Next Article in Journal / Special Issue
IgE Antibodies: From Structure to Function and Clinical Translation
Previous Article in Journal
Preferential Identification of Agonistic OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized Mouse-Derived Yeast Surface Display Libraries
Open AccessReview

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

by Alain Beck 1,* and Hongcheng Liu 2,*
Biologics CMC and developability, IRPF, Center d’immunologie Pierre Fabre, St Julien-en-Genevois CEDEX, 74160 Saint-Julien en Genevois, France
Anokion, 50 Hampshire Street, Suite 402, Cambridge, MA 02139, USA
Authors to whom correspondence should be addressed.
Antibodies 2019, 8(1), 18;
Received: 22 December 2018 / Revised: 19 January 2019 / Accepted: 13 February 2019 / Published: 19 February 2019
(This article belongs to the Special Issue Structure and Function of Antibodies)


Recombinant monoclonal antibodies (mAbs) intended for therapeutic usage are required to be thoroughly characterized, which has promoted an extensive effort towards the understanding of the structures and heterogeneity of this major class of molecules. Batch consistency and comparability are highly relevant to the successful pharmaceutical development of mAbs and related products. Small structural modifications that contribute to molecule variants (or proteoforms) differing in size, charge or hydrophobicity have been identified. These modifications may impact (or not) the stability, pharmacokinetics, and efficacy of mAbs. The presence of the same type of modifications as found in endogenous immunoglobulin G (IgG) can substantially lower the safety risks of mAbs. The knowledge of modifications is also critical to the ranking of critical quality attributes (CQAs) of the drug and define the Quality Target Product Profile (QTPP). This review provides a summary of the current understanding of post-translational and physico-chemical modifications identified in recombinant mAbs and endogenous IgGs at physiological conditions.
Keywords: critical quality attributes; comparability; developability; glycosylation; quality target product profile; mass spectrometry; post-translational modifications; proteoforms; safety critical quality attributes; comparability; developability; glycosylation; quality target product profile; mass spectrometry; post-translational modifications; proteoforms; safety

1. Introduction

Recombinant monoclonal antibodies are heterogeneous due to post-translational modifications (PTMs) and physico-chemical transformations that could occur during their entire life-span. Understanding of the mechanisms and the ways to control the heterogeneity are essential to the successful clinical development of monoclonal antibody (mAb) therapeutics. Based on International Conference on Harmonization (ICH) Q6B, mAb variants can be classified as either “Product-related substances” or “Product-related impurities”. Product-related substances are defined as “Molecular variants of the desired product formed during manufacturer and/or storage which are active and have no deleterious effect on the safety and efficacy of the drug product. These variants possess properties comparable to the desired product and are not considered impurities.” Product-related impurities are defined as “Molecular variants of the desired products (e.g., precursors, certain degradation products arising during manufacture and/or storage) which do not have properties comparable to those of the desired product with respect to activity, efficacy, and safety.” Therefore, mAb variants are required to be thoroughly characterized to determine their chemical nature and impact on stability, activity, efficacy, and safety.
Because process changes are inevitable during process development, optimization and scale-up, a thorough understanding of mAb variants is also critical to demonstrating comparability between batches. The acceptance criteria to establish comparability for product-related impurities are more stringent than that of product-related substances (ICH Q5E). Failure to demonstrate the presence of the same type of modifications at comparable levels in post-change materials may require additional preclinical or clinical studies, due to safety concerns. Furthermore, mAb variants with different modifications might impact long-term stability and, thus, shelf-life, efficacy, and safety.
Therapeutic mAbs have evolved from a murine origin, to chimeric, and humanized or fully human to reduce immunogenicity, based on amino acid sequence homology. Generally, human-like modifications, identified as such by their presence in natural Immunoglobulin Gs (IgGs), pose a lower risk of immunogenicity.
This review focuses on the current understanding of the various types of modifications of mAbs, that can occur during manufacturing, storage, and post-administration in vivo or during clinical trials. Known modifications of human endogenous IgGs are also discussed. An overall comparison between the different modifications found in mAbs versus natural IgGs is presented in Table 1.

2. N-Terminal Modifications

N-terminal pyroglutamate (pyroGlu) is a common mAb modification resulting mainly from a non-enzymatic cyclization of N-terminal glutamine (Gln) [1,2,3,4,5]. At a much lower rate, N-terminal glutamate (Glu) can also be converted to pyroGlu [6,7,8]. Various environmental factors, such as buffer composition, pH, and temperature during cell culture and purification, can impact the conversion rates, which accounts for the varied levels of N-terminal pyroGlu found in mAbs [1,2,3,4,5]. Conversion of Glu to pyroGlu does not contribute as extensively to N-terminal heterogeneity as does the more commonly observed Gln to pyroGlu conversion because of the dramatic difference in the conversion rates. Cyclization of N-terminal Gln or Glu to pyroGlu reduces the molecular weight of a mAb by 17 Da or 18 Da, respectively. MAbs with the original Gln are more basic than those with pyroGlu [1,2,9], though, the presence of N-terminal pyroGlu has no impact on mAb structure and function [2,8]. The same conversion from Gln to pyroGlu is expected for mAbs in circulation because of the non-enzymatic nature of this reaction. N-terminal Glu has also been shown to be converted to pyroGlu in circulation [6].
Another common N-terminal modification is the incomplete removal of light chain or heavy chain signal peptides, which results in mAbs with truncated signal peptides of varying sizes [2,9,10,11,12,13,14]. The presence of signal peptides with different number and type of amino acids adds mass heterogeneity to mAbs. Interestingly, mAbs with signal peptides have been detected mainly as basic species [2,9,12,13,15], but rarely as acidic species [16]. The presence of low levels of signal peptides has no impact on potency [2,9,13] and pharmacokinetics (PK) in rats [13].
Although it is less common, N-terminal truncation has been reported. A combination of a murine signal peptide and antibody lambda light chain causes an alternative cleavage of the signal peptide resulting in a mAb with the loss of three amino acids from the light chain [17]. With the use of a specific tag to label N-terminal primary amine in combination with liquid chromatography mass spectrometry (LC-MS), an mAb variant with the loss of one amino acid from the light chain was observed [11].
Natural IgG contains approximately 1.8-mole pyroGlu/mole IgG [6]. Based on the different reaction rates, it is expected that most of the pyroGlu originates from N-terminal Gln, rather than Glu. Assuming that the stress condition for massive production of mAbs from ex vivo expression in host cell lines is the cause of N-terminal signal peptides and truncation, they are not expected to occur and have not been reported for natural IgGs.

3. Asn Deamidation

Asparagine (Asn) deamidation is almost a ubiquitous modification of mAbs and has been well studied because of its contribution to heterogeneity, and its potential impact on potency and immunogenicity. Asn residues in the complementarity determining regions (CDRs) are inherently susceptible to deamidation because of their relatively higher flexibility and exposure to solvents than at other locations [2,18,19,20,21,22]. Deamidation in CDRs can cause a substantial loss of potency [20,22,23,24]. In addition to deamidation in the CDRs, deamidation also occurs in susceptible Asn residues in the constant region. The most widely observed deamidation site is located in the fragment crystallizable (Fc) region within the amino acid sequence of SNGQPENNY [2,25,26,27,28,29]. Deamidation in the constant regions other than within the commonly observed sequence has also been reported [25,30]. When measured by differential scanning calorimetry, the fragment antigen binding (Fab) fragment with the deamidation product, isoaspartate (isoAsp), is less stable compared to Fab with the original Asn residue [22]. Deamidation increases the molecular weight of mAbs by 1 Da and generates acidic species [2,12,13,20,21,22,31,32]. Variants containing deamidation products are less hydrophobic than those with the original Asn residues [20,32]. Deamidation does not impact in vivo clearance [21,26]. Deamidation of Asn residues continues to occur in vivo in the CDRs [19,21,33,34] and Fc region [26] of mAbs. The in vivo deamidation kinetics can be fully predicted via in vitro stress studies under physiological conditions [19,26], which indicates the same non-enzymatic mechanism. It should be noted that it is important to optimize digestion procedures and distinguish procedure-induced artifact versus real for the accurate determination of Asn deamidation levels.
Natural human IgG has 23% deamidation at the conserved site in the Fc region, which is consistent with the molecules’ in vivo half-life [26]. The presence of high levels of deamidation in natural human IgG suggests that deamidation, at least at the conserved site, is not foreign to the immune system and, therefore, would not present an increased risk of immunogenicity.

4. Asp Isomerization

Aspartate (Asp) isomerization has been commonly observed in mAbs in CDRs due to higher levels of flexibility and exposure [15,18,20,35,36,37,38,39,40,41]. Isomerization of Asp in CDRs has been shown to cause a decrease in antigen binding affinity [20,35,36,39,42]. Since there is no charge difference between Asp and isoAsp, the observed decrease in potency is probably caused by conformational changes due to the introduction of a methyl group into the peptide backbone. Isomerization does not change mAb molecular weight; however, depending on the specific location, isomerization can either generate acidic [12] or basic [20,43] species. Similarly, isomerization could result in mAbs or their Fab fragments becoming either more [15,35,37,44] or less [20,45] hydrophobic. Isomerization is a non-enzymatic reaction with an optimal pH of around 5 [23,41]. Under physiological conditions, isomerization was not found to increase for 34 days [26]. Therefore, the level of isomerization is expected to be low in natural IgGs.

5. Succinimide

Succinimide is the reaction intermediate of both Asn deamidation and Asp isomerization and is commonly detected in CDRs [15,20,23,34,35,37,41,42,46]. The presence of succinimide in the CDR has been demonstrated to cause a decrease in potency [23,34,35,42]. Succinimide as a deamidation intermediate has also been detected in the conserved susceptible Asn deamidation site [25,28]. MAb variants containing succinimide from Asp isomerization have been shown to become more acidic [43] or basic [15,20,23,41], due to direct charge difference and conformational changes. Similarly, mAb or Fab with succinimide as isomerization intermediate could be more [35,37,44] or less hydrophobic [15]. It is worth mentioning that mAb variants containing succinimide, which alters the molecular weight difference by only 18 Da, have been reported to appear as a back shoulder of the main peak by size exclusion chromatography (SEC) [34,47], suggesting a substantial conformational difference. It has been shown that the succinimide residue contained in mAb was converted to Asp and isoAsp after administration to monkeys [34]. Due to its instability under physiological pH, succinimide is not expected to be detected in natural human IgG.

6. Oxidation

Methionine (Met) residue is the most commonly observed amino acid that is susceptible to oxidation in mAbs. Studies have shown oxidation of Met in the heavy chain CDR2 [39] or the frame work region [48]. Met oxidation did not show a negative impact on antigen binding in either case. Two conserved Met residues close to the heavy chain constant domain 2 (CH2)-CH3 domain interface have been shown to be susceptible to oxidation [48,49,50,51]. The addition of one oxygen atom increases mAb molecular weight by 16 Da. As expected, mAb variants with oxidized Met are less hydrophobic compared to the non-oxidized molecules [44,45,52,53]. Interestingly, one mAb with the Fc conserved Met oxidized appeared to be more basic [51], while, another mAb with an oxidized Met in the Fab region appeared to be more acidic [31]. Oxidation of the two conserved Met residues in the Fc region caused conformational changes mainly in the CH2 domain [49,54] along with a host of negative impacts, including decreased thermal stability, [48,49,50,55] increased aggregation [49,55], decreased complement-dependent cytotoxicity (CDC) [48], decreased binding affinity to neonatal Fc receptor (FcRn) [48,56,57] and shorter in vivo half-life [58].
Oxidation has also been observed at tryptophan (Trp) residues in mAbs [59,60,61,62]. Trp residues in CDRs are more susceptible to oxidation due to a higher level of solvent exposure [63]. Oxidation of Trp generates a number of species, the major ones having molecular weight increases of 16 Da, and 32 Da [62,64]. MAb variants with oxidized Trp are less hydrophobic [52]. Oxidation of Trp residues in the CDRs can lead to reduced potency, decreased thermal stability, and increased aggregation propensity [59,60,61]. Trp oxidation has also been demonstrated to cause yellow coloration of the mAb solution, [64] due to kynurenine formation.
Oxidation of Met and several other amino acids has been detected in natural human IgG [65,66]. Oxidative stress under various pathological conditions and the resulting reactive oxygen species are expected to cause oxidation of susceptible residues in natural IgGs, as one of the most abundant proteins in circulation.

