Structure, heterogeneity and developability assessment of therapeutic antibodies

Figures & data

Figure 1. Major components of mAb developability assessment.

Table 1. Quality attributes of a panel of FDA/EMA approved and clinical stage mAb products.

Structural features	Ranges	References
Size variants
● High molecular weight (HMW) species	<2% for most of the tested mAbs	^Citation7
● Low molecular weight (LMW) species	<0.9%	^Citation7
Met Oxidation	28% with oxidation in the Fab part of the heavy chain and 17% with oxidation in the light chain based on evaluation of 121 late clinical stage mAbs after treatment with 0.1% hydrogen peroxide for 24 hours	^Citation8
Charge variants
● pI	6.1–9.4	^Citation9
● Acidic	18–60% (cIEF), 5–36% (WCX),	^Citation9
● Main	20–70% (cIEF), 25–92% (WCX)	^Citation9
● Basic	1–40% (cIEF), 3–40 % (WCX)	^Citation9
Hydrophobicity	58% tested mAbs with low to moderate hydrophobicity	^Citation7
	33% tested mAbs with moderate to high hydrophobicity
	8% tested mAbs with very high hydrophobicity
GlycosylationMajor forms
● G0F	30–85%	^Citation10–Citation16
● G1F	10–55%	^Citation10–Citation16
● G2F	1–15%	^Citation10–Citation16
Minor forms
● G0, G0F-GlcNAC	<5%	^Citation10–Citation16
● High mannose	<15%	^Citation10–Citation16
● Afucosylation	<20%	^Citation10–Citation16
● Sialylation	<15% (up to 35% with Fab glycan)	^Citation10,Citation12,Citation14–Citation16
∘ NANA	<12%	^Citation12,Citation14
∘ NGNA	<2% Low (murine cell lines)	^Citation12,Citation15
● Alpha 1,3-gal	<3%	^Citation10,Citation12,Citation13,Citation15
● Bisecting	<5%	^Citation10,Citation13
● Hybrid	<10%	^Citation12,Citation13,Citation15

Figure 2. Workflow of structural and CMC liabilities assessment.

Table 2. Categories of mAb attributes.

Categories	Structural features	Rational
Problematic (Especially in CDRs)
Negatively impact safety or efficacy	● Asn deamidation	● Decreased affinity and immunogenicity
Nonhuman	● Asp isomerization	● Decreased affinity and immunogenicity
Degradation products	● Met oxidation	● Decreased affinity
	● Trp oxidation	● Decreased affinity
	● Variable domain glycosylation	● Unpredictable impact on affinity, increased immunogenicity possibility and higher heterogeneity
	● Unpaired Cys	● Decreased affinity and stability. Increased heterogeneity
Unnecessary
Causing product heterogeneity without	● N-terminal Gln	● Increased heterogeneity
impacting safety and efficacy	● C-terminal Lys	● Increased heterogeneity
Further considerations
Improving product quality	● Immunogenicity motif	● Immunogenicity
	● Aggregation prone regions	● Aggregation and clearance
	● Mutations to increase half-life	● Patient convenience

Table 3. mAb quality attributes evaluated during developability assessment.

Attributes	Objectives and objectives
Primary structure and PTMs	Determine the primary structure and PTMs. The intended primary structure needs to be confirmed. PTMs may impact efficacy and safety
FcRn	Rank ordering mAb candidates based on their binding affinities. Higher binding affinity correlates with longer in vivo half-life
Thermal stability	Rank ordering mAb candidates based on their thermal stability. Higher thermal stability correlates with lower tendency of unfolding and aggregation
Solubility	Rank ordering mAb candidates based on their solubility. MAbs with lower solubility pose challenges for process development, especially for high concentration formulation
Viscosity	Rank ordering mAb candidates based on their solubility. MAbs with higher viscosity pose challenges for process development, especially for high concentration formulation
Hydrophobicity	Rank ordering mAb candidates based on their hydrophobicity. MAbs with lower hydrophobicity correlate with lower tendency to aggregation, lower viscosity and higher solubility
Aggregation propensity	Rank ordering mAb candidates based on their aggregation propensity. Aggregates are the common product-related impurity potentially causing immunogenicity
Charge variants	Compare the charge profiles of mAb candidates. If no difference in safety and efficacy, it is desirable to select mAb with lower level of heterogeneity
Free thiols	Avoid mAbs with abnormally high level of free thiols. A high level of free thiols correlates with lower thermal stability, increased tendency to form aggregates and potentially lower antigen biding affinity.
Protein-protein interactions	Rank ordering mAb candidates based on self- or unspecific interactions. Strong self-interaction correlates with higher aggregation propensity, lower solubility and higher viscosity. Strong non-specific interactions may cause shorter half-life.

