Problematic attributes decreased complement-dependent cytotoxicity (CDC),50

decreased binding affinity to FcRn50,54,55 and shorter in vivo

Problematic attributes of mAb candidates mainly encompass
half-life.56 Oxidation of Trp residues in the CDRs has been
摘要 PTMs in CDRs that can negatively impact potency, immuno-
genicity and stability. MAbs are subject to PTMs and degradation
reported, which can lead to reduced potency, decreased thermal
stability and increased aggregation propensity.57-59 Trp oxida-
during cell culture, purification, storage and even after adminis-
qiujun在第2页标注了12 处
tration. Exposure to various stress conditions during manufac-
tion has also been shown to cause a yellow coloration to the mAb x12
solution,60 due to the formation of kynurenine. Because higher
turing, such as elevated temperature (e.g., cell culture, process
Trp oxidation correlates with higher solvent exposure,26 Trp
hold steps), extreme pH (e.g., low pH protein A chromatography
residues in CDRs are expected to be more susceptible to oxida-
elution, or virus inactivation), agitation (e.g., cell culture, pump-
tion. Overall, Met and Trp in CDRs should be carefully evaluated
ing, mixing, filtration, or shipping), shear forces (e.g.,UF/DF)
to determine their susceptibility, and implement an appropriate
and ambient light can accelerate degradation.
244 Y. XU ET AL. control strategy during processing and formulation, if necessary.
Asn deamidation is one of the most commonly encountered
Though rare, mAbs may have an unpaired cysteine (Cys)
degradation pathways in mAbs, especially for Asn residues in
be evaluated residue in CDRs. The unpaired Cys can be toreadily modified
CDRs. 17
Asnwithout
followed any byobservable
the small and differences.
flexible On the other
glycine (Gly) be on those modifications that correspond
61,62
the potentially
hand, most by free cysteine in cellFor culture
thosemedium, which has been
residue (NG PTMs motif) is arehighly
highly dependent
susceptible to on cell line and
deamidation. cell
3,17,32-37 problematic attributes. PTMs that are
61
highly depen-
culture conditions, such as pH, temperature, and cell culture shown
dent onto cell decreaselines,antigen binding using
re-evaluation affinity.materials Candidates fromwith the
Additionally,
244 Y. XUprotein
ET AL. structures can have a substantial impact on
duration. Those cell unpaired
stable cellCys in throughout
the CDRs orthe other regions should be elimi-
Asn deamidation, andline-dependent attributes should
this has been demonstrated be evalu-
in cases where line development is necessary.
ated nated due to of thethe highly reactive nature is ofbased
the Cys on side chain.
Asn using
not in materials
NG motif produced from the stable
are susceptible to cell lines that 17,37
deamidation, will Comparison different candidates the nature
#1
be
be evaluated
used forcases
whereas,
hand, most PTMs
without
clinical any
and/or
where
observable
Asn commercial
in NG motif
differences.
manufacturing.
are highly dependent on cell line and cell
On the
are resistant to
other be on those
However,
of modifications
problematic
themodifications
formation
and theirofrelative
attributes. For those
that correspond
a disulfide bond
percentages. to the
frompotentially
two Cys p.4
residues in the heavy chain CDRsPTMs offersthat some areunique
highly epitopedepen-
deamidation.34,37 Asn deamidation in CDRs may cause
culture conditions, such as pH, temperature, and cell culture dent on cellproperties,
recognition
Modifications lines, re-evaluation
with safety though nousing
or efficacy impact materials
concerns on stability from and the
a decrease in antigenconfirmation
Primary binding affinity 33,35-37 ; therefore, deami-
duration. structure
Those cell line-dependentand sequence
attributes should variants
be evalu- stablegroup
solubility
This cellhas line
ofbeen observed.63the
throughout
modifications are development
known to beislinked necessary.with
dation in the CDRs continues to occur in circulation after
ated
The using materials
confirmation produced
of the intended from the stable cell lines that 3 will Comparison
It isand
safety of the issues.
not efficacy
uncommon different
for These
mAbscandidates
to haveisa based
modifications consensus on the
correspond nature
sequence to
administrated to humans, 32,33
resultingamino in lossacid sequence
of potency. is
For
be used for clinical
athisprerequisite for and/or
further commercial
analysis manufacturing.
and development of of modifications
for N-glycosylation
Problematic and
attributes their
(NXS/T,listed relative
X in
cannot percentages.