7. Cysteine and Disulfide Bond

Theoretically, all cysteine residues of mAbs should be involved in the formation of either intra- or inter-chain disulfide bonds in a well-defined linkage pattern. However, several variants that deviate from the well-established IgG disulfide bond structure have been discovered. These variants include the presence of free cysteine (Cys) residues, alternative disulfide bond linkage (scrambling), trisulfide bonding, the formation of thioether, and cysteine racemization.
The presence of free cysteine can be classified into three scenarios. The first scenario is the widely-reported occurrence of free cysteine residues [67,68,69,70,71]. These free cysteines have been shown to lower thermal stability [67] and increase the formation of reducible covalent aggregates [72,73,74]. The second scenario is the detection of relatively high levels of free Cys often due to the incomplete formation of a particular disulfide bond, mainly in the heavy chain variable domain [4,37,44,75,76] or the disulfide bond between the light chain and heavy chain [13,77]. The incomplete variable domain disulfide bond reduces the potency of one mAb [44], but has no impact on a different mAb [75]. MAb variants with the incomplete heavy chain variable domain disulfide bond were separated as acidic species in one case [75], but basic species in the other case [4], indicating that a structural change was likely the cause of different chromatographic behaviors. MAb variants with the incomplete variable domain disulfide bond are more hydrophobic [15,37,44]. MAb variants without the disulfide bond between the light chain and heavy chain are enriched in the acidic species [13,77]. The incomplete heavy chain variable domain disulfide bond can be reformed in vivo [4]. In the third scenario, the mAbs contained an extra non-canonical cysteine residue, mostly in the CDRs. The extra Cys in mAbs can be modified by small thiol containing compounds, such as free cysteines [78,79,80,81], and glutathione, [80,81] or oxidized to form cysteine sulfinic or sulfonic acid [81]. Modification of Cys introduces molecular weight heterogeneity. In addition, mAbs with modified Cys are less hydrophobic compared to unmodified molecules [81]. Cysteinylation increases mAb molecular weight, causes the formation of acidic species, and decreases antigen binding [78]. Modification of the extra Cys residue also causes lower expression titer, decreased thermal stability, and higher propensity towards aggregation [78,80].
The alternative disulfide bond linkage was first discovered in IgG4 molecules, where the formation of two inter-heavy chain disulfide bonds is in equilibrium with the formation of two intra-chain disulfide bonds [82,83,84,85]. The direct outcome of this equilibrium is the formation of bispecific antibodies, which has been reported for both recombinant monoclonal antibodies and natural antibodies [82,84]. Mutation of the IgG4 hinge region amino acid sequence, CPSC, to the IgG1 amino acid sequence, CPPC, can eliminate the Fab-exchange phenomena [85,86,87], which has been employed as a strategy to create stable mAb therapeutics based on the IgG4 framework. Later, the alternative disulfide bond linkage in the hinge region of IgG2 antibodies was discovered, both in recombinant and in natural human IgG2 [88]. Different IgG2 isoforms showed a subtle difference in structure and thermal stability [89]. While having no difference in molecular weights, the three disulfide isoforms, A, B, and A/B, can be differentiated using several analytical methods. By ion-exchange chromatography, the B isoform appeared to be more acidic than A/B, followed by A [43,88]. By reversed-phase chromatography, the B isoform eluted from columns earlier than A/B, followed by A [89,90,91]. By capillary electrophoresis sodium dodecyl sulfate (CE-SDS), the A isoform migrated faster than A/B, whereas the B isoform migrated the slowest [88]. Depending on the specific molecule, different isoforms may or may not have an impact on potency [89]. The conversion from A to B through the A/B isoform continues in mAbs in circulation [91].
The thioether linkage was first discovered in an IgG1 antibody as a non-reducible species using reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and CE-SDS [92] Later, it was found that the thioether between the light and heavy chains can also be formed in mAbs in vivo and in human natural IgGs [93]. The rate of thioether formation in IgG1 containing the lambda chain is faster than the conversion rate in IgG1 containing the kappa chain [93]. The formation of thioether reduces mAb molecular weight by 32 Da due to the loss of a sulfur atom.
Trisulfide bond was first discovered in an IgG2 mAb [94]. It was later found that trisulfide bond occurs in all classes of mAbs and natural human IgGs, mainly between the light chain and heavy chain [93,95,96]. Trisulfide bond formation can be controlled by changing the feeding strategy [97] or removed by a cysteine wash step during the protein A chromatography step [98]. Trisulfide bond increases mAb molecular weight by 32 Da. A mAb variant with a trisulfide bond appeared to be more acidic compared to mAb with the typical disulfide bond pattern [94]. The presence of a trisulfide bond has no impact on antigen binding [95,98] or thermal stability [94].
The cysteine residues located in the heavy chains that are involved in the formation of the light and heavy chain disulfide bonds were also found to exist in the D form [99]. A detailed study showed that racemization occurred in both the heavy and light chain cysteine residues in IgG1 lambda, but only the heavy chain in IgG1 kappa in both mAbs and human natural IgG1 [100]. The level of cysteine racemization is much lower in IgG2 [100]. As both thioether and racemization are catalyzed by basic condition and involve the same disulfide bonds, a general base-catalyzed mechanism was proposed, where beta elimination of the disulfide bond results in the formation of a dehydroalanine residue and the dehydroalanine residue can either form a thioether bond or revert to the disulfide bond, where chirality is regained to result in a mixture of D- and L- cysteine residues [100].
Most of the modifications identified in mAbs, including free cysteine [67,101,102], alternative disulfide bond linkage for IgG4 [82,84] and IgG2 [88], thioether [93], trisulfide bond [93,95,96], and D-Cys from racemization [100] have also been reported in natural human IgGs. However, cysteinylation and the presence of incomplete disulfide bonds have not been reported in natural human IgGs. Given all the negative impacts of cysteinylation, this modification may have been eliminated from natural IgG during evolution. The same could be true for the presence of a single pair of incomplete disulfide bonds.

8. Glycosylation

Similar to natural IgG molecules, mAbs are N-glycosylated at the conserved Asn residues in the CH2 domain. In addition, mAbs may have N-linked oligosaccharides in the Fab region [103,104,105]. Heterogeneity related to these oligosaccharides arises mainly from galactosylation, fucosylation, and sialylation of the biantennary complex oligosaccharides. The presence of low abundance oligosaccharides, such as high mannose (Man), hybrid and bisecting oligosaccharides adds further heterogeneity to mAbs.
The three major glycoforms are the core-fucosylated structures with either zero (G0F), one (G1F) or two (G2F) galactose [104,106,107,108,109,110,111]. Galactose adds an additional mass of 162 Da. However, galactosylation has not been reported to cause mAb heterogeneity in charge or hydrophobicity. The slight separation of mAb variants with different levels of galactosylation is probably caused by conformational differences since galactose should not change the charge properties [77]. Galactosylation can cause subtle conformational changes around the glycosylation site [54,112,113,114,115]. Conflicting results have been reported regarding biological functions, but it is generally agreed that galactose might slightly impact CDC, but not antibody-dependent cellular cytotoxicity (ADCC) [106,113,114,115,116,117,118,119,120,121,122]. Galactosylation has no impact on mAb stability [113,114,123,124], nor half-life [103,117,125,126,127,128].
Because the absence of the core-fucose can result in enhanced ADCC [122,129], the level of core-fucose has attracted great attention for the development of mAb therapeutics, especially to establish comparability or biosimilarity. The attachment of fucose adds a mass of 146 Da. Besides mass heterogeneity, fucose has not been reported to have an impact on charge and hydrophobicity. The addition of a fucose only has a subtle impact on mAb structure [54,130,131]. For mAbs without the core-fucose, animal studies on half-life have shown conflicting results [132,133]. However, the half-life was found to be as expected in human studies [125,127]. The level of core-fucose needs to be evaluated based on the target and therapeutic goal to balance the risk versus benefit [134].
Besides the complex oligosaccharides, high mannose oligosaccharides have been commonly observed in mAbs [108,109,135]. In addition to mass heterogeneity, mAbs with high mannose oligosaccharides demonstrate a slightly different chromatographic separation when using Protein A or Protein G columns [136]. High mannose oligosaccharides cause a subtle conformational change and increase the flexibility of the CH2 domain [137,138]. Although high mannose decreased the thermal stability of mAbs, it had no impact on long-term stability [131], or aggregation propensity under accelerated conditions [124]. High mannose shows increased Fc gamma receptor binding and ADCC, due to the absence of core-fucose [139]. IgG with high mannose showed reduced activities mediated by the first subcomponent of the C1 complex (C1q) binding [140,141]. High mannose oligosaccharides with greater than five mannose residues are rapidly converted into a structure with only 5 mannose residues (Man5) in human circulation [125]. MAbs with high mannose are cleared at a faster rate compared to those with complex oligosaccharides in animals and humans [126,127,139,140,142].
Sialic acid and alpha 1,3-galactose are two low abundant oligosaccharides that require special attention primarily because of safety concerns. In general, the level of sialic acid of mAbs that are associated with the conserved Fc glycosylation site is low [103,109,135]. However, substantial amounts of sialic acid have been found in mAbs containing a Fab glycosylation site [103,135]. Sialic acid adds mass heterogeneity and generates acidic species [2,13,27], but does not impact antigen binding [2,13,117,119,143,144] and clearance [13,125,128]. Sialic acid has been shown to cause subtle conformational changes that are local to the glycosylation sites [137,138,145,146,147]. Studies have demonstrated that sialic acid exerts no or a negative impact on ADCC and CDC [117,119,143,144]. Among the two types of sialic acids, N-Acetylneuraminic acid (NANA) and N-Glycolylneuraminic acid (NGNA), the latter, which is commonly found in mAbs from murine cell lines [2,103,104,108], has been linked to immunogenicity [148]. Similar to sialic acid, mAbs expressed in murine cell lines may contain low levels of alpha 1,3 galactose when associated with the Fc [3,103,104,108,109,149] and at relatively higher levels for mAbs containing Fab glycosylation [135]. Alpha1,3 galactose is also considered immunogenic [150] when associated with Fab [151].
Several other types of oligosaccharides including hybrid, bisecting, and smaller structures, such as those lacking outer arm N-acetylglucosamine (GlcNAc) residues, are present in mAbs at extremely low levels. Hybrid, bisecting, and smaller oligosaccharides could cause a subtle conformational change [138] and have a minimal impact on ADCC [118,122,137,152] and clearance [125,126]. Because of their extremely low levels, these types of oligosaccharides are not expected to have a substantial impact on mAb therapeutic development from the safety and efficacy point of view.
The absence of oligosaccharides also contributes to mAb heterogeneity, though, at low levels [153,154,155,156]. MAbs lacking oligosaccharides showed significant conformational changes [157,158], decreased thermal stability [112,113,131,159,160], and increased aggregation propensity [123,161]. The absence of oligosaccharides has a substantial impact on ADCC and CDC [113,119,157]. Initially, animal studies showed that the absence of oligosaccharides either caused a faster clearance [157,162,163] or had no impact on half-life [127,157,160,164,165]. However, later human trials demonstrated that aglycosylated mAbs had a normal half-life [164].
Human IgG contains similar major oligosaccharide structures but higher structural diversity [108,109,166]. The levels of bisecting and sialic acid are higher in natural human IgGs compared to mAbs [108,109,166], while, high mannose oligosaccharides in natural human IgG are extremely low at approximately 0.1% [109,167]. Human IgGs have also been shown to have less than 0.2% aglycosylation [167]. NGNA and alpha 1,3 galactose, are absent from natural human IgGs [108,109,166,168].

9. Glycation

Glycation is a non-enzymatic reaction between reducing sugars and the primary amine of the lysine (Lys) side chain or the N-terminus of the light chain or heavy chain [169,170,171]. Glycation mainly occurs during cell culture as sugars are used as nutrients [169], and, to a lesser degree, during storage or accelerated conditions due to decomposition of the non-reducing sugars used in formulation [172,173]. A slightly increased level of glycation has been reported during the course of administration when a diluent containing sugars is used [174]. Glycation increases mAb molecular weight by 162 Da with each site of glycation and generates acidic species due to loss of positive charges of Lys side chains or N-termini [13,27,43,169,171]. Glycation also increases the aggregation propensity under accelerated condition [173]. Glycation in the CDRs has not been shown to decrease antigen binding [13,169,171], and even a substantial level of glycation does not affect Fc gamma, FcRn, and protein A binding [175]. Advanced glycation end products (AGEs) contribute to product coloration [176]. The presence of glycation does not impact PK in rats [13]. Glycation of mAbs continues to occur in circulation in humans at a rate that can be predicted via in vitro incubation under physiological conditions [175].
As expected, glycation has been detected in endogenous human IgG [175], further supporting the simple reaction mechanism between circulating IgGs and sugars in vivo.

