Protein aggregation is a common phenomenon that can be encountered during various stages of a commercial antibody manufacturing process. For example, aggregates can form during the fermentation, purification, final formulation operations, or as a result of the storage of the drug substance or final drug product. Usually aggregation results from intermolecular associations of partially denatured protein chains, however it may also result from chemical degradation and subsequent exposure of hydrophobic surfaces, or from disulfide bond scrambling. Mono-dispersity of a therapeutic monolconal antibody is important in terms of both efficacy and safety. Molecular heterogeneity in size (e.g., aggregation) can compromise the biological activity of an antibody or result in a partial or total loss of its therapeutic properties. (Nti-Gyabaah, US 14/355014). 

Aggregation not only leads to loss of product but also has adverse effects such as reduced efficacy and immunological reactions. The role of aggregates in development of immune response to proteins is well reported. This can occur by cross-linked of the B-cell receptor, thus activating the B cells to proliferate and target proteins to the lysosomal pathway, eliciting the T cell response for antibody response. Aggregates can also present epitopes in an array form, evoking T cell independent antibody induction. Furthermroe, aggregates are readily taken up by antigen-prsenting cells and drive dendritic cell maturation, thus enhancing the thymus-dependent immune response. T (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205). 

Protein aggregates can be classified in several ways, including soluble/insoluble, covalent/non-covalent/reversible/non-reversible and native denatured. Soluble aggregates are not visible and may not be removed by a 0.22 um filter. Conversely, insoluble aggregates may be removed by filtraiton and are often visible. (Nti-Gyabaah, US 14/355014).

What causes Aggregation of antibodies?

It has been suggested that IgG aggregation may be mediated primarily by unfolding of the CH2 domain or Fab domain, depending on which mAb is being considered. Haixia Ru “PH and temperature-dependent mechaisms of non-native aggregation of anti-CD40 IGG1” Dissertion, Fall 2015. 

In addition to aggregation prone domains known to occur in mAbs, both full length and Fab portions, aggregation domains are also found in antibody fusion molecules, such as, for example, the anti-VEGF fusion protein alfibercept, which is a fusion of the VEGF0binding portions form the extracellular domains of human VEGF receptors 1 and 2 to the Fc portion of a human IgG1 immunoglobulin as well as etanercept, which is a fusion of the TNF receptor to the Fc poriton of a human IgG1. Bevacizumab contains 4 aggregation prone domains in its light chains. (Kraft, US 2020/0031917). 

Effect of Buffer Conditions:

The underlying mechanism that results in the aggregation due to changes in buffers and salts is complex and not well understood. The complexity is compounded by the inability to definitively differentiate between the various protein stabilization mechanisms and the samll free energies of stabilization of globular proteins. T (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205).

At both 4 and 30C, it has been shown that the increase in aggregate level is minimal even up to 7 days in buffers without salt. The only exception to this is the citrate buffer where appreciable amount of aggregation is observed even in the absence of salt. It can be concluded that the bomination of salt and high termpature results in a significant incrase in aggregation and that the citrate buffer is prone to higher aggregation as compared to acetate adn glycine buffers. T (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205).

Low pH leads to Mab aggregation. At high pH (pH 8, close to pI of Mab), increase in charge variants of the Mab is observed. The decrease in main peak content is due to an increase in the acidic variants. Deamidation of asparigine residue is a common cause of increase in acidic variants leading to degradation and decrease in effiacy of Mabs. This is why this is more significant for CEX and AEX. T (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205).

Aggregation in Fc Fusion Proteins

In addition to aggregation prone domains known to occur in mAbs, both full lenght and Fab portions, aggregation domains are also found in antibody fusion molecules, such as, for example, the anti-VEGF fusion protein alibercept, which is a fusion of the VEGF-binding poritions from teh extracellular domains of human VEGF receptors 1 and 2 to the Fc portion of a human IgG1 immunoglobulin as well as etanercept, which is a fusion of the TNF receptor to teh Fc potion of a human IgG1 immunoglobulin. Bevacizumab (AVASTIN) contains 4 aggregation prone domins in its light cahins. Ranibizumab (LUCENTIS), having the identical light chain, shares the same 4 aggregation prone domains in its light chains. Bevacizumab also cotnains another three aggregation prone doamins in its heavy chains past the inger (amino acid sequences SVFLFP, VVSVLTVL and GSFFL). The sequences are also found in the fusion protein aflibercept due to it including the Fc portion of a human IgG1 immunoglobulin. Aggregation prone domains are known to occr in bother biopharmaceuticals including albumin, somatotropin, insulin Factor VIII and glucagon. (Kraft, US 2020/0031917)

Wang (US 16/259,095, published as US 2019/0234959) disclsoes the Fc fusion protein Aflibercept haivng about 0.05% Fab2-Fab2 (Fab complexes). 

Particular Types of Purification Methods for Aggregates (just brief review from perspective of the aggregates — see particular schemes in outline for more detailed steps/parameters)

Protein A:

A few methods to reduce the risk of aggregation during protein A chroamtography have been disclosed. Most have centered on moderating the pH of elution for antibodies. Sodium chlorid (0-1M) has been disclosed as an elution buffer additive to increase the elution pH. Hydrophobic competitors such as ethylene glycol have been used to weaken hydrophobic interactions and thus increase elution pH from protein A columns. Urea in the concentration of 1-2M has been used as a mild denaturant to facilitate elution. A different approach has been to engineer the protein A ligan to allow for milder elution conditions.  (Shukla, “strategies to address aggregation during protein A chormotography” 36 BioProcess Technical. May 2005).

