Fc gamma RIII receptor:

Mcdonnell (WO2010/0483130 disclsoes a method of purifying Fc containing proteins using a soluble neonatal Fc receptor (sFcRn) linked to a support surface. In one embodiment that FcR is one which binds an IgG antibody and includes receptors of the FcyRi, FcyRII, and FcyRII subclasses.

Sondermann (US7,074,896 and WO. 00/32767) teaches recombinant soluble Fc receptors such as FcyRs which are characterized by the absence of transmembrane domains, signal peptides and glycosylation for the purification of antibodies. The Fc receptors can easily be obtained by expressing respective nucleic acids in prokaryotic host cells and renaturation of the obtained inclusion bodies. 

For separating antibody glycoforms:  See also glycan variants under “Antibody Purification”

Bolton (US2013/0084648) disclsoes methods for separating polypeptide glycoforms using a medium that includes an Fc receptor such as an extracellular portion of an Fc gamma RIII receptor. The separation method can be used in conjunction with other separation methods such as IEX, HA, protein A medium, HIC, a lectin or a combination thereof. The method can further include analyzing a characteristic of polypeptide eluted from the medium. In some embodiments, oligosaccharides form the polypeptide are anlayzed (e.g., N-linked oligosaccharides are analyzed by cleaving N-oligosaccharides from the polypeptide labeling the oligosaccharides and detecting labeled oligoscaccharide species.  Bolton teaches that Fc receptors that can be used include receptorss that preferentially bind to one or more polypeptide glycoforms. In some embodiments, an Fc receptro preferentially binds to glycoforms lacking or with reduced levels of fucose (e.g., glycoforms with low levels or the absence of core N-fucosylation, e.g., antibody glycoforms lacking fucose on one or both heavy chains. For example, the Fc receptor can include an extracellular porition of an Fc gamma RIII polypeptide (e.g., an Fc gamma RIIIa polypeptide or an Fc gamma RIIb polypeptide). 

–For separation of fucosylated antibodies:

The term “fucosylation” refers to the presence of fucose residues within the oligosaccharides attached to the peptide backbone of an antibody. Specifically, a fucosylated antibody includes alpha(1.6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or both of N-linked oligosaccharides attached to the antibody Fc region, e.g., at position Asn 297 of teh human IgG1 Fc domain. Asn 297 may also be located about 3 amino acis upstream or downstream of position 297, e.e., between positions 294 and 300, due to minor sequence variations in immunoglobulins. The “degree of fucosylation” is the percetnage of fucosylated oligosaccharides relative to all ogligosacccharides identified in an N-glycosidase F treated antibody sample by MALDI TOF MS. In a sample of a “fully fucosylated antibody” essentially all oligosaccharides include fucose residues. Accordingly, an individual antiboy in such a sample typically includes fucose residues in each of teh two N-linked ogligosaccharides in the Fc region. Conversely, in a sample of a “fully non-fucosylated” antibody essentially none of the oligosaccharides are fucosylated and an individual antibody in such a sample includes fucose residues in neitehr of teh two N-linked oligosaccahrides in the Fc region. In a sample of a “partially fucosylated antibody” only part of the oligosaccharides includes fucose. An antibody in such a sample can include residues in none, one or both of teh N-linked ogligosaccharides in the Fc region. Antibodies can be glycoengineered to contain different degrees of fucosylation such as by modifying the amino acid sequence of the side chain group of individual amino acids or of the oligosaccharide strutures. Glycoengineering can also include metabolic engineering of the clycosylation machinery of a cell, including genetic manipulations of teh oligosaccharide synthesis pathways to acheive altered glycosylation of glycoproteins expressed in the cell. For example, a glycoengineered antibody can result from an alteration in glycosyltranferase activity in the hose cell producing the antibody. An antibody with an increased proportion of non-fucosylated oligosaccharides in  its Fc region can be obtained by producing an antibody in a host cell having increased beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity or having decreased alpha(1,6) fucosyltransferase activity.  (Freimoser-Grundschober (US14/352411, published as US 9994610 and US 15/991,853, published as US 2018/0265544)

Freimoser-Grundschober (US14/352411, published as US 9994610 and US 15/991,853, published as US 2018/0265544) discloses separation of antibodies having different degrees of fucosylation, by Fc receptor affinity chromatography. The Fc receptors used to separate diffrently fucsylated include FcRyIIIa (V158). The method is based on the fact that binding affinity of the Rc receptor for the antibodies decreases with the degree of fucosylation of the antibodies (i.e., more fucosylation means less binding to the Fc receptor). 