10. C-Terminal Modifications

Mostly, mAbs are synthesized with the heavy chain C-terminal Lys, which can be removed during cell culture due to carboxypeptidase activity [177]. Incomplete removal results in mAbs with either zero, one or two C-terminal lysine at various levels [2,3,12,178,179]. When analyzed by mass spectrometry, heterogeneity caused by C-terminal lysine is reflected by peaks that differ in molecular weight by 128 Da. C-terminal Lys is a common cause of the generation of basic species [1,9,12,43,178,179,180]. The presence of C-terminal Lys results in the formation of less hydrophobic mAb variants [35,45]. C-terminal Lys does not impact mAb structure, stability, or biological functions including PK [2,9,13,180,181,182], though, one study demonstrated that the removal of C-terminal Lys is required for optimal CDC [183]. Interestingly, inclusion of the C-terminal Lys codon may impact mAb titer of cell culture [184]. C-terminal Lys can be rapidly removed from mAbs during circulation with a half-life of 62 minutes [185].
C-terminal amidation was first discovered in a recombinant monoclonal IgG1 antibody [186]. Later, it was found that C-terminal amidation is as common as C-terminal Lys removal [187]. C-terminal amidation is catalyzed by peptidylglycine alpha-amidating monooxygenase (PAM) [187]. The level of C-terminal amidation can be modulated by changing the copper concentration in the cell culture media [188] or via genetic engineering to reduce PAM activity [189]. Compared to mAbs without C-terminal Lys, a loss of glycine and conversion of the newly exposed amino acid carboxyl group to an amide group results in a net molecular weight decrease of 58 Da. MAbs with C-terminal amidation are separated as basic species [43,186,188]. MAb variants without C-terminal Lys or without both Lys and Gly showed no difference in structure, stability, function, and PK [181].
The overall level of C-terminal amidation in natural human IgG is extremely low, approximately 0.02% or lower [185,187].

11. Uncommon Modifications

Several of the reported modifications only contribute to mAb heterogeneity at very low levels or in only a limited number of cases.
Low level of sequence variation has been observed for several mAbs [190,191,192,193,194,195,196,197,198], which is expected to be the norm rather than the exception because of the inherent errors in protein transcription and translation. An mAb variant with the heavy chains containing amino acids that were coded by part of the intron sequence was also found [3]. Recombination between light chain and heavy chain sequences has been reported to result in a minor mAb species where the heavy chain containing a portion of the light chain sequence [199].
Aside from the few cases of amino acid variation, several rare chemical modifications can occur at various stages. Methylglyoxal generated during cell culture has been shown to modify an mAb at arginine (Arg) residues, resulting in molecular weight increases by 54 Da or 72 Da and generation of acidic species [200]. Metals can catalyze oxidative carbonylation of several surface-exposed residues including Arg, Proline (Pro), Lys, and Thr [201]. When exposed to light, histidine (His) can be oxidized [202], which can further lead to His–His cross-linking [203]. Cysteinylation, which frequently occurs at non-canonical cysteine residues, has also been reported at canonical cysteine residues in IgG2 [12,96] and is probably due to the relative instability of the IgG2 disulfide bond linkage around the hinge region. The presence of tyrosine sulfation resulted in the formation of a distinct acidic peak for a mAb expressed in Chinese hamster ovary (CHO) cells [204]. Modification of light chain and heavy chain N-termini by maleuric acid has been detected in a mAb expressed in transgenic goats [205]. During storage, the N-terminal primary amine or lysine side chain of mAbs can be modified by citric acid or its degradation products [206,207]. In addition to glycosylation of the conserved Asn residues in the Fc region or glycosylation of Asn in the consensus sequence in the variable domains, O-fucosylation of a serine residue in the light chain CDR1 [208] and N-glycosylation of Asn in non-consensus sequence and Gln [209,210] have also been reported.
MAbs expressed in mammalian cell lines have been extensively characterized. However, novel modifications are expected whenever new cell culture media or formulations are used. The use of alternative expression systems is also expected to lead to novel modifications that are specific to the selected organism. Novel and non-clinically qualified modifications naturally bear higher safety risks, and, thus, requires thorough evaluation.

12. Heterogeneity in the Broader Scheme

12.1. Stability

ICH Q6B states that “degradation of drug substance and drug product, which may occur during storage, should be considered when establishing specifications.” ICH Q6B also discusses the concept of “Release limits vs shelf-life limits”, where tighter release limits will ensure that product at the end of shelf life can meet the acceptance criteria to maintain safety and efficacy.
Regarding stability, the aforementioned PTMs can be classified into two categories. The first category includes modifications that are catalyzed by enzymatic reactions. Those modifications include signal peptides, various glycoforms, C-terminal Lys removal, and C-terminal amidation. These types of modifications are not expected to continue during storage because of the lack of their respective enzymes in the drug substance and drug product. However, the levels of these modifications can potentially impact other degradation pathways, and, thus, stability. For example, the subtle conformational difference in mAbs with various oligosaccharides and the substantial conformational difference caused by the lack of oligosaccharides are expected, at least in theory, to impact other modifications by modulating surface exposure and inter-molecule interactions. The second category includes modifications that are dependent only on environmental factors, such as pH, temperature, and light exposure. Modifications in this category include N-terminal Gln and Glu cyclization, deamidation, isomerization, succinimide intermediate formation, oxidation, cysteine and disulfide bond related modifications, and glycation. Modifications in this category are expected to continue to occur during storage.
Overall, PTMs are, either indirectly or directly, linked to mAb stability. Detailed characterization of drug substances at the time of lot release and understanding of the degradation pathways derived from forced degradation, and stability studies can ensure mAb stability during shelf-life for consistent safety and efficacy.

12.2. Comparability and Biosimilarity

Comparability is required when process changes are introduced, which is inevitable during development. Q5E states that “The demonstration of comparability does not necessarily mean that the quality attributes of the pre-change and post-change product are identical, but that they are highly similar and that the existing knowledge is sufficiently predictive to ensure that any differences in quality attributes have no adverse impact upon safety or efficacy of the drug product”. Scientific understanding of the chemical nature of PTMs and their impact on safety and efficacy is critical to establishing comparability, especially when a quality attribute is outside of the historical range.
MAb heterogeneity is also central to the development of biosimilar products. Given the requirement that the primary sequence of the originator and a biosimilar product should be identical, it becomes clear that similarity is mainly dependent on various PTMs.
In-depth characterization of mAb heterogeneity plays an essential role in establishing comparability and biosimilarity. The National Institute of Standards and Technology mAb (NISTmAb) tryptic peptide spectral library can be used as a good reference for those detailed comparisons [211], as it contains an extensive list of modifications, including the commonly observed analytical artifacts, which should be differentiated from true modifications.

12.3. Antibody-Drug Conjugate

Antibody-drug conjugates (ADCs) take advantage of the specificities of mAbs to deliver functional molecules to targets, and commonly, high toxicity compounds, to cancer cells [212]. MAb heterogeneity, thus, becomes an integral characteristic of ADCs, and exerts similar impact on structure, and stability. The microenvironment of the conjugation sites including solvent accessibility and charges has been demonstrated to have a substantial impact on the in vivo stability and activity of ADCs [213]. The presence of trisulfide bonds, for example, has also been shown to affect conjugation and the resulting drug-to-antibody ratio (DAR) [16,214]. Higher levels of heterogeneity have been reported for ADCs based on IgG2 mAbs, which are known for their various disulfide bond isoforms and difference in disulfide bond accessibility [215].

13. Conclusions

Heterogeneity is recognized as a common feature of mAbs due to modifications that cause IgG variants or proteoforms that differ in molecular weight, charge or hydrophobicity. MAb variants are required to be evaluated to establish their structure–function and safety relationships. In addition, different variants may have (or not) different impacts on stability, which is ultimately linked to safety and efficacy.
A wealth of information has been accumulated over the past decades. Such knowledge can be generally used to define the quality target product profile and applied to the assessment of developability of clinical candidates during the early phase of pharmaceutical development. Later in development, molecule-specific modifications are observed and managed throughout the lifecycle of the selected mAb.


This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest. Alain Beck is an employee of Institut de Recherche Pierre Fabre. Hongcheng Liu is an employee of Anokion.


ADCAntibody-drug conjugate
ADCCAntibody-dependent cellular cytotoxicity
AGEAdvanced glycation end product
C1qFirst subcomponent of the C1 complex
CDCComplement-dependent cytotoxicity
CDRComplementarity determining region
CE-SDSCapillary electrophoresis sodium dodecyl sulfate
CH2Heavy chain constant domain 2
CHOChinese hamster ovary
CQACritical quality attribute
FabFragment antigen binding
FcFragment crystallizable
FcRnNeonatal Fc receptor
ICHInternational Conference on Harmonization
IgGImmunoglobulin G
LC-MSLiquid chromatography mass spectrometry
mAbMonoclonal antibody
NANAN-Acetylneuraminic acid
NGNAN-Glycolylneuraminic acid
PAMPeptidylglycine alpha-amidating monooxygenase
PTMPosttranslational modification
QTPPQuality target product profile
SDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresis


