MAbs undergo several posttranslational modifications such as disulfide bond formation, oxidation, deamidation, truncation and cyclization of N terminal glutamine residues to pyroglutamate. The heterogeneity observed in mAbs is often moniteored by weak cation exchange (WCX)-high performance liquide chromatography (HPLC). Resolution on the WCX column depends on the surface charge of the molecule, which in turn determines the apparent isoelectripoint (pI) of the molecules. Regions of the chromatography before and after the main species are often referred to as the acidic and basic regions. Lowering of the apparent pI of the main molecule due to modification such as deamidation as asparagine residues, C terminal truncation of lysine residues and cyclization of glutamine residues will result in migration toward the acidic region wereas an increase in the pI of the molecule due to modification such as methionine oxidation and proline amidation will result in migration toward the basic region (Ouellette, Analytical Biochemistry, 397 (2010) 37-47). 

Acidic and basic species are commonly observed when recombinant mAbs are produced. Charge variants may substantially affect the in vitro and in vivo properties of antibodies. It has been demonstrated using chemically modified antibodies that charge variation can alter binding to proteins or cell membrane targets, thus affecting the tissue penetration, tissue distribution and pharmacokinetics (PK) of the antibodies. The impact on biological functions is highly dependent o the sites and levels of modificaitons. For modifications that are localized in teh Fc regions, even the presence of higher levels may not have direct effects on the binding affinity. Complete elimination of acidic and basic species is unrealistic and unnecessary. (Du, “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, mAbs 4-5, 578-585, 2012)

The extent of charge heterogeneity in Mabs also regulates their structure, stability, binding affinity, chemical properties and hence their biological reactivity. During manufacturing, various forms of microheterogeneities are formed with respoct to size, charge, and other attributes. The reason could be enzymatic processes, spontaneous degradation and other modificaitons. Common forms of charge hterogeneities that have ben obvserved include structural isoforms, glycosylation, glycation, degradation, deamidation, isomerization and peptide clevage. Charge heterogeneity resutls in modificaiton of the isoelectric point (pI) of the moelcule. It has been demonstrated that modifying the pI of an antibody by about one pH unit can lead to significant differences in the pharmacokinetcs of an intact Mab. (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205)

Acidic variants: 

An acidic variants is more acidic than the main species antibody. An acidic variant has gained negative charge or lost positive charge relative to the main species antibody. Acidic variants of a main species antibody elute earlier than the main peak upon separation by cation exchagne chromatography (CEX) or later than the main peak from AEX. 

Sialylation and C-terminal lysine cleavage or deamidation increase net negative cahrge and produce acidic variants, which elute earlier during CEX. (Kim, Arch. Pharm. Res. (2016) 38: 1472-1481). 

Causes of Acidic Variants: Some of the main causes for the formation of acidic species are the following: 

Deamidated antibodies: is one in which one or more asparagine residues have been derivitized, e.g., to an aspartic acid, a succinimide, or an iso-aspartic acid. Deamidation occurs both in the variable domains as well as in the constant domains. Deamidation of Asn residues in the CDR region is almost guaranteed to result in the generation of acidic species. (Du, “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, mAbs 4-5, 578-585, 2012). 

Deamidation is a non-enzymatic chemcial reaction in which an amide functional group is removed. The reaction is important in the degradation of proteins because it alters the amide containing side chains of the maino acids asparagine and glutamine. In an example of the reaction, the side chain of an asparagine attacked the adjacent peptide group, forming a symmetric succinimide intermediate. The symmetry of the intermediate results in two hydrolysis products, either asparatate or isoaspartate. This process is considered a deamidation reaction becasue the amide in the asparagine side chain is replaced by a carboxylate group. A similar reaciton can occur in asparatate side chains, yielding a partical conversion to isoaspartate. In the case of glutamine, the rate of deamidation is generally ten fold less than asparagine, however, the mechanisms is essentially the same, requiring only water molecules to proceed. (Ram US 2011/0130544).

–Deamidation of asparagine (Asn or N):  The deamidation of asparagine residues in mAbs can be a major route of degradation, and it can limit shelf-life if appropriate formulation and storgage condtiions are not selected. In general, the Asn deamidation mechaisms has been well characterized. At alkaline pH, deamidation is a peredominantly base ctalyzed reaction that proceeds via a nucleophilic attack of the N+1 nitrogen of the protein backbone on the carbonyl carbon of the Asn side chain, forming a cyclic imide intermediate, succinimide. Spontaneous hydrolysis occurs at eitehr of the succinimide alpha or beta carbonyl groups to form isoaspartic acid or aspartic acid in a ratio of about 3:1. These deamidation producs result in the formation of antibody acidic variants because of the generation of additional carboxylic acid groups. (Pace “Asparagine deamidation depenence on buffer type, pH and temperature” J. Pharmaceutical Sciences, 10296) (2013)

–How measured:

Protein deamidation can be measured using the ProPac WCX-10 column (see Dionex, application Note 125 “Monitoring Protein Deamidation by Cation-Exchange Chromatography”).

