or more disulfide bonds linking a heavy chain and the light chain of the intact antibody, or dissociating an intact light chain and a heavy chain fragment from the antibody fragment by cleaving disulfide bonds linking the heavy chain fragment and the light chain of the antibody fragment;
• determining a mass of the light chain of the intact antibody from a mass spectroscopy spectrum of a product ion of the light chain of the intact antibody, or determining a mass of the light chain of the antibody fragment from a mass spectroscopy spectrum of a product ion of the light chain of the antibody fragment and determining a mass of the heavy chain fragment from a mass spectroscopy spectrum of a product ion of the heavy chain fragment of the antibody fragment;
• comparing the identified mass of the light chain and the mass of the intact antibody with a database comprising amino acid sequences of light chain sequences and heavy chain sequences or comparing the identified mass of the light chain and the mass of the heavy chain fragment with a database comprising amino acid sequences of light chain sequences and heavy chain sequences;
• and
• determining heavy chain and light chain pairing of the intact antibody or antibody fragment.