  1. Moorhouse, K.G.; Nashabeh, W.; Deveney, J.; Bjork, N.S.; Mulkerrin, M.G.; Ryskamp, T. Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion. J. Pharm. Biomed. Anal. 1997, 16, 593–603. [Google Scholar] [CrossRef]
  2. Lyubarskaya, Y.; Houde, D.; Woodard, J.; Murphy, D.; Mhatre, R. Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity. Anal. Biochem. 2006, 348, 24–39. [Google Scholar] [CrossRef] [PubMed]
  3. Beck, A.; Bussat, M.C.; Zorn, N.; Robillard, V.; Klinguer-Hamour, C.; Chenu, S.; Goetsch, L.; Corvaïa, N.; Van Dorsselaer, A.; Haeuw, J.F. Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2005, 819, 203–218. [Google Scholar] [CrossRef] [PubMed]
  4. Ouellette, D.; Alessandri, L.; Chin, A.; Grinnell, C.; Tarcsa, E.; Radziejewski, C.; Correia, I. Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule. Anal. Biochem. 2010, 397, 37–47. [Google Scholar] [CrossRef] [PubMed]
  5. Dick, L.W., Jr.; Kim, C.; Qiu, D.; Cheng, K.C. Determination of the origin of the N-terminal pyro-glutamate variation in monoclonal antibodies using model peptides. Biotechnol. Bioeng. 2007, 97, 544–553. [Google Scholar] [CrossRef] [PubMed]
  6. Liu, Y.D.; Goetze, A.M.; Bass, R.B.; Flynn, G.C. N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies. J. Biol. Chem. 2011, 286, 11211–11217. [Google Scholar] [CrossRef] [PubMed]
  7. Chelius, D.; Jing, K.; Lueras, A.; Rehder, D.S.; Dillon, T.M.; Vizel, A.; Rajan, R.S.; Li, T.; Treuheit, M.J.; Bondarenko, P.V. Formation of pyroglutamic acid from N-terminal glutamic acid in immunoglobulin gamma antibodies. Anal. Chem. 2006, 78, 2370–2376. [Google Scholar] [CrossRef] [PubMed]
  8. Yu, L.; Vizel, A.; Huff, M.B.; Young, M.; Remmele, R.L., Jr.; He, B. Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development. J. Pharm. Biomed. Anal. 2006, 42, 455–463. [Google Scholar] [CrossRef] [PubMed]
  9. Meert, C.D.; Brady, L.J.; Guo, A.; Balland, A. Characterization of antibody charge heterogeneity resolved by preparative immobilized pH gradients. Anal. Chem. 2010, 82, 3510–3518. [Google Scholar] [CrossRef]
  10. Ying, H.; Liu, H. Identification of an alternative signal peptide cleavage site of mouse monoclonal antibodies by mass spectrometry. Immunol. Lett. 2007, 111, 66–68. [Google Scholar] [CrossRef]
  11. Ayoub, D.; Bertaccini, D.; Diemer, H.; Wagner-Rousset, E.; Colas, O.; Cianferani, S.; Van Dorsselaer, A.; Beck, A.; Schaeffer-Reiss, C. Characterization of the N-terminal heterogeneities of monoclonal antibodies using in-gel charge derivatization of alpha-amines and LC-MS/MS. Anal. Chem. 2015, 87, 3784–3790. [Google Scholar] [CrossRef] [PubMed]
  12. Neill, A.; Nowak, C.; Patel, R.; Ponniah, G.; Gonzalez, N.; Miano, D.; Liu, H. Characterization of Recombinant Monoclonal Antibody Charge Variants Using OFFGEL Fractionation, Weak Anion Exchange Chromatography, and Mass Spectrometry. Anal. Chem. 2015, 87, 6204–6211. [Google Scholar] [CrossRef] [PubMed]
  13. Khawli, L.A.; Goswami, S.; Hutchinson, R.; Kwong, Z.W.; Yang, J.; Wang, X.; Yao, Z.; Sreedhara, A.; Cano, T.; Tesar, D.; et al. Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats. MAbs 2010, 2, 613–624. [Google Scholar] [CrossRef][Green Version]
  14. Kotia, R.B.; Raghani, A.R. Analysis of monoclonal antibody product heterogeneity resulting from alternate cleavage sites of signal peptide. Anal. Biochem. 2010, 399, 190–195. [Google Scholar] [CrossRef] [PubMed]
  15. Sreedhara, A.; Cordoba, A.; Zhu, Q.; Kwong, J.; Liu, J. Characterization of the isomerization products of aspartate residues at two different sites in a monoclonal antibody. Pharm. Res. 2012, 29, 187–197. [Google Scholar] [CrossRef] [PubMed]
  16. Liu, H.; Ren, W.; Zong, L.; Zhang, J.; Wang, Y. Characterization of recombinant monoclonal antibody charge variants using WCX chromatography, icIEF and LC-MS/MS. Anal. Biochem. 2018, 564–565, 1–12. [Google Scholar] [CrossRef] [PubMed]
  17. Gibson, S.J.; Bond, N.J.; Milne, S.; Lewis, A.; Sheriff, A.; Pettman, G.; Pradhan, R.; Higazi, D.R.; Hatton, D. N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains. Biotechnol. Bioeng. 2017, 114, 1970–1977. [Google Scholar] [CrossRef] [PubMed]
  18. Sydow, J.F.; Lipsmeier, F.; Larraillet, V.; Hilger, M.; Mautz, B.; Molhoj, M.; Kuentzer, J.; Klostermann, S.; Schoch, J.; Voelger, H.R.; et al. Structure-based prediction of asparagine and aspartate degradation sites in antibody variable regions. PLoS ONE 2014, 9, e100736. [Google Scholar] [CrossRef]
  19. Tran, J.C.; Tran, D.; Hilderbrand, A.; Andersen, N.; Huang, T.; Reif, K.; Hotzel, I.; Stefanich, E.G.; Liu, Y.; Wang, J. Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate in Vivo Deamidation of Therapeutic Antibodies. Anal. Chem. 2016, 88, 11521–11526. [Google Scholar] [CrossRef]
  20. Harris, R.J.; Kabakoff, B.; Macchi, F.D.; Shen, F.J.; Kwong, M.; Andya, J.D.; Shire, S.J.; Bjork, N.; Totpal, K.; Chen, A.B. Identification of multiple sources of charge heterogeneity in a recombinant antibody. J. Chromatogr. B Biomed. Sci. Appl. 2001, 752, 233–245. [Google Scholar] [CrossRef]
  21. Huang, L.; Lu, J.; Wroblewski, V.J.; Beals, J.M.; Riggin, R.M. In vivo deamidation characterization of monoclonal antibody by LC/MS/MS. Anal. Chem. 2005, 77, 1432–1439. [Google Scholar] [CrossRef] [PubMed]
  22. Vlasak, J.; Bussat, M.C.; Wang, S.; Wagner-Rousset, E.; Schaefer, M.; Klinguer-Hamour, C.; Kirchmeier, M.; Corvaïa, N.; Ionescu, R.; Beck, A. Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody. Anal. Biochem. 2009, 392, 145–154. [Google Scholar] [CrossRef] [PubMed]
  23. Yan, B.; Steen, S.; Hambly, D.; Valliere-Douglass, J.; Vanden Bos, T.; Smallwood, S.; Yates, Z.; Arroll, T.; Han, Y.; Gadgil, H.; et al. Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain. J. Pharm. Sci. 2009, 98, 3509–3521. [Google Scholar] [CrossRef] [PubMed]
  24. Yang, X.; Xu, W.; Dukleska, S.; Benchaar, S.; Mengisen, S.; Antochshuk, V.; Cheung, J.; Mann, L.; Babadjanova, Z.; Rowand, J.; et al. Developability studies before initiation of process development: Improving manufacturability of monoclonal antibodies. MAbs 2013, 5, 787–794. [Google Scholar] [CrossRef] [PubMed]
  25. Chelius, D.; Rehder, D.S.; Bondarenko, P.V. Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. Anal. Chem. 2005, 77, 6004–6011. [Google Scholar] [CrossRef] [PubMed]
  26. Liu, Y.D.; van Enk, J.Z.; Flynn, G.C. Human antibody Fc deamidation in vivo. Biologicals 2009, 37, 313–322. [Google Scholar] [CrossRef] [PubMed]
  27. Xiao, Z.; Yin, X.; Han, L.; Sun, B.; Shen, Z.; Liu, W.; Yu, F. A comprehensive approach for evaluating charge heterogeneity in biosimilars. Eur. J. Pharm. Sci. 2018, 115, 19–24. [Google Scholar] [CrossRef]
  28. Sinha, S.; Zhang, L.; Duan, S.; Williams, T.D.; Vlasak, J.; Ionescu, R.; Elizabeth, M.T. Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody. Protein Sci. 2009, 18, 1573–1584. [Google Scholar] [CrossRef][Green Version]
  29. Gaza-Bulseco, G.; Li, B.; Bulseco, A.; Liu, H.C. Method to differentiate asn deamidation that occurred prior to and during sample preparation of a monoclonal antibody. Anal. Chem. 2008, 80, 9491–9498. [Google Scholar] [CrossRef]
  30. Zhang, Y.T.; Hu, J.; Pace, A.L.; Wong, R.; Wang, Y.J.; Kao, Y.H. Characterization of asparagine 330 deamidation in an Fc-fragment of IgG1 using cation exchange chromatography and peptide mapping. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2014, 965, 65–71. [Google Scholar] [CrossRef]
  31. Ponniah, G.; Kita, A.; Nowak, C.; Neill, A.; Kori, Y.; Rajendran, S.; Liu, H. Characterization of the acidic species of a monoclonal antibody using weak cation exchange chromatography and LC-MS. Anal. Chem. 2015, 87, 9084–9092. [Google Scholar] [CrossRef] [PubMed]
  32. King, C.; Patel, R.; Ponniah, G.; Nowak, C.; Neill, A.; Gu, Z.; Liu, H. Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2018, 1085, 96–103. [Google Scholar] [CrossRef] [PubMed]
  33. Bults, P.; Bischoff, R.; Bakker, H.; Gietema, J.A.; van de Merbel, N.C. LC-MS/MS-Based Monitoring of In Vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and Its Deamidation Products in Human Plasma. Anal. Chem. 2016, 88, 1871–1877. [Google Scholar] [CrossRef] [PubMed]
  34. Ouellette, D.; Chumsae, C.; Clabbers, A.; Radziejewski, C.; Correia, I. Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule. MAbs 2013, 5, 432–444. [Google Scholar] [CrossRef] [PubMed]
  35. Cacia, J.; Keck, R.; Presta, L.G.; Frenz, J. Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: Identification and effect on binding affinity. Biochemistry 1996, 35, 1897–1903. [Google Scholar] [CrossRef] [PubMed]
  36. Rehder, D.S.; Chelius, D.; McAuley, A.; Dillon, T.M.; Xiao, G.; Crouse-Zeineddini, J.; Vardanyan, L.; Perico, N.; Mukku, V.; Brems, D.N.; et al. Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma2 antibody highlights the role avidity plays in antibody activity. Biochemistry 2008, 47, 2518–2530. [Google Scholar] [CrossRef] [PubMed]
  37. Wakankar, A.A.; Borchardt, R.T.; Eigenbrot, C.; Shia, S.; Wang, Y.J.; Shire, S.J.; Liu, J.L. Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies. Biochemistry 2007, 46, 1534–1544. [Google Scholar] [CrossRef] [PubMed]
  38. Wakankar, A.A.; Liu, J.; Vandervelde, D.; Wang, Y.J.; Shire, S.J.; Borchardt, R.T. The effect of cosolutes on the isomerization of aspartic acid residues and conformational stability in a monoclonal antibody. J. Pharm. Sci. 2007, 96, 1708–1718. [Google Scholar] [CrossRef] [PubMed]
  39. Yan, Y.; Wei, H.; Fu, Y.; Jusuf, S.; Zeng, M.; Ludwig, R.; Krystek, S.R., Jr.; Chen, G.; Tao, L.; Das, T.K. Isomerization and Oxidation in the Complementarity-Determining Regions of a Monoclonal Antibody: A Study of the Modification-Structure-Function Correlations by Hydrogen-Deuterium Exchange Mass Spectrometry. Anal. Chem. 2016, 88, 2041–2050. [Google Scholar] [CrossRef] [PubMed]
  40. Xiao, G.; Bondarenko, P.V. Identification and quantification of degradations in the Asp-Asp motifs of a recombinant monoclonal antibody. J. Pharm. Biomed. Anal. 2008, 47, 23–30. [Google Scholar] [CrossRef] [PubMed]
  41. Chu, G.C.; Chelius, D.; Xiao, G.; Khor, H.K.; Coulibaly, S.; Bondarenko, P.V. Accumulation of succinimide in a recombinant monoclonal antibody in mildly acidic buffers under elevated temperatures. Pharm. Res. 2007, 24, 1145–1156. [Google Scholar] [CrossRef] [PubMed]
  42. Valliere-Douglass, J.; Jones, L.; Shpektor, D.; Kodama, P.; Wallace, A.; Balland, A.; Bailey, R.; Zhang, Y. Separation and characterization of an IgG2 antibody containing a cyclic imide in CDR1 of light chain by hydrophobic interaction chromatography and mass spectrometry. Anal. Chem. 2008, 80, 3168–3174. [Google Scholar] [CrossRef] [PubMed]
