Isotypes as Particular Types of Antibodies Purified and Techniques Used:

Antibody Fragments: (for Single Domain Binding Polypeptides and Nanobodies see “domain antibodies” in outline)

–Advantages of Using Antibody Fragments: 

Small genetically engineered immuoglobical constructs are being developed industry wide for a growing range of in vivo applications. Examples include Fab, F(ab’)2, single-chain (sc) Fv, bis-scFV, diabodies, minibodies and single domain antibodies. Their small size potentially tives them access to tissues that are poorly accessible by intact antibodies; rpaid clearance form blood and neotargeted tissues, lower immunogenic response and eye-drop inhalent or oral administration. (Gagnon, “Minibodies and Multimodal chromatography methods” BioProcess Interational Feb 2010).

–Purification by pH gradient

Slough (US Patent Application 15/566231, published as US 2018/010007) discloses the isolation of antibodies that lack an Fc region using a pH gradient, such as pH 6-2.1. As binding to Protein A by antibody fragments that lack an Fc region is known due to binding to the human VH3 variable framework subclass of the V regions, multimeric spcies will have increased avidity for Protein A because they have mroe VH3 regions, requiring a lower pH for elution. In one partciular embodiment a dsFv antibody was purified via a pH gradient elution. 

–Purification using CEX-AEX:

Spitali (US 2013/0184439) discloses purification of an antibody fragemtn from a periplasmic cell extract using a first CEX and a second AEX step. 

—-with Reducing agent (glutathione) See also purification of particular contaminants and the section there entied “disulfide bond variants”

Slough (US 15/538282, published as US 20170342105) discloses purifying of a Fab by expressing the Fab in E coli into the periplasmic space, clarification, addition of glutathione, loading onto CEX with loading, washing and eluting in the presence of glutathione followed by AEX equilibrated with buffer containing glutathione, washing with Tris buffer containing glutathione to recover the Fab in the flow thorugh of the loading and washing steps. 

Immunoglobulin homodimers:

(Christei (US 16/497,788, published as US 20200033363) discloses a method of producing/screening/purifying immunoglobulin homodimers which include identical light chain variable regions (VL) monomers which includes expressing each light chain variable domain monomer in a bacteriophage, performing an affinity screening step and a dimerization screening step. In one example the light chain variable domain (VL) maby be a VL from a kappa light chain. In another example, the VL may be a VL from a lambda light chain. The affinity screening step includes contacting the immunoglobulin homodimers with an antigen and selecting those immunoglobulin homodimers that bind specifically to the antigen. The dimerization screening step includes determining the stochiometric ratio (relative numbers of VL monomers and antigen molecuels in the immunoglobulin-antigen complex) of immunoglobulin monomer to antigen. The stoichimoetric ratio may be determind using for example measuring the molecular weight of proteins such as by size exclusion chromatography coupled with multi-angle laser light scattering (SEC MALLS). The MWs of the complex, immunoglobulin alone and antigen alone can then be compared. Variants of the light chain variable domain (VL) monomers can be geenrated for example by modification of V and J gene segments through general mutagenesis. Sequence of VLs for the V and J gene segments can be obtainable for example from public databases. The VL monomers may be capable of dimerizing to form immunoglobulin homodimers. In one example, the method includes the use of a phage display technique which includes expressing multiple copies of a VL monomer joined to a filamentous pahge protin via a linker protein. The linker protein can include a sufficient number of amino acids to allow sufficient confomational flexibility to enable VL monomer dimiriziation. 

Bispecific Antibodies (see outline)

Catalytic Antibodies (Abzymes)

Natural Abzymes: from the sera of patients are usually polyclonal in origin and are products of different immuno-competent cells. Their purification is one of the most complicated aspects of their study. Affinity chromatography on immobilized hapten usually produces homogeneous monoclonal Abzs, but polyclonal Abs from patients are very heterogeneous and their affinity for an immobilized substrate analogs can differ by several orders of magnitude. The first step of purificaiton should spearate Abs efficiently from other proteins so that a fraction with high affinity for potential substrates is obtained. In the first step, affinity chromatogrpahy on adsorbents bearing anti-IgG, anti-IgM or anti-IgA Abs is often used in conditions to remove non-specificaly bound proteins. However, absdorbents bearing immobilzied Abs are often not convenient for Abx purificaiton and protein A-Sepharose is an optimal resin for the first step because it has a higher affinity for Abzs than for Abs without catalytic activity. (Nevinsky, “Natural Catalytic Antibodies – Abzymes, Chapter 19 from Catalytic Antibodies, 2005, pp. 505-569). 