–With C Domain:

Bian (US 14/768,254, published as US 2016/0122305) discloses using the C domain of Protein A and either a pH gradient or pH step elution which elutes at least 30% of the protein aggregates (such as dimer, trimer, tetramer) prior to the elution of the Fc containing protein in addition to removal of protein aggregates after elution of the F containing protein. In one embodiment, the seris of pH changes steps are in the order of pH 5.0 to pH 3.0 or a pH gradient ranging from pH 7 to 3. 

Ion-Exchange:  See also CEX for the pufication of antibodies

The fact that aggregates will generally carry more charge than the product at the working pH range and thus bind more strongly to ion exchangers than the monomeric form of the can be exploited for purificaiton. (Nti-Gyabaah, US 14/355014)

Hydroxyapatite

HA and FA have been shown to be effective for removal of aggregates from many antibody preprations. Antibody aggregates usually elute after antibodies but may eoelute with antibodies to varying degrees. Aggregate removal is important because aggregates are known to contribute to nonspecific interactions that reduce the shelf stability, sensitivity, accuracy and reproducibility of analytical results in conjunction with in vitro diagnostic applications. (Gagnon, US 7,999,085). 

HIC

HIC is an established purification step for aggregate removal with the majority of aggregates eluting either on the tail portion of the native antibody peak or as a distinct peak. Aggregation increases the hydrophobicity of the resulting molecular form relative to the hydrophobicity of the monomeric mAb. Phenyl sepharose HP is usually the HIC resin of choice because of the particle size and known surface accessility of aromatic residues on the monoclonal antibody structure. (Nti-Gyabaah, US 14/355014, published as US 2014/0288278)

–bind & elute mode:

(Nti-Gyabaah US14/355014, published as US 2014/0288278) disclsoes showed using HIC in a bind and elute mode as a final polishing step to resolve heterogenous aggregates from anti TNF mAb by using Pehnyl Sepharose HP at pH 7 and conductivity 140 mS/cm. A linear elution gradient was employed with decreasing ammonium sulfate to separate the monomer from the aggregates by their hydrophbicity. The eluate was fractionated and tested for charge heterogeneity via HP-IEX and monomer purity via HP-SEC showing one main peak followed by a secondary small peak during the gradient elution. The main peak consisted entirely of high purity monomer while the secondary smaller peak contained increased levels of aggregate which included a mixture of both acidic and basic variatns. 

—-To separate three-light chain species:

Gentofte (US 15/114,655, published as 2016/0347833) discloses antibody purification using HIC where a “pre-monomer” that includes an antibody  monomer and a light chain non-valently bonded thereto is separated from the the main peak of the monomeric antibody when analysed by SEC-HPLC. A gradient with decreasing ammonium sulphate concentrations was used for the elution step on a Pehnyl Sepharose HP resin. Pre-monomer together with other aggregates elutes at the back end of the monomer peak. 

Wollacott, Monoclonal antibodies, 2013, 5(6), pp. 925-935) disclose a novel mAb species which eluted between the monomeric and dimeric species of a mAb as shown by size exclusion high performance liquid chromatography (SE-HPLC). The species, referred to as “shoulder” indicated that it was a mAb containig an extra light chain and had a MW of about 175 kDA. The extra light cahin was found to be non-covalently assocaited with the Fab portion of the protein. The species was idnetified as a mAb monomoer with an additional light chain covalently associated trhoguh a disulfide bond formed between a light cahin cysteine and an engineered cysteine in either the H or L chaims of the molecule. Wollacott discloses using HIC in place of CEX to exploit the subtle differences in the hydrophobicity between the monomer and shoulder. Using HIC, the shoulder species was reduced from 3% to less than 0.5% with good step recovery. More hydrophobic HIC resins such as GE Butly Sepharose HP were best at discrimiating between mAb-X monomer and shoulder, dimer and high MW species, resulting in product containing 99.7% monomer and less than 0.1% shoulder by the end of the elution by a lear gradient from 1.2-0 M ammonium sulfate in 20 mM citrate, pH 6. The Phenyl Sepharose resin also produced veyr pure monomer (99.2%), however, monomer did not bind as tightly to the resin. 

–Flow through mode:

(Nti-Gyabaah US14/355014, published as US 2014/0288278) discloses using HIC in a flowthorugh mode as a final polishing step to resolve heterogeneous aggreagates from TNF mAb. Anti-TNF mAb mixture was loaded onto a Phenyl Sepharose HP column at pH 7 and conductivity 110 mS/cml. After loading, the column was washed with an equilvaent pH and conductivity buffer to recvoer unbound anit-TNF mAb B. Fractions were taken at various loading points. Product pools in the flowthrough mode contained solely 100% pure monomter with an 85% yield. 

Size Exclusion Chromatography (SEC): 

Size exclusion chromatography (SEC): has been reproted to provide an effective process for reducing the level of aggregates in a partially purified builk product. The sucess of this type of chromatography exploites the fact that the monmeric mAb has a smaller size than aggregates and thus elutes earlier. (Nti-Gyabaah US14/355014, published as US 2014/0288278).

Thio-heterocylic cations:

Gagnon (US 14/766123; see also 14/769098) discloses a method of reducing aggregate content in a protein preparation having a target protein such as an antibody by contacting the preparation with a thio-heterocyclic cation such as methylene blue or methylene green. The cation is then removed by contacting the resulting mixture with a first functionalized solid such as a negatively charged solid or HIC.  In some embodiments, the thio-hterocyclic cation may be used in combination with allantoin. For example, allantoin can be added to a cell culture harvest in an amount of 1%, methylene blue is added to a concentration of 0.025%. Particles bearing an electropositive metal affinity ligand tris(2-aminoethyl)amine (TREN) are added in an amount of 2-5%.The mixture is incubated and stirred for 4 hours and the solids removed. (Gagnon 14/769098).