Neonatal Fc receptor (sFcRn):

Human FcRn is a heterodimeric protein consisting of two polypeptides, a 48-52 kDa glycosylated class I major histocompatibility coplex-like protein (alpha-FcRn) containing a single N-glycan moiety and a B2-microglobulin (beta2 min) subunit of about 14 kDa. FdRn binds with high affinity to the CH2-CH3 poriton of the Fc domain of IgG. (Papadimitrio, “Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies” MABS, July 1, 2013, vol 5, No. 4, pp. 576-586). 

Falkenstein (US14/378808, published as US 2015/0018241; see also US 16/258,294, published as US 2019/0276492) discloses the use of an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as affinity chromatography ligand. Specifically, the soluble extracellular domain of FcRn for human FcRn with a C-terminal His-Avi Tag was co-expressed with beta2-microglobulin in mammalian cells. The non-covalent FcRn-microglobulin complex was then biotinylated and loaded onto streptavidin derivatized sepharose. The ligand can be used to separate antibody variants (closely related antibody species differing in a single or a limited number ofa mino acid residues) using a linear pH gradient. An antibody having a modified Fc region with reduced FcRn binding has a rentention time that is smaller whereas an antibody having a modified Fc region with enhanced FcRn binding has a retention time that is larger. This allows the analysis of the itneraction between FcRn and IgG in vitro and can provdie insight into the structural and functional integrity of therapeutic IgG regarding pharmacokinetics in vivo. A stanrdarized pH gradeint FcRn affinit liquid chromatography can for example be used with conditions closely resembling the mechanism of interaction between IgG and FcRn in vivo. Human FcRn is immobilized on the column as affinity ligand and a liner pH gradient, e.g., from 5.5 to 8.8 can be applied. Fc region variants with increased affinit for the FcRn (i.e., increased retention time on an FcRn column but still eluting before a pH value of pH 7.4 are predicted to have longer serum half-lives compared to those with decreased affinity for the FcRn. This has application in methods of treating humans where long half-life of the adminsitered antibody is desired. The method can also distinguish 1) the same IgG with differnet Fab fragments, oxidized IgG forms form non-oxidized IgG forms, aggregates form monomers and antibodies with variations in the Fc region. 

Farrington (WO2005/047327) discloses a fusion construct of the extracellular domain of a neonatal Fc recptor with the Fc region of an IgG1 antibody. Briefly, human beta-2-microboulin and the alpha chain of human FcRN and an Fc region from a human IgG1 antibody were fused and the construct was used for purification of antibodies containing altered Fc regions.

Gastinel (US5,623,053) discloses a novel Fc receptor construct which is a fusion of the heavy chain of FcR without the transmembrane domain the domain responsible for attachment of the FcR to the cell membrane so it can now become soluble)  and the light chain of FcR (beta2 microglobulin). The construct binds to the Fc porition of antibodies at a pH of 6-65 and relases the antibodies at a pH ranging from 7.5-8.0.

Mcdonnell (WO2010/048313 discloses a method of purifying Fc containing proteins using a soluble neonatal Fc receptor (sFcRn) linked to a support surface. In one embodiment, one or both heavy chain (alpha-chain) or light chain (beta2m) domains are modified to modulate Fc tonatining protein binding. Covalently joining the two subunits as by an amino acid linker to form a single chain sFcRn protein can also result in greater stability.

Papadimitrio, “Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies” MABS, July 1, 2013, vol 5, No. 4, pp. 576-586) discloses a FcRn column chromatography with a linear pH gradietn from pH 5.5 to 8.8 to study interactions between IgG and FcRn. The column was prepared by expressing  cDNAs encoding the extracellular domain of human FcRn alpha-chain and human Beta2-microglobulin. Between 1.2 mg and 12 mg FcRn/beta2-microglobulin in 5 ml 20 mM sodium citrate buffer, pH 5.5 were biotinlated  and coupled to streptavidin sepharose.