  43. Ponniah, G.; Nowak, C.; Neill, A.; Liu, H. Characterization of charge variants of a monoclonal antibody using weak anion exchange chromatography at subunit levels. Anal. Biochem. 2017, 520, 49–57. [Google Scholar] [CrossRef]
  44. Harris, R.J. Heterogeneity of recombinant antibodies: Linking structure to function. Dev. Biol. (Basel) 2005, 122, 117–127. [Google Scholar]
  45. Valliere-Douglass, J.; Wallace, A.; Balland, A. Separation of populations of antibody variants by fine tuning of hydrophobic-interaction chromatography operating conditions. J. Chromatogr. A 2008, 1214, 81–89. [Google Scholar] [CrossRef]
  46. Huang, H.Z.; Nichols, A.; Liu, D. Direct identification and quantification of aspartyl succinimide in an IgG2 mAb by RapiGest assisted digestion. Anal. Chem. 2009, 81, 1686–1692. [Google Scholar] [CrossRef] [PubMed]
  47. Nowak, C.; Ponniah, G.; Neill, A.; Liu, H. Characterization of succinimide stability during trypsin digestion for LC-MS analysis. Anal. Biochem. 2017, 526, 1–8. [Google Scholar] [CrossRef] [PubMed]
  48. Mo, J.; Yan, Q.; So, C.K.; Soden, T.; Lewis, M.J.; Hu, P. Understanding the Impact of Methionine Oxidation on the Biological Functions of IgG1 Antibodies Using Hydrogen/Deuterium Exchange Mass Spectrometry. Anal. Chem. 2016, 88, 9495–9502. [Google Scholar] [CrossRef]
  49. Liu, D.; Ren, D.; Huang, H.; Dankberg, J.; Rosenfeld, R.; Cocco, M.J.; Li, L.; Brems, D.N.; Remmele, R.L. Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation. Biochemistry 2008, 47, 5088–5100. [Google Scholar] [CrossRef]
  50. Liu, H.; Gaza-Bulseco, G.; Xiang, T.; Chumsae, C. Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody. Mol. Immunol. 2008, 45, 701–708. [Google Scholar] [CrossRef]
  51. Chumsae, C.; Gaza-Bulseco, G.; Sun, J.; Liu, H. Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2007, 850, 285–294. [Google Scholar] [CrossRef] [PubMed]
  52. Boyd, D.; Kaschak, T.; Yan, B. HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2011, 879, 955–960. [Google Scholar] [CrossRef] [PubMed]
  53. Lam, X.M.; Yang, J.Y.; Cleland, J.L. Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. J. Pharm. Sci. 1997, 86, 1250–1255. [Google Scholar] [CrossRef] [PubMed]
  54. Houde, D.; Peng, Y.; Berkowitz, S.A.; Engen, J.R. Post-translational modifications differentially affect IgG1 conformation and receptor binding. Mol. Cell Proteom. 2010, 9, 1716–1728. [Google Scholar] [CrossRef] [PubMed]
  55. Zhang, A.; Hu, P.; MacGregor, P.; Xue, Y.; Fan, H.; Suchecki, P.; Olszewski, L.; Liu, A. Understanding the conformational impact of chemical modifications on monoclonal antibodies with diverse sequence variation using hydrogen/deuterium exchange mass spectrometry and structural modeling. Anal. Chem. 2014, 86, 3468–3475. [Google Scholar] [CrossRef] [PubMed]
  56. Bertolotti-Ciarlet, A.; Wang, W.; Lownes, R.; Pristatsky, P.; Fang, Y.; McKelvey, T.; Li, Y.; Li, Y.; Drummond, J.; Prueksaritanont, T.; et al. Impact of methionine oxidation on the binding of human IgG1 to Fc Rn and Fc gamma receptors. Mol. Immunol. 2009, 46, 1878–1882. [Google Scholar] [CrossRef] [PubMed]
  57. Pan, H.; Chen, K.; Chu, L.; Kinderman, F.; Apostol, I.; Huang, G. Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A. and FcRn. Protein Sci. 2009, 18, 424–433. [Google Scholar] [CrossRef]
  58. Wang, W.; Vlasak, J.; Li, Y.; Pristatsky, P.; Fang, Y.; Pittman, T.; Roman, J.; Wang, Y.; Prueksaritanont, T.; Ionescu, R. Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies. Mol. Immunol. 2011, 48, 860–866. [Google Scholar] [CrossRef]
  59. Dashivets, T.; Stracke, J.; Dengl, S.; Knaupp, A.; Pollmann, J.; Buchner, J.; Schlothauer, T. Oxidation in the complementarity-determining regions differentially influences the properties of therapeutic antibodies. MAbs 2016, 8, 1525–1535. [Google Scholar] [CrossRef][Green Version]
  60. Qi, P.; Volkin, D.B.; Zhao, H.; Nedved, M.L.; Hughes, R.; Bass, R.; Yi, S.C.; Panek, M.E.; Wang, D.; Dalmonte, P. Characterization of the photodegradation of a human IgG1 monoclonal antibody formulated as a high-concentration liquid dosage form. J. Pharm. Sci. 2009, 98, 3117–3130. [Google Scholar] [CrossRef]
  61. Wei, Z.; Feng, J.; Lin, H.Y.; Mullapudi, S.; Bishop, E.; Tous, G.I.; Casas-Finet, J.; Hakki, F.; Strouse, R.; Schenerman, M.A. Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus. Anal. Chem. 2007, 79, 2797–2805. [Google Scholar] [CrossRef] [PubMed]
  62. Nowak, C.; Ponniah, G.; Cheng, G.; Kita, A.; Neill, A.; Kori, Y.; Liu, H. Liquid chromatography-fluorescence and liquid chromatography-mass spectrometry detection of tryptophan degradation products of a recombinant monoclonal antibody. Anal. Biochem. 2016, 496, 4–8. [Google Scholar] [CrossRef] [PubMed]
  63. Sharma, V.K.; Patapoff, T.W.; Kabakoff, B.; Pai, S.; Hilario, E.; Zhang, B.; Charlene, L.; Oleg, B.; Robert, F.K.; Ilya, C.; et al. In silico selection of therapeutic antibodies for development: Viscosity, clearance, and chemical stability. Proc. Natl. Acad. Sci. USA 2014, 111, 18601–18606. [Google Scholar] [CrossRef] [PubMed][Green Version]
  64. Li, Y.; Polozova, A.; Gruia, F.; Feng, J. Characterization of the degradation products of a color-changed monoclonal antibody: Tryptophan-derived chromophores. Anal. Chem. 2014, 86, 6850–6857. [Google Scholar] [CrossRef] [PubMed]
  65. Jasin, H.E. Oxidative modification of inflammatory synovial fluid immunoglobulin G. Inflammation 1993, 17, 167–181. [Google Scholar] [CrossRef] [PubMed]
  66. Lunec, J.; Blake, D.R.; McCleary, S.J.; Brailsford, S.; Bacon, P.A. Self-perpetuating mechanisms of immunoglobulin G aggregation in rheumatoid inflammation. J. Clin. Investig. 1985, 76, 2084–2090. [Google Scholar] [CrossRef]
  67. Lacy, E.R.; Baker, M.; Brigham-Burke, M. Free sulfhydryl measurement as an indicator of antibody stability. Anal. Biochem. 2008, 382, 66–68. [Google Scholar] [CrossRef]
  68. Chumsae, C.; Gaza-Bulseco, G.; Liu, H. Identification and localization of unpaired cysteine residues in monoclonal antibodies by fluorescence labeling and mass spectrometry. Anal. Chem. 2009, 81, 6449–6457. [Google Scholar] [CrossRef]
  69. Xiang, T.; Chumsae, C.; Liu, H. Localization and quantitation of free sulfhydryl in recombinant monoclonal antibodies by differential labeling with 12C and 13C iodoacetic acid and LC-MS analysis. Anal. Chem. 2009, 81, 8101–8108. [Google Scholar] [CrossRef]
  70. Cheng, Y.; Chen, M.T.; Patterson, L.C.; Yu, X.C.; Zhang, Y.T.; Burgess, B.L.; Chen, Y. Domain-specific free thiol variant characterization of an IgG1 by reversed-phase high-performance liquid chromatography mass spectrometry. Anal. Biochem. 2017, 519, 8–14. [Google Scholar] [CrossRef]
  71. Zhang, W.; Czupryn, M.J. Free sulfhydryl in recombinant monoclonal antibodies. Biotechnol. Prog. 2002, 18, 509–513. [Google Scholar] [CrossRef] [PubMed]
  72. Brych, S.R.; Gokarn, Y.R.; Hultgen, H.; Stevenson, R.J.; Rajan, R.; Matsumura, M. Characterization of antibody aggregation: Role of buried, unpaired cysteines in particle formation. J. Pharm. Sci. 2010, 99, 764–781. [Google Scholar] [CrossRef] [PubMed]
  73. Huh, J.H.; White, A.J.; Brych, S.R.; Franey, H.; Matsumura, M. The identification of free cysteine residues within antibodies and a potential role for free cysteine residues in covalent aggregation because of agitation stress. J. Pharm. Sci. 2013, 102, 1701–1711. [Google Scholar] [CrossRef] [PubMed]
  74. Van Buren, N.; Rehder, D.; Gadgil, H.; Matsumura, M.; Jacob, J. Elucidation of two major aggregation pathways in an IgG2 antibody. J. Pharm. Sci. 2009, 98, 3013–3030. [Google Scholar] [CrossRef] [PubMed]
  75. Zhang, T.; Zhang, J.; Hewitt, D.; Tran, B.; Gao, X.; Qiu, Z.J.; Tejada, M.; Gazzano-Santoro, H.; Kao, Y.H. Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody. Anal. Chem. 2012, 84, 7112–7123. [Google Scholar] [CrossRef]
  76. Chaderjian, W.B.; Chin, E.T.; Harris, R.J.; Etcheverry, T.M. Effect of copper sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed. Biotechnol. Prog. 2005, 21, 550–553. [Google Scholar] [CrossRef]
  77. Miao, S.; Xie, P.; Zou, M.; Fan, L.; Liu, X.; Zhou, Y.; Zhao, L.; Ding, D.; Wang, H.; Tan, W.S. Identification of multiple sources of the acidic charge variants in an IgG1 monoclonal antibody. Appl. Microbiol. Biotechnol. 2017, 101, 5627–5638. [Google Scholar] [CrossRef]
  78. Banks, D.D.; Gadgil, H.S.; Pipes, G.D.; Bondarenko, P.V.; Hobbs, V.; Scavezze, J.L.; Kim, J.; Jiang, X.R.; Mukku, V.; Dillon, T.M. Removal of cysteinylation from an unpaired sulfhydryl in the variable region of a recombinant monoclonal IgG1 antibody improves homogeneity, stability, and biological activity. J. Pharm. Sci. 2008, 97, 775–790. [Google Scholar] [CrossRef]
  79. Gadgil, H.S.; Bondarenko, P.V.; Pipes, G.D.; Dillon, T.M.; Banks, D.; Abel, J.; Kleemann, G.R.; Treuheit, M.J. Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis. Anal. Biochem. 2006, 355, 165–174. [Google Scholar] [CrossRef]
  80. Buchanan, A.; Clementel, V.; Woods, R.; Harn, N.; Bowen, M.A.; Mo, W.; Popovic, B.; Bishop, S.M.; Dall’Acqua, W.; Minter, R.; et al. Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression. MAbs 2013, 5, 255–262. [Google Scholar] [CrossRef][Green Version]
  81. McSherry, T.; McSherry, J.; Ozaeta, P.; Longenecker, K.; Ramsay, C.; Fishpaugh, J.; Allen, S. Cysteinylation of a monoclonal antibody leads to its inactivation. MAbs 2016, 8, 718–725. [Google Scholar] [CrossRef] [PubMed]
  82. Schuurman, J.; Van Ree, R.; Perdok, G.J.; Van Doorn, H.R.; Tan, K.Y.; Aalberse, R.C. Normal human immunoglobulin G4 is bispecific: It has two different antigen-combining sites. Immunology 1999, 97, 693–698. [Google Scholar] [CrossRef] [PubMed]
  83. Aalberse, R.C.; Schuurman, J. IgG4 breaking the rules. Immunology 2002, 105, 9–19. [Google Scholar] [CrossRef] [PubMed][Green Version]
  84. van der Neut Kolfschoten, M.; Schuurman, J.; Losen, M.; Bleeker, W.K.; Martinez-Martinez, P.; Vermeulen, E.; den Bleker, T.H.; Wiegman, L.; Vink, T.; Aarden, L.A.; et al. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science 2007, 317, 1554–1557. [Google Scholar] [CrossRef] [PubMed]
  85. Schuurman, J.; Perdok, G.J.; Gorter, A.D.; Aalberse, R.C. The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds. Mol. Immunol. 2001, 38, 1–8. [Google Scholar] [CrossRef]
  86. Bloom, J.W.; Madanat, M.S.; Marriott, D.; Wong, T.; Chan, S.Y. Intrachain disulfide bond in the core hinge region of human IgG4. Protein Sci. 1997, 6, 407–415. [Google Scholar] [CrossRef]
  87. Angal, S.; King, D.J.; Bodmer, M.W.; Turner, A.; Lawson, A.D.; Roberts, G.; Pedley, B.; Adair, J.R. A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody. Mol. Immunol. 1993, 30, 105–108. [Google Scholar] [CrossRef]