Table 4. Known PTMs of mAbs identified by LC-MS.

Mono. mass	Ave. mass	Composition	Site/location	Modifications	Recommendation/Rationale	Ref.
+ various	+ various	Various amino acids	N-terminal	Partial leader sequence	Select candidates without or with low level of leader sequence because of unknown risks	^Citation116,Citation124-Citation127
307.0903	307.26	H(17)C(11)NO(9)	N-linked glycan	Sialylation by Neu5Gc	Select candidates with low amounts because of potential immunogenicity	^Citation12,Citation72
305.0681	305.31	H(15)C(10)N(3)O(6)S	Cys	Glutathionylation	Select candidates without or with low amount because it indicates free cysteine/broken disulfides	^Citation128
291.0954	291.26	H(17)C(11)NO(8)	N-linked glycan	Sialylation by Neu5Ac	Not highly relevant because of low abundance, however, high levels could indicate variable domain glycosylation	^Citation12
174.0164	174.11	C(6)H(6)O(6)	N-terminal primary amine	Citric acid modification	Not highly relevant because it only occurs after long-term storage in formulation containing citric acid	^Citation129
162.0528	162.14	H(10)C(6)O(5)	Lys and N-terminal primary amine	glycation	Select candidates with low levels of glycation because it is a modification product between protein amines and reducing sugars	^Citation130-Citation138
		H(10)C(6)O(5)	N-linked glycan	Alpha 1,3-galactose	Select candidates without or with low amounts because of potential immunogenicity	^Citation12,Citation13,Citation71
		H(10)C(6)O(5)	N-linked glycan	Galactosylation	Not highly relevant to manufacturability, but high levels could be due to glycosylation in variable domain. High level in Fc may correlate with higher CDC	^Citation10,Citation12,Citation139
156.0059	156.09	H(4)C(6)O(5)	N-terminal primary amine	Citric acid modification	Not highly relevant to manufacturability because it occurs after long-term storage in formulation containing citric acid	^Citation129
148.0372	148.12	H(8)C(5)O(5)	N-terminal; K	Modification by xylosone – degradation product of ascorbate – to form hemiaminal product	Selects candidates with low amounts because of uncertainty towards safety and efficacy	^Citation140
146.0579	146.14	H(10)C(6)O(4)	N-linked glycan	N-linked core fucosylation	Low fucose results in high ADCC. Highly relevant to mAb efficacy if ADCC is involved in MOA. Relevant to mAb safety if ADCC is not involved in MOA, but if the mAb recognizes cell surface target	^Citation141-Citation144
146.0579	146.14	H(10)C(6)O(4)	S	O-linked fucosylation	Select candidates without O-linked fucosylation because of uncertainty towards safety and efficacy	^Citation145
144.042	144.13	H(10)C(6)O(4)	R	Advanced Glycation end product 3-deoxyglucosane hydroxyimidazolone	Select candidates with low amount because the presence of this indicate susceptible Arg towards glycation	^Citation138
140.022	140.10	H(4)C(5)N(2)O(3)	N-terminal; K	Modification by maleuric acid	Not highly relevant because its only present in mAb expressed from transgenic goats	^Citation146
130.0266	130.10	H(6)C(5)O(4)	N-terminal; K	Modification by xylosone – degradation product of ascorbate – to form Schiff base product	Not highly relevant due to low frequency	^Citation140
128.0950	128.17	H(12)C(6)N(2)O	C-terminal K	Addition of Lys	Mutate C-terminal Lys out to decrease heterogeneity;	^Citation147,Citation148
					Not very relevant because of lack of biological functions
119.0041	119.14	H(5)C(3)NO(2)S	C	Cysteinylation	Avoid candidates with extra Cys in general because modification of CDR extra Cys causes decreased affinity. Select candidates with low level of cysteinylation because of unknown risks and potential for higher heterogeneity	^Citation61,Citation62,Citation149,Citation150
79.9568	80.06	O(3)S	Y	Sulfation	Not highly relevant because of low frequency of occurrence	^Citation151
72.0211	72.06	H(4)C(3)O(2)	K	Advanced glycation end product, carboxyethyllysine (CEL)	Select candidates with lower amounts because its presence indicates susceptible Arg towards glycation	^Citation138
72.0211	72.06	H(4)C(3)O(2)	R	Glycation degradation to dihydroxyimidazolidine Or direct modification by methylglyoxal in cell culture	Select candidates with low amounts because its presence indicates susceptible Arg towards glycation	^Citation138,Citation152
58.0055	58.04	H(2)C(2)O(2)	K	Advanced glycation product, Carboxymethyllysine (CML)	Selects candidates with low amounts because its presence indicates susceptible Lys towards glycation, advanced glycation end product	^Citation138
56.0262	56.06	H(4)C(3)O	N-terminal; K	Modified by citric acid photo degradation product	Not highly relevant because it occurs under harsh conditions in formulation buffers containing citric acid	^Citation153
54.0106	54.05	H(2)C(3)O	R	Advanced end Glycation degradation product-methyl glyoxal hydroimidazolone or direct modification by methylglyoxal in cell culture	Select candidates with low amounts because its presence indicates susceptible Arg towards glycation	^Citation138,Citation152