Pyro-glutamate Variants:

Many of the human IgG1 or IgG2 types of antibodies contain a glutamic acid (Glu) and/or a glutamine (Gln) residue at the N-temrinus of either the light or heavy chain or both. Such N-temrinal glutamic acid and/or glutamine residues may undergo cyclization to form pyro-glutamate (pGlu) . During cyclization of glutamine, the N-temrinal primary amine (positively charged at a neutral pH) is converted to a neutral amide, resulting in a change of the net charge of the antibody. The lack of cyclization may be detected as basic variatns by CEX since the main peak is typical the fully cyclized species or as late-eluting peaks by reversed-phase HPLC tdo to the increased hydrophobicity after the loss of the N-terminal amine. Difffernt levels of completeness of the process are a common source of heterogeneity. (Van Schravendijk, US 14/780964, published as US 2016/0060349)

The role of N-terminal cyclization and the enzymes involved is not entirely known. However, it is speculated to provide a protective function against protein degradation from extracellular amino peptidases. Formation of pyroglutamate from glutamine results in the loss of mass of 17 Da due to the release of ammonia. The pyroglutamate variant is more acidic because of the loss of the primary amine and, thus elutes as a more acidic species. (Ouellette, Analytical Biochemistry, 397 (2010) 37-47).

–Methods to promote pyro-glutamate formation to reduce heterogeneity

—-Enzymatic conversion

Xu (Analytical Biochemistry 436 (2013) 10-12) discloses a method to reduce charge heterogeneity by adding glutaminyl-peptide cyclotransferase, which is specific in catalyzing protein N-terminal Gln to PyroGlu. Thus charge variants of terminal Gln and PyroGlu can be identified without fraction collection. In addition, treatment of mAb antibodies can assist fraction collection for the characterization of other antibody variants. 

—-Non-enzymatic conversion

Van Schravendijk, (US 14/780964, published as US 2016/0060349) discloses a method for the conversio f an N-temrinal glutamine and/or glutamic acid risude ofn antibody to pyro-glutamic acid within a purification process such as one including protien A chromatography and IEX by incubating the antibody under conditions to promote cyclization of the N-terminal glutamine and/or glutamic acid residue. In one embodiment, the conditions include a termpature of 20-45C with a pH in the range of 3.5-9.0 for 4-120 hours. 

—-Changing N-mterin Aln residues for other residues

Rasmussen (US 2008/0131882) discloses that charge heterogeneity may result from the N-tmerinal clockage by pyroglutamic acid (PyroGlu) resulting from cyclization of N-tmerinal glutamine residues (deamidation) which results in charge heterogeneity giving a coplex IEX pattern. The problem cannot be solved by the use of the specific enzyme, pyroglutamate aminopeptidase because deblocking has to be peormed on reduced and alklyated antibodies in order to obtain high yeidls of the deblocked antibodies not compatible with a subsequent IEX analysis and second because it is not possible to obtain a 100% cleavage for all the antibodies. Rasmussen discloses instead ensuring that no polypeptide chain contains a N-temrinal glutamine by chaning N-termianl glutamine residue to another amino acid. 

Production Methods to Reduce Acidic Variants:

Cell Culture Methods:

(Subramanian US9,150,645) discloses a method of producing adalimumab having less than 10% total acidc species by culturing a mammalian cell producing adalimumb with basic amino acids selected from arginine, lysine, ornithin and histidine.

Addition of stabilizers

–addition of acid:  Santora (US2004/0162414) discloses a method of identifying modifications of anti-TNF antibody such as acidic modification as detected using a weak cation exchange columnn (WCX-10) as well as a method for reducing modifications such as acidification by adding acid to a transgenic goat milk solution containing the antibody.

Glycated variants: See outline

Basic Variants

Basic species are variants with higher apparent pI when analyzed using isoelectric focusing methods.  Basic species elute later than the main peak from CEX or ealirer than the mean peak from AEX.

C-terminal lysines, glycine amidation, succinimide formation, amino acid oxidation, asparate isomerization and removal of sialic acid increase net positive charge and produce basic varitns, which elute latter. (Kim, Arch. Pharm. Res. (2016) 38: 1472-1481)

Similar to acidic species, the location s of modifications that form basic speceis are critical to whether or not the modifications have any effect on the structure, stability and biological functions. Modifications of either N termini or C termini of antibodies are not expected to have substantial effects on antibody structure, stability and functions because these regions are highly exposed and not part of any ligand binding sites. 