Table be2,P)which
in variableincludesdomains,dea-
reason, Asn followed by Gly, and to a lesser degree, Asn
a mAb lead candidate. Modern mass in additionisomerization,
to the conserved Met N-glycosylation site inunpairedthe Fc
followed by serine (Ser), threonine (Thr)spectrometry
in the CDRs, should (MS) has be midation, and Trp oxidation,
the capability of accurately measuring the molecular weight of Modifications
region.
cysteine The with safety
variable
and additional domain or efficacy
glycosylation inconcerns
glycosylation showeddomains.
the variable variable
Primary structure confirmation
highlighted during in silico assessment and evaluated and sequence variants during
an IgG at characterization
approximately 150 ≤2 Da. This group
effects on antigenof binding,64-69 are
of modifications but known
no impact to forbe in linked with
vivo half-
extended andkDa withdegradation
forced the accuracytoofconfirm This group
67,70
modifications should be carefully examined
The confirmation
The ability obtainofand
toliability. the intended the amino acid sequence is safety
life. and efficacy
Variable issues. glycosylation
domain These modifications
andadds correspond
another level toof
deamidation IgG confirm
also contains monoisotopic
susceptible deamidationmolecular during extended characterization forced degradation
prerequisite
aweights of mAbs for
at further
the subunit analysis
level and development
provides strong evidence of Problematic
uncertainty
studies. attributes
with regardlisted in Table
to potency and2,comparability
which includes dea-
later in
sites in the so-called “PENNY” loop 107 peptide in the Fc. 38,39
a mAb
for lead candidate.
confirming Modern mass spectrometry (MS)
the has midation,
development. isomerization, higherofMet
The types level and Trp oxidation,
of terminal galactoseunpaired
α1,3-Gal, of Fab
Deamidation at thethisprimary structure.
site continues to occur Ultimately,
with mAbs full
in There are three oligosaccharides, NGNA
the
primarycapability
sequenceof accurately
can be measuring
confirmed 40 the
by molecular
liquid weight of
chromatogra- cysteine
and highand
glycosylation additional
increases
mannose, thattheglycosylation
likelihood
should forin further
be evaluated the variable domains.
galactosylation
carefully. As dis-
humans and to endogenous IgG. Because this region is con-
an IgG
phy at approximately
(LC)-MS MS/MS150 kDa with the accuracy of ≤2 Da. Thisα1,3-galactose
by
cussed group of modifications
previously, α1,3-Gal
and and should
sialylation. NGNA be immunogenic.
Fab-associated
are carefully examined
oligosacchar- High
served and notand associated peptide
with mapping.
negative impact, deamidation at
TheSeveral
abilitystudies
to obtain andshown
confirm the monoisotopic molecular during
ides withextended
the been
addition of α1,3-galactose
characterization and(Gal) forced degradation
havehalf-life
been shown 185-191
this site should nothave
be a concern the
for presence
developabilityof lowassessment.
abundance mannose has shown to cause shorter
71
in vivo
weights
sequence of mAbs
variants. at the
108-112 subunit
Detection level provides
and strong evidence
identification of low studies.
to cause
and immunogenicity.