47.9847	48.00	O(3)	C	Oxidation of Cys to form sulfonic acid	Not highly relevant because of low frequency of occurrence	^Citation150
38.0156	38.048	H2C3	N-terminal	Modified by citric acid photodegradation product	Not highly relevant because it occurs under harsh conditions in formulation buffers containing citric acid	^Citation153
37.979	38.00	C(2)H(−2)O(1)	R	Arg modification to form glyoxal hydroxyimidazolone	Select candidates with low amounts because its presence indicates susceptible Arg towards glycation	^Citation138
31.9898	32.00	O(2)	M	Double oxidation to form methionine sulfone	Not highly relevant because of low frequency of occurrence under very harsh condition	^Citation138
		O(2)	W	Oxidation to N-formylkynurenine	Select candidates with low amounts because presence of this modification indicates Trp susceptibility	^Citation60
31.9721	32.06	S	C	Trisulfide	Select candidates with low amounts because its degradation product with unknown risks	^Citation154,Citation155
		O(2)	C	Oxidation of Cys to form sulfinic acid	Not highly relevant because of low frequency of occurrence	^Citation150
19.9898	19.99	C(−1)O(2)	W	Oxidation to 3-hydroxykynurenine	Select candidates with low amounts because presence of this modification indicates Trp susceptibility	^Citation60
15.9949	16.00	O	M	Oxidation to methionine sulfoxide	Select candidates with low amounts because Met oxidation may cause decreased affinity when in CDRs and decreased binding to FcRn and shorter half-life when in the constant domains	^Citation50,Citation54,Citation55,Citation138,Citation156
		O	K	hydroxylysine	Not highly relevant because of low frequency	^Citation157
		O	W	Oxidized to hydroxytryptophan	Select candidates with low amounts because the presence of this modification indicates Trp susceptibility	^Citation57,Citation59,Citation60,Citation138,Citation158
		O	P	Oxidative carbonylation to glutamic semialdehyde	Not highly relevant because it of low frequency	^Citation159
13.9792	13.98	H(−2)O	H	His-His cross-linking	Not highly relevant unless the candidates are extremely sensitive to light	^Citation160
3.9949	3.99	C(−1)O	W	Oxidation to kynurenine	Select candidates with low amounts because the presence of this modification indicates Trp susceptibility	^Citation60,Citation138
2.0157	2.02	H(2)	C	Incomplete disulfide bonds	Select candidates without or with low amounts of this modification because it decreased antigen affinity when in CDRs and stability when in other domains.	^Citation161–Citation163
0.9840	0.98	H(−1)N(−1)O	N	deamidation	Select candidates with low amount of this modification because deamidation decreases potency when in CDRs and in general causes potential immunogenicity heterogeneity	^Citation33,Citation36-Citation39,Citation49,Citation75,Citation164,Citation165
0	0	-	D	Asp isomerization	Select candidates with low amount because isomerization decreases affinity when in CDR and the formation of isoAsp causes concerns for immunogenicity	^Citation41-Citation43
		-	C	Racemization	Not very relevant because of low frequency	^Citation121,Citation122
		-	S	Racemization	Not very relevant because of low frequency	^Citation123
−1.0316	−1.03	H(−3)N(−1)O	K	Oxidative carbonylation to aminoadipic semialdehyde	Not highly relevant because of low frequency	^Citation159
−2.0157	−2.02	H(−2)	W	Trp oxidation product	Not very relevant because of low frequency	^Citation59
		H(−2)	T	Oxidative carbonylation to keto-threonine	Not very relevant because of low frequency	^Citation159
−17.0265	−17.03	H(−3)N(−1)	Q	Pyro-Glu	Mutate N-terminal Gln to lower heterogeneity. If present, it has no impact except causing heterogeneity	^Citation74,Citation75,Citation162,Citation166
		H(−3)N(−1)	N	Succinimide as Asn deamidation intermediate	Select candidates without or with low amount of succinimide because it indicates the presence of susceptible deamidation sites	^Citation49,Citation167
−18.0106	18.02	H(−2)O(−1)	E	Pyro-E	Not highly relevant because of lower rate of conversion	^Citation168,Citation169
		H(−2)O(−1)	D	Succinimide as Asp isomerization intermediate	Select candidates without or with low amount of succinimide because it indicates the presence of susceptible deamidation sites	^Citation41,Citation43,Citation47,Citation48,Citation170-Citation172
−31.9721	−32.06	S(−1)	C	Thioether	Select candidates with low amount of thioether because its presence indicates disulfide bond degradation	^Citation173,Citation174
−33.9877	−34.08	H(−2)S(−1)	C	Dehydroalanine	Not highly relevant because it of low frequency	^Citation173
−43.0534	−43.07	H(−5)N(−3)C(−1)O	R	Oxidative carbonylation to glutamic semialdehyde	Not highly relevant because it of low frequency	^Citation159
−58.0055	−58.04	H(−2)C(−2)O(−2)	C-terminal P	C-terminal amidation	Not highly relevant because of low frequency	^Citation175,Citation176
−203.0793	−203.20	H(−13)C(−8)N(−1)O(−5)	N-linked glycan	Loss of N-acetylglucosamine	Select candidates with low amount because it indicates incomplete glycan processing or degradation	^Citation10,Citation177-Citation184