IgA (see outline)

IGM

–Chromatography methods:

Gagnon (WO2009/149067) discloses that IgMs have characteristics that can limit the application of standard antibody purification tools. IgMs tend to be less soluble than IgGs and more susceptible to denaturation (precipitation, including aggregate formation) at extremes of pH, and under conditions of low conductivity. IgMs are generally tolerant of high salt concentrations, which can be useful for IEX, but are susceptible to denaturation from exposure to strongly hydrophobic surfaces, which can limit the usefulness of HIC. Although IgMs can be eluted from modernately hydrophobic supports for HIC in a well definited peak at reasonably low salt concentraiton, IgMs will precipitate at the higher salt concentraitons that are preferred to support good capacity on moderately hydrophobic mediation. Because IgMs are typically more charged than IgGs, IgMs bind more strongly than IgGs to IEX and hydroxyapatite and often bind much more strongly than most contaminants. The large size of IgMs can be a challenge for purificaiton, due to slow diffusion constants, which can be a problem for porous particle based chromatogrpahy media dependent on diffusion for mass transport. Purification of clinical grade IgM can generally be acheived with 3 bind elute chromatography steps on hydroxyapatite, AEX and CEX. Omitting an affinity step is a positive contribution as well as avoiding intermediate diafiltration by using in-line dilution to load samples. 

Gagnon (WO2009/149067) discloses using a first chromatography step such as HA or IEX with the use of a nonionic polymer for removal of the aggregates of the protein product and then a step of combining a solubility enhacning additive such as a switterion, a urea compound and an alkylene glycose and a second chromatography step comprising the use of IEX. The method is particularly useful for purificaiton of IgM from a mixture. Buffers containing nonionic polymers to enhance aggregate removal can be introduced direclty into the zqitterion contianing compositions that enhance protein solubility and substantially avoid aggregate formation, thereby avoiding additional manipulations such as desalting, polymer removal or buffer exchange that could affect the yield and or quality of the purified protein product. 

–Affinity chromatography

—-Secretory component as the ligand:

Secretory component has high affinity for IgM (Bouvet, Scand. J. Immunol. 31, 437-441, 1990).

Jones (J Immunol Methods 1987 104(1-2): 237-43) discloses human secretory component bound covalently to sepharose 4B used as an affinity adsorbetn to isolate and purify polymeric immunoglobulin M from cell culture supernatants.

——–Isolation of secretory component itself:

Corthesy (US2015/0056180) discloses that secretory component itself can be isolated by attaching a hexa-histidine tag to the SC which can further include a cleavable linker so that the tag may be cleaved off prior to use.

Kraehenbuhl (WO98/20141) discloses purification of (SC(IpaB)6xHis by Ni2+ chelate affinity chromatography).

Underdown (Immunochemistry, 1977, 14, pp. 11-118) discloses that SC can be isoalted from human whey by affinity chromatography on IgM sepharose adsorbents.

IgG

–IgG2:

(Dillon, US14/363735) discloses a method of produing an IgG2 antibody enriched for at least one of the several IgG2 structural variants which differ by disulfide connectivity in the hinge region, comprising contacting a solution containing a recombinantly produced IgG2 antibody with either a strong cation exchange (SCX) matrix, an IgG2 affinity matrix or a Protein L matrix and eluting two or more first elution fractions from the matrix where the IgG2 antibody solution elutes off the matrix as two or ore separate forms corresponding to two or more IgG2 structural variantswhich differ by disulfide connectivity in the hinge region. 

IGE

Procedures for different Isotopes: It has been shown theat the constant domain fragment (Fc) of IgE is more sensitive to low pH/high salt conditions that the IgG1 Fc. These results indicate that purification and formulation strategies commonly used for IgG antibodies are not amendable to IgE. “GE HealthcareLife Sciences, Application note 28-9870-45AA “Better guidance in antibody therapeutics process development using differential scanning calorimetry” 2011, pp. 1-4.

Anti-amyloid beta and anti-infectious disease IGs

–By CEX:

Hofbauer (US14/213,585 and US2014/0271669) (see also Gnauer (US 2017/0218051) discloses using CEX for purification of immunoglobulin enriched in anti-infectious disease immunoglobulins uisng CEX by providing a plasma composition comprising IgG immunoglobulins, binding the IgG to a CEX, eluting a majority of the bound IgG immunoglobulins form the CEX in a first elution step and then eluting the remaining IgG bound to the CEX using a second cation elution buffer having a higher pH or conductivity from the first elution step. Hofbauer discloses that anti-brain disease related protein IgG antibodies present in human plasma such as anti-amyloid beta bind to CEX with higher affinity than do most IgG antibodies present in the plasma. By using an extreme ionic strengh conditions for the second elution (e.g., with a solution containing 2 M sodium chloride), the targetted IgG is obtained.

–By Thiophilic Affinity Chromatography

Relkin (US 2012/0183527) discloses using theiophilic chromatography to detect and isolate antibodies that are specific for various forms of amylid, including monomeric, oligomeric and fibrillar amyloid proteins. The resulting population of amyloid beta specific antibodies is hereogenous but binds to amyloid beta with high affinity. The thiophilic chromaotgraphy is used to enrich the antiboedies prior to detection. Under the proecdure a biological sample such as plasma is contacted with a thiophilic resin to bind an anti-amyloid antibody present in the sample, the antibody is eluted from the resin at a non-denaturing pH to form an eluate (an advantage over say Protein A), and the eluate is contacted with an amyloid anitgen to form a complex between the amyloid anitgen and the anti-amyloid antibody and this complex is detected as by ELISA. In an advantageous embodiment, a chaotropic wash step is performed to reduce the signal assocaited with non-specific and low-avidity anti-amyloid antigen binding.