  88. Wypych, J.; Li, M.; Guo, A.; Zhang, Z.; Martinez, T.; Allen, M.J.; Fodor, S.; Kelner, D.N.; Flynn, G.C.; Liu, Y.D.; et al. Human IgG2 antibodies display disulfide-mediated structural isoforms. J. Biol. Chem. 2008, 283, 16194–16205. [Google Scholar] [CrossRef]
  89. Dillon, T.M.; Ricci, M.S.; Vezina, C.; Flynn, G.C.; Liu, Y.D.; Rehder, D.S.; Plant, M.; Henkle, B.; Li, Y.; Deechongkit, S.; et al. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J. Biol. Chem. 2008, 283, 16206–16215. [Google Scholar] [CrossRef]
  90. Dillon, T.M.; Bondarenko, P.V.; Rehder, D.S.; Pipes, G.D.; Kleemann, G.R.; Ricci, M.S. Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability. J. Chromatogr. A 2006, 1120, 112–120. [Google Scholar] [CrossRef]
  91. Liu, Y.D.; Chen, X.; Enk, J.Z.; Plant, M.; Dillon, T.M.; Flynn, G.C. Human IgG2 antibody disulfide rearrangement in vivo. J. Biol. Chem. 2008, 283, 29266–29272. [Google Scholar] [CrossRef] [PubMed]
  92. Tous, G.I.; Wei, Z.; Feng, J.; Bilbulian, S.; Bowen, S.; Smith, J.; Strouse, R.; McGeehan, P.; Casas-Finet, J.; Schenerman, M.A. Characterization of a novel modification to monoclonal antibodies: Thioether cross-link of heavy and light chains. Anal. Chem. 2005, 77, 2675–2682. [Google Scholar] [CrossRef] [PubMed]
  93. Zhang, Q.; Schenauer, M.R.; McCarter, J.D.; Flynn, G.C. IgG1 thioether bond formation in vivo. J. Biol. Chem. 2013, 288, 16371–16382. [Google Scholar] [CrossRef] [PubMed]
  94. Pristatsky, P.; Cohen, S.L.; Krantz, D.; Acevedo, J.; Ionescu, R.; Vlasak, J. Evidence for trisulfide bonds in a recombinant variant of a human IgG2 monoclonal antibody. Anal. Chem. 2009, 81, 6148–6155. [Google Scholar] [CrossRef] [PubMed]
  95. Gu, S.; Wen, D.; Weinreb, P.H.; Sun, Y.; Zhang, L.; Foley, S.F.; Kshirsagar, R.; Evans, D.; Mi, S.; Meier, W.; et al. Characterization of trisulfide modification in antibodies. Anal. Biochem. 2010, 400, 89–98. [Google Scholar] [CrossRef] [PubMed]
  96. Kita, A.; Ponniah, G.; Nowak, C.; Liu, H. Characterization of Cysteinylation and Trisulfide Bonds in a Recombinant Monoclonal Antibody. Anal. Chem. 2016, 88, 5430–5437. [Google Scholar] [CrossRef] [PubMed]
  97. Kshirsagar, R.; McElearney, K.; Gilbert, A.; Sinacore, M.; Ryll, T. Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture. Biotechnol. Bioeng. 2012, 109, 2523–2532. [Google Scholar] [CrossRef]
  98. Aono, H.; Wen, D.; Zang, L.; Houde, D.; Pepinsky, R.B.; Evans, D.R. Efficient on-column conversion of IgG1 trisulfide linkages to native disulfides in tandem with Protein A affinity chromatography. J. Chromatogr. A 2010, 1217, 5225–5232. [Google Scholar] [CrossRef]
  99. Amano, M.; Hasegawa, J.; Kobayashi, N.; Kishi, N.; Nakazawa, T.; Uchiyama, S.; Fukui, K. Specific racemization of heavy-chain cysteine-220 in the hinge region of immunoglobulin gamma 1 as a possible cause of degradation during storage. Anal. Chem. 2011, 83, 3857–3864. [Google Scholar] [CrossRef]
  100. Zhang, Q.; Flynn, G.C. Cysteine racemization on IgG heavy and light chains. J. Biol. Chem. 2013, 288, 34325–34335. [Google Scholar] [CrossRef]
  101. Gevondyan, N.M.; Volynskaia, A.M.; Gevondyan, V.S. Four free cysteine residues found in human IgG1 of healthy donors. Biochemistry (Mosc.) 2006, 71, 279–284. [Google Scholar] [CrossRef] [PubMed]
  102. Schauenstein, E.; Dachs, F.; Reiter, M.; Gombotz, H.; List, W. Labile disulfide bonds and free thiol groups in human IgG. I. Assignment to IgG1 and IgG2 subclasses. Int. Arch. Allergy Appl. Immunol. 1986, 80, 174–179. [Google Scholar] [CrossRef] [PubMed]
  103. Huang, L.; Biolsi, S.; Bales, K.R.; Kuchibhotla, U. Impact of variable domain glycosylation on antibody clearance: An LC/MS characterization. Anal. Biochem. 2006, 349, 197–207. [Google Scholar] [CrossRef] [PubMed]
  104. Qian, J.; Liu, T.; Yang, L.; Daus, A.; Crowley, R.; Zhou, Q. Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion. Anal. Biochem. 2007, 364, 8–18. [Google Scholar] [PubMed]
  105. Lim, A.; Reed-Bogan, A.; Harmon, B.J. Glycosylation profiling of a therapeutic recombinant monoclonal antibody with two N-linked glycosylation sites using liquid chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer. Anal. Biochem. 2008, 375, 163–172. [Google Scholar] [CrossRef] [PubMed]
  106. Raju, T.S.; Jordan, R.E. Galactosylation variations in marketed therapeutic antibodies. MAbs 2012, 4, 385–391. [Google Scholar] [CrossRef][Green Version]
  107. Schiestl, M.; Stangler, T.; Torella, C.; Cepeljnik, T.; Toll, H.; Grau, R. Acceptable changes in quality attributes of glycosylated biopharmaceuticals. Nat. Biotechnol. 2011, 29, 310–312. [Google Scholar] [CrossRef]
  108. Maeda, E.; Kita, S.; Kinoshita, M.; Urakami, K.; Hayakawa, T.; Kakehi, K. Analysis of nonhuman N-glycans as the minor constituents in recombinant monoclonal antibody pharmaceuticals. Anal. Chem. 2012, 84, 2373–2379. [Google Scholar] [CrossRef]
  109. Stadlmann, J.; Pabst, M.; Kolarich, D.; Kunert, R.; Altmann, F. Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides. Proteomics 2008, 8, 2858–2871. [Google Scholar] [CrossRef]
  110. Kamoda, S.; Ishikawa, R.; Kakehi, K. Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies. J. Chromatogr. A 2006, 1133, 332–339. [Google Scholar] [CrossRef]
  111. Giorgetti, J.; D’Atri, V.; Canonge, J.; Lechner, A.; Guillarme, D.; Colas, O.; Wagner-Rousset, E.; Beck, A.; Leize-Wagner, E.; François, Y.-N. Monoclonal antibody N-glycosylation profiling using capillary electrophoresis—Mass spectrometry: Assessment and method validation. Talanta 2018, 178, 530–537. [Google Scholar] [CrossRef] [PubMed]
  112. Ghirlando, R.; Lund, J.; Goodall, M.; Jefferis, R. Glycosylation of human IgG-Fc: Influences on structure revealed by differential scanning micro-calorimetry. Immunol. Lett. 1999, 68, 47–52. [Google Scholar] [CrossRef]
  113. Mimura, Y.; Church, S.; Ghirlando, R.; Ashton, P.R.; Dong, S.; Goodall, M.; Lund, J.; Jefferis, R. The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: Properties of a series of truncated glycoforms. Mol. Immunol. 2000, 37, 697–706. [Google Scholar] [CrossRef]
  114. Mimura, Y.; Sondermann, P.; Ghirlando, R.; Lund, J.; Young, S.P.; Goodall, M.; Jefferis, R. Role of oligosaccharide residues of IgG1-Fc in Fc gamma RIIb binding. J. Biol. Chem. 2001, 276, 45539–45547. [Google Scholar] [CrossRef] [PubMed]
  115. Yamaguchi, Y.; Nishimura, M.; Nagano, M.; Yagi, H.; Sasakawa, H.; Uchida, K.; Shitara, K.; Kato, K. Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy. Biochim. Biophys. Acta 2006, 1760, 693–700. [Google Scholar] [CrossRef] [PubMed]
  116. Raju, T.S. Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr. Opin. Immunol. 2008, 20, 471–478. [Google Scholar] [CrossRef] [PubMed]
  117. Wright, A.; Morrison, S.L. Effect of C2-associated carbohydrate structure on Ig effector function: Studies with chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells. J. Immunol. 1998, 160, 3393–3402. [Google Scholar]
  118. Hodoniczky, J.; Zheng, Y.Z.; James, D.C. Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol. Prog. 2005, 21, 1644–1652. [Google Scholar] [CrossRef]
  119. Boyd, P.N.; Lines, A.C.; Patel, A.K. The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. Mol. Immunol. 1995, 32, 1311–1318. [Google Scholar] [CrossRef]
  120. Nimmerjahn, F.; Anthony, R.M.; Ravetch, J.V. Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity. Proc. Natl. Acad. Sci. USA 2007, 104, 8433–8437. [Google Scholar] [CrossRef][Green Version]
  121. Groenink, J.; Spijker, J.; van den Herik-Oudijk, I.E.; Boeije, L.; Rook, G.; Aarden, L.; Smeenk, R.; van de Winkel, J.G.; van den Broek, M.F. On the interaction between agalactosyl IgG and Fc gamma receptors. Eur. J. Immunol. 1996, 26, 1404–1407. [Google Scholar] [CrossRef] [PubMed]
  122. Shinkawa, T.; Nakamura, K.; Yamane, N.; Shoji-Hosaka, E.; Kanda, Y.; Sakurada, M.; Uchida, K.; Anazawa, H.; Satoh, M.; Yamasaki, M.; et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem. 2003, 278, 3466–3473. [Google Scholar] [CrossRef] [PubMed]
  123. Onitsuka, M.; Kawaguchi, A.; Asano, R.; Kumagai, I.; Honda, K.; Ohtake, H.; Omasa, T. Glycosylation analysis of an aggregated antibody produced by Chinese hamster ovary cells in bioreactor culture. J. Biosci. Bioeng. 2014, 117, 639–644. [Google Scholar] [CrossRef] [PubMed]
  124. Lu, Y.; Westland, K.; Ma, Y.H.; Gadgil, H. Evaluation of effects of Fc domain high-mannose glycan on antibody stability. J. Pharm. Sci. 2012, 101, 4107–4117. [Google Scholar] [CrossRef] [PubMed]
  125. Chen, X.; Liu, Y.D.; Flynn, G.C. The effect of Fc glycan forms on human IgG2 antibody clearance in humans. Glycobiology 2009, 19, 240–249. [Google Scholar] [CrossRef] [PubMed]
  126. Alessandri, L.; Ouellette, D.; Acquah, A.; Rieser, M.; Leblond, D.; Saltarelli, M.; Radziejewski, C.; Fujimori, T.; Correia, I. Increased serum clearance of oligomannose species present on a human IgG1 molecule. MAbs 2012, 4, 509–520. [Google Scholar] [CrossRef] [PubMed][Green Version]
  127. Goetze, A.M.; Liu, Y.D.; Zhang, Z.; Shah, B.; Lee, E.; Bondarenko, P.V.; Flynn, G.C. High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology 2011, 21, 949–959. [Google Scholar] [CrossRef] [PubMed][Green Version]
  128. Millward, T.A.; Heitzmann, M.; Bill, K.; Langle, U.; Schumacher, P.; Forrer, K. Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice. Biologicals 2008, 36, 41–47. [Google Scholar] [CrossRef]
  129. Shields, R.L.; Lai, J.; Keck, R.; O’Connell, L.Y.; Hong, K.; Meng, Y.G.; Weikert, S.H.; Presta, L.G. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biol. Chem. 2002, 277, 26733–26740. [Google Scholar] [CrossRef]
  130. Matsumiya, S.; Yamaguchi, Y.; Saito, J.; Nagano, M.; Sasakawa, H.; Otaki, S.; Satoh, M.; Shitara, K.; Kato, K. Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1. J. Mol. Biol. 2007, 368, 767–779. [Google Scholar] [CrossRef]
  131. Zheng, K.; Yarmarkovich, M.; Bantog, C.; Bayer, R.; Patapoff, T.W. Influence of glycosylation pattern on the molecular properties of monoclonal antibodies. MAbs 2014, 6, 649–658. [Google Scholar] [CrossRef] [PubMed][Green Version]
  132. Junttila, T.T.; Parsons, K.; Olsson, C.; Lu, Y.; Xin, Y.; Theriault, J.; Crocker, L.; Pabonan, O.; Baginski, T.; Meng, G.; et al. Superior in vivo efficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancer. Cancer Res. 2010, 70, 4481–4489. [Google Scholar] [CrossRef]
  133. Kanda, Y.; Yamada, T.; Mori, K.; Okazaki, A.; Inoue, M.; Kitajima-Miyama, K.; Kuni-Kamochi, R.; Nakano, R.; Yano, K.; Kakita, S.; et al. Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: The high-mannose, hybrid, and complex types. Glycobiology 2007, 17, 104–118. [Google Scholar] [CrossRef] [PubMed]