Table 5. Modifications that form either acidic or basic species.

Modifications	References
Acidic
● Deamidation	^Citation33,Citation36,Citation37,Citation75,Citation116,Citation126,Citation264,Citation267-Citation270
● Glycation	^Citation126,Citation130
● Sialic acid	^Citation75,Citation126,Citation271-Citation273
● Incomplete disulfide bond	^Citation163
● Tyrosine sulfation	^Citation151
● Trisulfide bond	^Citation155
● IgG2A/B and IgG2B	^Citation256
● O-fucosylation	^Citation145
● Modification by maleuric acid	^Citation146
● Modification by methylglyoxal	^Citation152
● Cysteinylation	^Citation61
● Methionine oxidation	^Citation172,Citation264
● Asp isomerization intermediate succinimide	^Citation274
● Leader sequence	^Citation275
● Asp isomerization	^Citation116
Basic
● C-terminal Lys	^Citation75,Citation77,Citation126,Citation127,Citation166
● N-terminal remaining Gln	^Citation75,Citation127,Citation162,Citation166
● Succinimide intermediate from Asp	^Citation37,Citation43,Citation47,Citation49
● C-teminal amidation	^Citation116,Citation124,Citation175
● Asp isomerization	^Citation37,Citation274
● Leader sequence	^Citation75,Citation116,Citation126,Citation127
● Incomplete disulfide bond	^Citation162
● Met oxidation	^Citation265,Citation266
● Smaller oligosaccharides	^Citation263
● Cysteinylation	^Citation275

Table 6. Modifications causing HIC retention time shift.