enhanced antibody-dependent Sialylation could also addcytotoxicity
cell-meditated the immu-
Asp isomerization is another common 107degradation pathway
for confirming
levels of sequence the primaryarestructure. Ultimately, the full Theremoiety
nogenic
(ADCC) are three
due toofthe types
lack of of oligosaccharides,
core-fucose.187,190,191
N-glycolylneuraminic α1,3-Gal,
acid (NGNA). 12,72
NGNA
Additionally, It is
for mAbs. Similar variants
to deamidation, madeAsp possible
residuesby usingin CDRsLC-MS are
primary
/MS in sequence canwith
combination be confirmed
database17 by liquid
searches. chromatogra-
113-115
The pre- and
the afucosylation level must be monitored because of itsAs
worth high mannose,
mentioning that
that theshould
addition be of α-1,3
evaluated carefully.
Gal and NGNA dis-
corre- is
generally prone to isomerization, especially when followed by
phy (LC)-MS
sence andabundance
MS/MS peptide mapping. cussed
highly previously,
dependent α1,3-Gal
on cell line, and 12,13,72
141 NGNA
whichand aretheir
immunogenic.
be levels
either should Highbe
a Gly of very low 17,37,41-46 sequence ofvariants is likelybycaused
His42 lation with enhanced ADCC, could beneficial
#2
by
residue.
Several studies have shown
the47naturally
or Ser have alsooccurring
been
108-112
Isomerization
low
reported,
the
frequency
suggesting
Asp followed
presenceerrors of lowduring
the
abundance
involvement tran-
of
mannose
evaluated
or harmful has
usingbeen shownon
material
depending to cause
from the shortermechanism
themAb’s
intended instable
vivo half-life
cell of
185-191
line.action p.6
sequence and
scription variants.
translation. Detection
Selection and identification
of size
mAb candidates of and
low and enhanced
192
In silico evaluation,
(MOA). antibody-dependent
Higher levels especially cell-meditated
with the useare
of afucosylation cytotoxicity
of computational
beneficial for
other factors such as residue flexibility, and structure.17,26 187,190,191
levels ofwith
clones sequence
minimal variants are made possible by using LC-MS (ADCC)
methods, due
may to alsothe lack
assist inofidentifying
core-fucose. initiatingAdditionally,
less apparent problematic
Isomerization of Asp sequence
in CDRs may variantsalso iscause
made a possible
decrease by in mAbs targeting cell surface antigen and cell killing,
/MS in combination
extensive characterization. with database searches.113-115 The pre-
37,41,42,46,48 the
while afucosylation
attributes. The low
it is harmful level
for must
mAbsbe
expression monitored
thatandonlylowblock because
stabilitycell of the
of
surfaceits corre-
mAb
anti-
antigen binding affinity. Asp isomerization is favored 141
sence of very low abundance sequence variants is likely caused lation
candidate
gens. Thewithlevels
enhanced
was caused
of those ADCC, whichamino
byoligosaccharides
uncommon could
should be
acidseither beneficial
beidentified
re-evaluated by
around pH 5,47,49 making it challenging to formulate mAb in 73
by the naturally occurring low frequency errors during tran- or harmful
statistical
using material depending
sequence generated on
analysis.from thethe mAb’s
Hydrophobic
stable mechanism
cell amino
lines ofacids
that action
will in
be
liquid formulation
Posttranslational around
modifications this commonly used pH range. To 192
scription and translation. Selection of mAb candidates and (MOA).
CDRs
used forwere Higher
clinical andlevels
responsible of precipitation
for
commercial afucosylation
production.and are absorption
beneficial for of
reduce development risks, Asp followed by Gly, to a lesser
clones with minimal sequence variants is made possible by mAbs
mAb totargeting
filters duringcell surface antigen and
manufacturing shorter incell
andinitiating vivo killing,
half-
degree, followed by Asp or role
LC-MS plays an essential His, for developability
should be highlighted assessment
during
extensive
because ofcharacterization.
its high sensitivity, fast turnaround time and, most life.2 Yet,
while it isanother
Modifications harmful for mAbs
study
corresponding that
demonstrated only that
to degradation block cell surfacecharge
asymmetric anti-
in silico evaluation and further evaluated during extended
importantly, the and ability to obtain an in-depth level of informa- gens.