  134. Jiang, X.R.; Song, A.; Bergelson, S.; Arroll, T.; Parekh, B.; May, K.; Chung, S.; Strouse, R.; Mire-Sluis, A.; Schenerman, M. Advances in the assessment and control of the effector functions of therapeutic antibodies. Nat. Rev. Drug Discov. 2011, 10, 101–111. [Google Scholar] [CrossRef] [PubMed]
  135. Largy, E.; Cantais, F.; Van Vyncht, G.; Beck, A.; Delobel, A. Orthogonal liquid chromatography-mass spectrometry methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level. J. Chromatogr. A 2017, 1498, 128–146. [Google Scholar] [CrossRef] [PubMed]
  136. Gaza-Bulseco, G.; Hickman, K.; Sinicropi-Yao, S.; Hurkmans, K.; Chumsae, C.; Liu, H. Effect of the conserved oligosaccharides of recombinant monoclonal antibodies on the separation by protein A and protein G chromatography. J. Chromatogr. A 2009, 1216, 2382–2387. [Google Scholar] [CrossRef] [PubMed]
  137. Falck, D.; Jansen, B.C.; Plomp, R.; Reusch, D.; Haberger, M.; Wuhrer, M. Glycoforms of Immunoglobulin G Based Biopharmaceuticals Are Differentially Cleaved by Trypsin Due to the Glycoform Influence on Higher-Order Structure. J. Proteome Res. 2015, 14, 4019–4028. [Google Scholar] [CrossRef] [PubMed]
  138. Fang, J.; Richardson, J.; Du, Z.; Zhang, Z. Effect of Fc-Glycan Structure on the Conformational Stability of IgG Revealed by Hydrogen/Deuterium Exchange and Limited Proteolysis. Biochemistry 2016, 55, 860–868. [Google Scholar] [CrossRef]
  139. Yu, M.; Brown, D.; Reed, C.; Chung, S.; Lutman, J.; Stefanich, E.; Wong, A.; Stephan, J.P.; Bayer, R. Production, characterization, and pharmacokinetic properties of antibodies with N-linked mannose-5 glycans. MAbs 2012, 4, 475–487. [Google Scholar] [CrossRef]
  140. Wright, A.; Morrison, S.L. Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1. J. Exp. Med. 1994, 180, 1087–1096. [Google Scholar] [CrossRef][Green Version]
  141. Zhou, Q.; Shankara, S.; Roy, A.; Qiu, H.; Estes, S.; McVie-Wylie, A.; Culm-Merdek, K.; Park, A.; Pan, C.; Edmunds, T. Development of a simple and rapid method for producing non-fucosylated oligomannose containing antibodies with increased effector function. Biotechnol. Bioeng. 2008, 99, 652–665. [Google Scholar] [CrossRef] [PubMed]
  142. Liu, Y.D.; Flynn, G.C. Effect of high mannose glycan pairing on IgG antibody clearance. Biologicals 2016, 44, 163–169. [Google Scholar] [CrossRef] [PubMed]
  143. Naso, M.F.; Tam, S.H.; Scallon, B.J.; Raju, T.S. Engineering host cell lines to reduce terminal sialylation of secreted antibodies. MAbs 2010, 2, 519–527. [Google Scholar] [CrossRef] [PubMed]
  144. Scallon, B.J.; Tam, S.H.; McCarthy, S.G.; Cai, A.N.; Raju, T.S. Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol. Immunol. 2007, 44, 1524–1534. [Google Scholar] [CrossRef] [PubMed]
  145. Ahmed, A.A.; Giddens, J.; Pincetic, A.; Lomino, J.V.; Ravetch, J.V.; Wang, L.X.; Bjorkman, P.J. Structural characterization of anti-inflammatory immunoglobulin G Fc proteins. J. Mol. Biol. 2014, 426, 3166–3179. [Google Scholar] [CrossRef] [PubMed]
  146. Barb, A.W.; Meng, L.; Gao, Z.; Johnson, R.W.; Moremen, K.W.; Prestegard, J.H. NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation. Biochemistry 2012, 51, 4618–4626. [Google Scholar] [CrossRef] [PubMed]
  147. Crispin, M.; Yu, X.; Bowden, T.A. Crystal structure of sialylated IgG Fc: Implications for the mechanism of intravenous immunoglobulin therapy. Proc. Natl. Acad. Sci. USA 2013, 110, E3544–E3546. [Google Scholar] [CrossRef] [PubMed]
  148. Ghaderi, D.; Taylor, R.E.; Padler-Karavani, V.; Diaz, S.; Varki, A. Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat. Biotechnol. 2010, 28, 863–867. [Google Scholar] [CrossRef] [PubMed][Green Version]
  149. Sheeley, D.M.; Merrill, B.M.; Taylor, L.C. Characterization of monoclonal antibody glycosylation: Comparison of expression systems and identification of terminal alpha-linked galactose. Anal. Biochem. 1997, 247, 102–110. [Google Scholar] [CrossRef]
  150. Chung, C.H.; Mirakhur, B.; Chan, E.; Le, Q.T.; Berlin, J.; Morse, M.; Murphy, B.A.; Satinover, S.M.; Hosen, J.; Mauro, D.; et al. Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N. Engl. J. Med. 2008, 358, 1109–1117. [Google Scholar] [CrossRef]
  151. Lammerts van Bueren, J.J.; Rispens, T.; Verploegen, S.; van der Palen-Merkus, T.; Stapel, S.; Workman, L.J.; James, H.; van Berkel, P.H.; van de Winkel, J.G.; Platts-Mills, T.A.; et al. Anti-galactose-alpha-1,3-galactose IgE from allergic patients does not bind alpha-galactosylated glycans on intact therapeutic antibody Fc domains. Nat. Biotechnol. 2011, 29, 574–576. [Google Scholar] [CrossRef] [PubMed]
  152. Umana, P.; Jean-Mairet, J.; Moudry, R.; Amstutz, H.; Bailey, J.E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 1999, 17, 176–180. [Google Scholar] [CrossRef] [PubMed]
  153. Chen, X.; Tang, K.; Lee, M.; Flynn, G.C. Microchip assays for screening monoclonal antibody product quality. Electrophoresis 2008, 29, 4993–5002. [Google Scholar] [CrossRef] [PubMed]
  154. Rustandi, R.R.; Washabaugh, M.W.; Wang, Y. Applications of CE SDS gel in development of biopharmaceutical antibody-based products. Electrophoresis 2008, 29, 3612–3620. [Google Scholar] [CrossRef] [PubMed]
  155. Salas-Solano, O.; Tomlinson, B.; Du, S.; Parker, M.; Strahan, A.; Ma, S. Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies. Anal. Chem. 2006, 78, 6583–6594. [Google Scholar] [CrossRef] [PubMed]
  156. Smith, M.T.; Zhang, S.; Adams, T.; DiPaolo, B.; Dally, J. Establishment and validation of a microfluidic capillary gel electrophoresis platform method for purity analysis of therapeutic monoclonal antibodies. Electrophoresis 2017, 38, 1353–1365. [Google Scholar] [CrossRef] [PubMed]
  157. Tao, M.H.; Morrison, S.L. Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J. Immunol. 1989, 143, 2595–2601. [Google Scholar]
  158. Raju, T.S.; Scallon, B.J. Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain. Biochem. Biophys. Res. Commun. 2006, 341, 797–803. [Google Scholar] [CrossRef]
  159. More, A.S.; Toprani, V.M.; Okbazghi, S.Z.; Kim, J.H.; Joshi, S.B.; Middaugh, C.R.; Tolbert, T.J.; Volkin, D.B. Correlating the Impact of Well-Defined Oligosaccharide Structures on Physical Stability Profiles of IgG1-Fc Glycoforms. J. Pharm. Sci. 2016, 105, 588–601. [Google Scholar] [CrossRef]
  160. Hristodorov, D.; Fischer, R.; Joerissen, H.; Muller-Tiemann, B.; Apeler, H.; Linden, L. Generation and comparative characterization of glycosylated and aglycosylated human IgG1 antibodies. Mol. Biotechnol. 2013, 53, 326–335. [Google Scholar] [CrossRef]
  161. Kayser, V.; Chennamsetty, N.; Voynov, V.; Forrer, K.; Helk, B.; Trout, B.L. Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies. Biotechnol. J. 2011, 6, 38–44. [Google Scholar] [CrossRef] [PubMed]
  162. Wawrzynczak, E.J.; Cumber, A.J.; Parnell, G.D.; Jones, P.T.; Winter, G. Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the CH2 domains. Mol. Immunol. 1992, 29, 213–220. [Google Scholar] [CrossRef]
  163. Wawrzynczak, E.J.; Parnell, G.D.; Cumber, A.J.; Jones, P.T.; Winter, G. Blood clearance in the mouse of an aglycosyl recombinant monoclonal antibody. Biochem. Soc. Trans. 1989, 17, 1061–1062. [Google Scholar] [CrossRef] [PubMed][Green Version]
  164. Hristodorov, D.; Fischer, R.; Linden, L. With or without sugar? (A)glycosylation of therapeutic antibodies. Mol. Biotechnol. 2013, 54, 1056–1068. [Google Scholar] [CrossRef] [PubMed]
  165. Ng, C.M.; Stefanich, E.; Anand, B.S.; Fielder, P.J.; Vaickus, L. Pharmacokinetics/pharmacodynamics of nondepleting anti-CD4 monoclonal antibody (TRX1) in healthy human volunteers. Pharm. Res. 2006, 23, 95–103. [Google Scholar] [CrossRef] [PubMed]
  166. Routier, F.H.; Hounsell, E.F.; Rudd, P.M.; Takahashi, N.; Bond, A.; Hay, F.C.; Alavi, A.; Axford, J.S.; Jefferis, R. Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: A critical evaluation of different methods. J. Immunol. Methods 1998, 213, 113–130. [Google Scholar] [CrossRef]
  167. Flynn, G.C.; Chen, X.; Liu, Y.D.; Shah, B.; Zhang, Z. Naturally occurring glycan forms of human immunoglobulins G1 and G2. Mol. Immunol. 2010, 47, 2074–2082. [Google Scholar] [CrossRef]
  168. Raju, T.S.; Briggs, J.B.; Borge, S.M.; Jones, A.J. Species-specific variation in glycosylation of IgG: Evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 2000, 10, 477–486. [Google Scholar] [CrossRef]
  169. Quan, C.; Alcala, E.; Petkovska, I.; Matthews, D.; Canova-Davis, E.; Taticek, R.; Ma, S. A study in glycation of a therapeutic recombinant humanized monoclonal antibody: Where it is, how it got there, and how it affects charge-based behavior. Anal. Biochem. 2008, 373, 179–191. [Google Scholar] [CrossRef]
  170. Zhang, B.; Yang, Y.; Yuk, I.; Pai, R.; McKay, P.; Eigenbrot, C.; Dennis, M.; Katta, V.; Francissen, K.C. Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody. Anal. Chem. 2008, 80, 2379–2390. [Google Scholar] [CrossRef]
  171. Miller, A.K.; Hambly, D.M.; Kerwin, B.A.; Treuheit, M.J.; Gadgil, H.S. Characterization of site-specific glycation during process development of a human therapeutic monoclonal antibody. J. Pharm. Sci. 2011, 100, 2543–2550. [Google Scholar] [CrossRef]
  172. Gadgil, H.S.; Bondarenko, P.V.; Pipes, G.; Rehder, D.; McAuley, A.; Perico, N.; Dillon, T.; Ricci, M.; Treuheit, M. The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations. J. Pharm. Sci. 2007, 96, 2607–2621. [Google Scholar] [CrossRef] [PubMed]
  173. Banks, D.D.; Hambly, D.M.; Scavezze, J.L.; Siska, C.C.; Stackhouse, N.L.; Gadgil, H.S. The effect of sucrose hydrolysis on the stability of protein therapeutics during accelerated formulation studies. J. Pharm. Sci. 2009, 98, 4501–4510. [Google Scholar] [CrossRef] [PubMed]
  174. Fischer, S.; Hoernschemeyer, J.; Mahler, H.C. Glycation during storage and administration of monoclonal antibody formulations. Eur. J. Pharm. Biopharm. 2008, 70, 42–50. [Google Scholar] [CrossRef]
  175. Goetze, A.M.; Liu, Y.D.; Arroll, T.; Chu, L.; Flynn, G.C. Rates and impact of human antibody glycation in vivo. Glycobiology 2012, 22, 221–234. [Google Scholar] [CrossRef] [PubMed]
  176. Butko, M.; Pallat, H.; Cordoba, A.; Yu, X.C. Recombinant antibody color resulting from advanced glycation end product modifications. Anal. Chem. 2014, 86, 9816–9823. [Google Scholar] [CrossRef] [PubMed]
  177. Harris, R.J. Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture. J. Chromatogr. A 1995, 705, 129–134. [Google Scholar] [CrossRef]
  178. Dick, L.W., Jr.; Qiu, D.; Mahon, D.; Adamo, M.; Cheng, K.C. C-terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods and possible causes. Biotechnol. Bioeng. 2008, 100, 1132–1143. [Google Scholar] [CrossRef]
