A species identified as a mAb monomer with an additional light chain covalently associated through a disulfide bond formed between a L chain cystein and an engineered cystein in either the H or L chains is known. Genetech researchers also characterized mAb species containing variants with one and two additional light chains present at 0.2% when produced in CHO cells. They determined that the relative quantity of this species was related to the redox environment of the cell culture, which implicates disulfide bonding as the mode of binding for tehe additional light chains. In addition, a report characterizaing a mAb size variant using SE-HPLC coupled with multi-angle light scattering (MALS), matrix-assisted laser desportion/ionization-time of light (MALDI-TOF) mass spectrometry, capillary gel elctrophoresis and N-terminal sequences showed a mAb with incorporation of a third light chain; however the extra L chain was associated through non-covalent interactions. A cell based potency assay showed the reduced aiblity of the extra L chain species to neutralzie the target antigen. Wollacot (mAbs, 5(6): 925-935 (2013)

Jensen (US 2016/0347833) discloses an antibody pre-monomer impurity that includes two heavy chains (HC) and three light chains (LC) of which one is non-covalently attached (LC2HC2: LC). Based on the biophysical, spectroscopic and function characterists of LC2HC2:LC, the molecular structure is an antibody where an addition light chain has taken up the position normally occupied by a HC. The additional LC is bound via non-covalent itneractions to another LC, which in turn is covalently bound to HC. The C-terminal cysteine in the non-covalently attached LC is capped by forming a disulfide bond with either glutathione or cysteine. 

Challenges Posed in removing Extra Light chains 

Due to the similarities to the monomer species, the separation of H2L3 antibodeis generates a challenge during downstream purificaiton of monoclonal H2L2 cysteine-modified antibodies. (Liu US 2019/0112359). 

Purification Strategies for Intermediate HMW species:

CEX:

Liu (US 2019/0112359) discloses a purificaiton strategy to seperate triple light chain (H2L3) antibodies from double light chain (H2L2) antibodies using CEX. using optimzied POROS SC strong CEX, the amount of H2L3 antibody can be reduced from 11% to less than 1%. In one embodiment, an antibody composition is applied to the CEX so that H2L3 antibodies and H2L2 antibodies bind and eluting with a pH of about 3.8 to about 5 and collecting an H2L2 composition eluted form the resin. In some embodiments, no more than 0.5 of the antibodies in the H2L2 composition are H2L3 antibodies. 

HIC

Jensen (US 2016/0347833) disclosing a method for removing an antibody pre-monomer that includes two H chains and 3 L chains (LC2HC2:LC) which includes the step of purifying the monomeric antibodies using hydrophobic interaction chromatography (HIC). Antibody products after the method include anti-IL21 comprising at most 1% antibody pre-monomer aggregates. Elution from the HIC was done by decreasing the ammonium sulphage concetnration, such as by using alinear gradient of ammonium sulphage to 0 mol/kg. In one embodiment, the monomeric antibody componets elutes as the first antibody componentIn such examples, the antibody solution of interest may be obtained by collecting the fractions eluting prior to the fractions including the majority of pre-monomers and antibody dimers. Pre-monomer mayb be excluded form the antibody composition by deselecting subsequent eluate fractions. 

Wollacot (mAbs, 5(6): 925-935 (2013) disclsoes a robust purificaiton method to reduce an intermedaite HMW species using hydrophobic interaction chromatography (HIC) from about 3% to less than 0.5% with good step recovery. 

SEC:

Size-exclusion chromatogrpahy (SEC) is the most commonly used method to separate and quantify mAbs size variants. SEC analysis of MAb-A resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. Elctron spray ionization -time of light mass spectrometry (ESI-TOF MS) microfluidics capillary electrophoresis and sodium dodecyl sulfate-PAGE (SDS PAGE) results demonstrated that SEC Peak 1 contains two structural variants: MAb-A with one extra light chain (2H3L) and mAb-A with two extral light chains (2H4L). Teh C-terminal Cys of the extra light chain in Peak 1 variants is either a free thiol, capped by glutathione, cystein or another light chain. the Peak 1 fraction also shows two major peaks with calculated molecular weights of 175 kDa and 191 kDa which are the approximate sizes of 2H3L and 2H4L. (Liu, “Chracterization of monoclonal antibody size variants containing extra light chain” mAbs 5:1, 102-111, 2013).