Some of the more common reasons for basic variants are the following:

C-terminal Lys: 

One major reasons for the formation of basic species is incomplete removal of C terminal Lys. MAbs with heavy chain C terminal Lys are more basic than the main species due to the additional positive charges.  (Du, “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, mAbs 4-5, 578-585, 2012). The removal of the carboxy-terminal lysine form the H chain is routinely observed upon the characterizaiton of mAbs and is cuased by intracellular enzymes. From a regulatory aspect, this “lysine clipping” is not regarded as critical under the condition that a potency assay is available that proofs the quality of the mAb. (Antes, J Chromatogr B Analyt Technol Biomed Life Sci, 852(-12), 2007, 250-6). 

Santora (Analytical Biochemistry 275, 98-108 (1999) discloses the use of cation exchange liquid chromatography (CIEX) (WCX-10 CEX0 and capillary isoelectric focusing (cIEF) for determination of heavy chain C terminal variants of D2E7, a human anti9-tumor necrosis factor monoclonal antibody. In addition, exveral enzymatic treatments were used to assist in determining the idnetity of isoforms; deglycosylation of the antiobdy with PNGaseF, which hydrolyzes all types of N-glycan chains and CPB which specifically cleaves Lys and Arg residues form the C termini.

Subramanian (US9,181,337) discloses a composition comprising human anti-TNF alpha (adalimumab) where less than 65% of the lysine variant speceis in the composition have zero C-terminal lysines and where the lysine variant species include the main peak and peaks that elute at a releative residence time later than the main peak as detected using weak cation-exchange chromatography. Since lysine can carry a positive charge, antibodies lakcing the basic C temrinal lysin(s) differ in their charge state from ones that contain the lysine, so that the distribution of lysine variants (% Lys 0, % Lys1, % lys2 of the total lysine sum) can be detected using IEX such as ProPac WCX-10 Weak CEX.

–Removal of basic isoforms with Carboxypeptidase B (CPB):

Carboxypeptidase B (CPB) is thought to specifically remove C-terminal basic amino acid residues. Therefore, the properotion of C-terminal basic amino acids in a Mab can be analyzed through teh change in the proportion of charge variants following CPB treatment. (Kim, Arch. Pharm. Res. (2016) 38: 1472-1481)

Gadgil (US 16/340,822, published as US 2019/0263855) discloses a multi-column chromatography system such as a periodic counter current chromatography system (PCC) which includes carboxypeptidase B immobilized on sepharose.  The C-terminal lysine residues on H chain can be truncated by passing a harvest recovered form perfusion cell culture on a column which has CPB on sepharose. The CPB preferentially acts upon the basic amino acids, such as arginine and lysine and thus the resin can be used for removal of charged isoforms belowing to any class of antibodies. In one embodiment, a continuous process for reducing heterogeneity of an antibody includes a CPB-Sepharose column connect to a Protein A column. The flow through from the CPB-Sepharose column is directly loaded ontto the protein A column for capture step. 

As to Particular Antibodies

HER2: (Harris, US2009/0202546) discloses a composition comprising a main species HER2 antibody comprising varialbe light and heavy sequence and acidic variants of the main species, as identified by methods like WCX-10 CEX chromatography. Rat pharmacokinetic dats showed that although acidic variants are chmically different from the main peak, they have equivalent pharmacokinetics.

TNF-alpha: Fraunhoffer (US21009/0291062) discloses an adalimumab solution after DF/UF processing having 2.26% acidic region 1 and 11.81% acidic Region 2. 

Particular Purification Methods Used  (just brief review from perspective of variants — see the particular schemes in outline)

AEX: Linke (WO2012/015912) discloses methods for purifying a polypetpide from a solution containing the polypetpide and an acidic variant thereof such as a deamidated species by contacting the immunoconjugate wtih an AEX and eluting with a high salt buffer thereby separating the active immunoconjugate form the deamidated varient.

CEX: With CEX which allows for charge based elution, mostly acidic negatively charged species elute first and basic positively chard species elute toward the tail end of the antibody peak. The early eluting aggregates are negatively charged acidic varitns while the later eluting aggregates are positively chard basic variants. (Nti-Gyabaah, US 14/355014)

HIC: Acidic variants will elute first from the HIC column as they are less hydrophobic than basic charged aggregates. (Nti-Gyabaah, US 14/355014, published as US 2014/0288278) )