Modifications	References
Early shift
● C-terminal Lys	^Citation41,Citation172
● Deamidation	^Citation37,Citation270
● Asp isomerization	^Citation37,Citation172
● Met oxidation	^Citation156,Citation161,Citation172,Citation296
● Trp oxidation	^Citation296
● Fragments	^Citation172
● O-fucosylation	^Citation145
● Fab N-glycosylation	^Citation20
● Isomerization succinimide	^Citation43
● Cysteinylation	^Citation150
Late shift
● Asp isomerization	^Citation41,Citation43,Citation44,Citation161
● Variable domain incomplete disulfide bond	^Citation43,Citation44,Citation161,Citation297
● Succinimide as isomerization and deamidation intermediate	^Citation37,Citation41,Citation43,Citation44,Citation161,Citation167,Citation172
● Aggregates	^Citation172

Table 7. Forced degradation conditions, degradation pathways, and recommendations for developability evaluation.

Conditions	Objective	Degradation pathways and impact	Whether or not to include in developability assessment
Thermal	To accelerate degradation and increase detectability of degradants	Aggregation^Citation132,Citation164,Citation317Citation327	Include
		Fragmentation^Citation164,Citation317,Citation320,Citation324-Citation326,Citation328-Citation333	Thermal stress is the most relevant condition to product development because high temperature is the common stress encountered during processing, stability and in vivo. Thermal stress can accelerate degradation and thus increases the detectability of potential degradation pathways
		Asn deamidation ^Citation3,Citation33,Citation49,Citation164,Citation324,Citation334,Citation335
		Cyclization of N-terminal Glu^Citation164
		Met oxidation^Citation156,Citation324
		Asp isomerization^Citation41,Citation47,Citation48,Citation170,Citation171
		Disulfide bond degradation through β-elimination^Citation322,Citation323,Citation333
High pH	To probe for liable deamidation sites	Asn deamidation^Citation267,Citation276,Citation322	Include
		Aggregation^Citation322,Citation323	Deamidation is one of the most commonly observed modifications that can cause loss of potency and potentially immunogenicity
		Fragmentation^Citation322,Citation328,Citation332,Citation336,Citation337 Disulfide bonds
		degradation^Citation322,Citation337
Oxidation	To probe for liable oxidation sites	Met oxidation^Citation8,Citation54-Citation56,Citation324	Optional
		Trp oxidation^Citation60	Oxidation is one of the most commonly observed modifications that can cause loss of potency directly or indirectly by shortening half-life. However, highly susceptible residues are detectable during extended characterization or thermal stress.
		Aggregation^Citation323
Low pH	To determine low pH susceptibility towards aggregation, fragmentation, and increase detectability of succinimide from Asp isomerization	Aggregation^Citation322,Citation336,Citation338,Citation339	Optional
		Fragmentation^Citation330,Citation332,Citation333,Citation337	Low pH conditions can be encountered during purification such as protein A chromatography elution and low pH virus inactivation.
		Accumulation of succinimide intermediate^Citation41-Citation43,Citation170,Citation171,Citation259
Agitation	To determine susceptibility towards aggregation	Aggregation^Citation301,Citation319,Citation323,Citation336,Citation340-Citation344	Do not include
		Met oxidation^Citation324	Agitation is a common stress during cell culture, purification and shipping. However, agitation may not be a major concern of causing stability issues for mAb in general, unless, there are reasons to suspect a particular mAb is sensitive to agitation.
		Trp oxidation^Citation324
Freeze/thaw	To determine feasibility of freezing	Aggregation and precipitation^Citation321,Citation325,Citation327,Citation336,Citation345	Do not include
			Freeze/thaw is stress only when mAbs are stored or shipped under frozen condition. This condition is only included when exploring the option of frozen drug substance.
Light	To determine photo-catalyzed degradation mainly oxidation and cross-linking	Trp oxidation^Citation58,Citation59,Citation346,Citation347	Do not include
		Met oxidation^Citation58,Citation59,Citation156,Citation346,Citation348	Generally, light sensitivity is not a common concern unless there are Trp residues in CDRs
		Histidine (His) oxidation^Citation58
		Aggregation^Citation58,Citation346
		Fragmentation^Citation58,Citation346
		Asn deamidation^Citation58
		Covalent cross-linking^Citation160,Citation346

Figure 3. Recommended attributes for developability assessment.

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