  179. Santora, L.C.; Krull, I.S.; Grant, K. Characterization of recombinant human monoclonal tissue necrosis factor-alpha antibody using cation-exchange HPLC and capillary isoelectric focusing. Anal. Biochem. 1999, 275, 98–108. [Google Scholar] [CrossRef]
  180. Antes, B.; Amon, S.; Rizzi, A.; Wiederkum, S.; Kainer, M.; Szolar, O.; Fido, M.; Kircheis, R.; Nechansky, A. Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2007, 852, 250–256. [Google Scholar] [CrossRef]
  181. Jiang, G.; Yu, C.; Yadav, D.B.; Hu, Z.; Amurao, A.; Duenas, E.; Wong, M.; Iverson, M.; Zheng, K.; Lam, X.; et al. Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies. J. Pharm. Sci. 2016, 105, 2066–2072. [Google Scholar] [CrossRef] [PubMed]
  182. Liu, H.; Bulseco, G.G.; Sun, J. Effect of posttranslational modifications on the thermal stability of a recombinant monoclonal antibody. Immunol. Lett. 2006, 106, 144–153. [Google Scholar] [CrossRef] [PubMed]
  183. Van den Bremer, E.T.; Beurskens, F.J.; Voorhorst, M.; Engelberts, P.J.; de Jong, R.N.; van der Boom, B.G.; Cook, E.M.; Lindorfer, M.A.; Taylor, R.P.; van Berkel, P.H.; et al. Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation. MAbs 2015, 7, 672–680. [Google Scholar] [CrossRef][Green Version]
  184. Hu, Z.; Tang, D.; Misaghi, S.; Jiang, G.; Yu, C.; Yim, M.; Shaw, D.; Snedecor, B.; Laird, M.W.; Shen, A. Evaluation of heavy chain C-terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells. Biotechnol. Prog. 2017, 33, 786–794. [Google Scholar] [CrossRef] [PubMed]
  185. Cai, B.; Pan, H.; Flynn, G.C. C-terminal lysine processing of human immunoglobulin G2 heavy chain in vivo. Biotechnol. Bioeng. 2011, 108, 404–412. [Google Scholar] [CrossRef]
  186. Johnson, K.A.; Paisley-Flango, K.; Tangarone, B.S.; Porter, T.J.; Rouse, J.C. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal. Biochem. 2007, 360, 75–83. [Google Scholar] [CrossRef]
  187. Tsubaki, M.; Terashima, I.; Kamata, K.; Koga, A. C-terminal modification of monoclonal antibody drugs: Amidated species as a general product-related substance. Int. J. Biol. Macromol. 2013, 52, 139–147. [Google Scholar] [CrossRef]
  188. Kaschak, T.; Boyd, D.; Lu, F.; Derfus, G.; Kluck, B.; Nogal, B.; Emery, C.; Summers, C.; Zheng, K.; Bayer, R.; et al. Characterization of the basic charge variants of a human IgG1: Effect of copper concentration in cell culture media. MAbs 2011, 3, 577–583. [Google Scholar] [CrossRef][Green Version]
  189. Skulj, M.; Pezdirec, D.; Gaser, D.; Kreft, M.; Zorec, R. Reduction in C-terminal amidated species of recombinant monoclonal antibodies by genetic modification of CHO cells. BMC Biotechnol. 2014, 14, 76. [Google Scholar] [CrossRef]
  190. Harris, R.J.; Murnane, A.A.; Utter, S.L.; Wagner, K.L.; Cox, E.T.; Polastri, G.D.; Helder, J.C.; Sliwkowski, M.B. Assessing genetic heterogeneity in production cell lines: Detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody. Biotechnology (N. Y.) 1993, 11, 1293–1297. [Google Scholar] [CrossRef]
  191. Yu, X.C.; Borisov, O.V.; Alvarez, M.; Michels, D.A.; Wang, Y.J.; Ling, V. Identification of codon-specific serine to asparagine mistranslation in recombinant monoclonal antibodies by high-resolution mass spectrometry. Anal. Chem. 2009, 81, 9282–9290. [Google Scholar] [CrossRef] [PubMed]
  192. Wen, D.; Vecchi, M.M.; Gu, S.; Su, L.; Dolnikova, J.; Huang, Y.M.; Foley, S.F.; Garber, E.; Pederson, N.; Meier, W. Discovery and investigation of misincorporation of serine at asparagine positions in recombinant proteins expressed in Chinese hamster ovary cells. J. Biol. Chem. 2009, 284, 32686–32694. [Google Scholar] [CrossRef] [PubMed]
  193. Khetan, A.; Huang, Y.M.; Dolnikova, J.; Pederson, N.E.; Wen, D.; Yusuf-Makagiansar, H.; Chen, P.; Ryll, T. Control of misincorporation of serine for asparagine during antibody production using CHO cells. Biotechnol. Bioeng. 2010, 107, 116–123. [Google Scholar] [CrossRef] [PubMed]
  194. Yang, Y.; Strahan, A.; Li, C.; Shen, A.; Liu, H.; Ouyang, J.; Katta, V.; Francissen, K.; Zhang, B. Detecting low level sequence variants in recombinant monoclonal antibodies. MAbs 2010, 2, 285–298. [Google Scholar] [CrossRef] [PubMed][Green Version]
  195. Ren, D.; Zhang, J.; Pritchett, R.; Liu, H.; Kyauk, J.; Luo, J.; Amanullah, A. Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2011, 879, 2877–2884. [Google Scholar] [CrossRef] [PubMed]
  196. Fu, J.; Bongers, J.; Tao, L.; Huang, D.; Ludwig, R.; Huang, Y.; Qian, Y.; Basch, J.; Goldstein, J.; Krishnan, R.; et al. Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2012, 908, 1–8. [Google Scholar] [CrossRef] [PubMed]
  197. Feeney, L.; Carvalhal, V.; Yu, X.C.; Chan, B.; Michels, D.A.; Wang, Y.J.; Shen, A.; Ressl, J.; Dusel, B.; Laird, M.W. Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies. Biotechnol. Bioeng. 2013, 110, 1087–1097. [Google Scholar] [CrossRef]
  198. Guo, D.; Gao, A.; Michels, D.A.; Feeney, L.; Eng, M.; Chan, B.; Laird, M.W.; Zhang, B.; Yu, X.C.; Joly., J. Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells. Biotechnol. Bioeng. 2010, 107, 163–171. [Google Scholar] [CrossRef]
  199. Wan, M.; Shiau, F.Y.; Gordon, W.; Wang, G.Y. Variant antibody identification by peptide mapping. Biotechnol. Bioeng. 1999, 62, 485–488. [Google Scholar] [CrossRef]
  200. Chumsae, C.; Gifford, K.; Lian, W.; Liu, H.; Radziejewski, C.H.; Zhou, Z.S. Arginine modifications by methylglyoxal: Discovery in a recombinant monoclonal antibody and contribution to acidic species. Anal. Chem. 2013, 85, 11401–11409. [Google Scholar] [CrossRef]
  201. Yang, Y.; Stella, C.; Wang, W.; Schoneich, C.; Gennaro, L. Characterization of oxidative carbonylation on recombinant monoclonal antibodies. Anal. Chem. 2014, 86, 4799–4806. [Google Scholar] [CrossRef] [PubMed]
  202. Amano, M.; Kobayashi, N.; Yabuta, M.; Uchiyama, S.; Fukui, K. Detection of histidine oxidation in a monoclonal immunoglobulin gamma (IgG) 1 antibody. Anal. Chem. 2014, 86, 7536–7543. [Google Scholar] [CrossRef] [PubMed]
  203. Liu, M.; Zhang, Z.; Cheetham, J.; Ren, D.; Zhou, Z.S. Discovery and characterization of a photo-oxidative histidine-histidine cross-link in IgG1 antibody utilizing (1)(8)O-labeling and mass spectrometry. Anal. Chem. 2014, 86, 4940–4948. [Google Scholar] [CrossRef] [PubMed]
  204. Zhao, J.; Saunders, J.; Schussler, S.D.; Rios, S.; Insaidoo, F.K.; Fridman, A.L.; Li, H.; Liu, Y.H. Characterization of a novel modification of a CHO-produced mAb: Evidence for the presence of tyrosine sulfation. MAbs 2017, 9, 985–995. [Google Scholar] [CrossRef] [PubMed]
  205. Santora, L.C.; Stanley, K.; Krull, I.S.; Grant, K. Characterization of maleuric acid derivatives on transgenic human monoclonal antibody due to post-secretional modifications in goat milk. Biomed. Chromatogr. 2006, 20, 843–856. [Google Scholar] [CrossRef]
  206. Chumsae, C.; Zhou, L.L.; Shen, Y.; Wohlgemuth, J.; Fung, E.; Burton, R.; Radziejewski, C.; Zhou, Z.S. Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody. Anal. Chem. 2014, 86, 8932–8936. [Google Scholar] [CrossRef] [PubMed]
  207. Valliere-Douglass, J.F.; Connell-Crowley, L.; Jensen, R.; Schnier, P.D.; Trilisky, E.; Leith, M.; Follstad, B.D.; Kerr, J.; Lewis, N.; Vunnum, S. Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics. Protein Sci. 2010, 19, 2152–2163. [Google Scholar] [CrossRef][Green Version]
  208. Valliere-Douglass, J.F.; Brady, L.J.; Farnsworth, C.; Pace, D.; Balland, A.; Wallace, A.; Wang, W.; Treuheit, M.J.; Yan, B. O-fucosylation of an antibody light chain: Characterization of a modification occurring on an IgG1 molecule. Glycobiology 2009, 19, 144–152. [Google Scholar] [CrossRef]
  209. Valliere-Douglass, J.F.; Eakin, C.M.; Wallace, A.; Ketchem, R.R.; Wang, W.; Treuheit, M.J.; Balland, A. Glutamine-linked and non-consensus asparagine-linked oligosaccharides present in human recombinant antibodies define novel protein glycosylation motifs. J. Biol. Chem. 2010, 285, 16012–16022. [Google Scholar] [CrossRef]
  210. Valliere-Douglass, J.F.; Kodama, P.; Mujacic, M.; Brady, L.J.; Wang, W.; Wallace, A.; Yan, B.; Reddy, P.; Treuheit, M.J.; Balland, A. Asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the CH1 domain of human antibodies. J. Biol. Chem. 2009, 284, 32493–32506. [Google Scholar] [CrossRef]
  211. Dong, Q.; Liang, Y.; Yan, X.; Markey, S.P.; Mirokhin, Y.A.; Tchekhovskoi, D.V.; Bukhari, T.H.; Stein, S.E. The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization. MAbs 2018, 10, 354–369. [Google Scholar] [CrossRef] [PubMed]
  212. Beck, A.; Goetsch, L.; Dumontet, C.; Corvaia, N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug Discov. 2017, 16, 315–337. [Google Scholar] [CrossRef] [PubMed]
  213. Shen, B.Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K.L.; Tien, J.; Yu, S.F.; Mai, E.; et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 2012, 30, 184–189. [Google Scholar] [CrossRef] [PubMed]
  214. Cumnock, K.; Tully, T.; Cornell, C.; Hutchinson, M.; Gorrell, J.; Skidmore, K.; Chen, Y.; Jacobson, F. Trisulfide modification impacts the reduction step in antibody-drug conjugation process. Bioconjug. Chem. 2013, 24, 1154–1160. [Google Scholar] [CrossRef] [PubMed]
  215. Liu-Shin, L.; Fung, A.; Malhotra, A.; Ratnaswamy, G. Influence of disulfide bond isoforms on drug conjugation sites in cysteine-linked IgG2 antibody-drug conjugates. MAbs 2018, 10, 583–595. [Google Scholar] [CrossRef] [PubMed]
Table 1. Micro-heterogeneity natural IgGs and recombinant mAbs.
Table 1. Micro-heterogeneity natural IgGs and recombinant mAbs.
ModificationsNaturalRecombinantResulting Heterogeneity
N-terminal modifications
  PyroGlu  100% pyroGlu  Varied levels  Mass, charge for Gln to pyroGlu
  Truncation  Not expected  Rare and low  Mass
  Signal peptides  Not expected  Low  Mass and charge
Asn deamidation  Substantial level  Common, varied levels  Mass and charge
Asp isomerization  Not expected  Common, varied levels  Charge and hydrophobicity
Succinimide  Not expected  Common, varied levels  Mass, charge, and hydrophobicity
Oxidation  Low  Met, Trp, Cys, His   Mass and hydrophobicity
Cysteine related modifications
  Free cysteine  Low  Low  Mass, charge and hydrophobicity
  Alternative disulfide bond linkage  Common  Common  Charge
  Trisulfide bond  Extremely low  Low  Mass and charge
  Thioether  Low  Low  Mass
Glycosylation  Common  Common  Mass and charge
Glycation  Common  Common  Mass and charge
C-terminal modifications
  C-terminal Lys  Complete removal  Common, varied levels  Mass, charge and hydrophobicity
  C-terminal modifications  Not detected  Low varied levels  Mass and charge
Back to TopTop