This The levels
distribution,
group ofand oftothose
modifications oligosaccharides
a lesser degree
has not been should
hydrophobicity,reportedbe re-evaluated
contributes
to impact
characterization forced degradation. 26
tion. PTMs and degradation using
product material
significantlysafetyto or generated
the observed
efficacy, from
buthigh the stable
viscosity.
could cell
potentially lines
These cause that
hydrophobicwill be
immuno-
Posttranslational
Oxidation occurs frequentlyof
modifications withmAb lead candidates
methionine (Met) and can be
tryp-
obtained(Trp) fromresidues.
LC-MS Studies analysisdemonstrated
at intact, subunit or peptide used
or
genicityfor
charged clinical
patches
because and
these commercial
maymodifications production.
not be obviousare at not
the primary
present sequencein either
tophan that oxidation of
LC-MSLC-MS plays an essential roleintact
for developability assessment level,
human butendogenous
visible through IgG proper sequenceproducts.
or degradation analysis and Thisstructural
group of
alevels.
Met in the heavy analysischain at CDR2,
the 46
as level
well as enables
one indetection
the frame- of
because of
modifications its high sensitivity, fast turnaround time and, most Modifications
simulation
modifications using corresponding
includesvariousthe presence to degradation
computational of partial methods. Extended
leader sequence,
work region,50above did its notresolution
impact antigenand detection binding. limit, such as
However,
importantly, N-terminal
glycoforms, the ability to obtain anC-terminal in-depth level of informa- This group
characterization of modifications
and forcedand has not been
degradation To reported
can confirm
minimizethese tothisimpact
non-
a negative impact may bepyroGlu, expected with eitherLys, higher C-terminal
levels of trisulfide bond, thioether, glycation. risk,
tion. PTMs andglycation.
amidation, degradation of mAb leadatcandidates canlevel
be product
obvious
mAb lead safety
problematic or efficacy,
candidates withbut
attributes. thecould
lowestpotentially
levels ofcause theseimmuno-types of
oxidation orand at locations that LC-MSare more analysis
critical the subunit
to antigen bind-
obtained
or after from LC-MS
reduction into analysis
light and atheavy
intact, subunit
chains or peptide
localizes mod- genicity
modification because should these be modifications are not present
selected. Modifications in this in either
category
ing. Two conserved Met residues close to the CH2-CH3 domain
levels. LC-MS
ifications to eitheranalysis Fab, at theF(ab’)2,
intact Fc level enableslight detection of human alsoendogenous IgG or degradation products.
and cellThis group of
interface and part ofthe the neonatal Fc receptor regions,
(FcRn), Protein chain A, or may
Unnecessary
be highly dependent
attributes
on cell lines culture para-
modifications
heavy chain. aboveInitsaddition
8,116,117 resolution to andtraditionally
the detection limit, used such
papain, as modifications
meters such includes as the presence
temperature, pH, ofmedia partialcomposition
leader sequence, and
and Protein G binding sites have been shown to be susceptible to
glycoforms,
more specific
50-52N-terminal
digestion pyroGlu,
can beC-terminal
achieved Lys,
usingC-terminal
limited trisulfide bond, thioether, and glycation. To minimize this risk,
formulation.
oxidation. Oxidation of these Met residues results in Some sequence and structural features of mAbs cause mAb
amidation,
Lys-C117,118and glycation.
or Ides enzyme LC-MS50-53 analysis at theThe
107,119,120 subunit level mAb lead candidates with the lowest levels of these types of
decreased thermal stability,digestion. increased aggregation, combina- 51,53 heterogeneity, but not linked to any biological functions, and
or
tion of IdeS digestion and reduction decreases localizes
after reduction into light and heavy chains the molecularmod- modification
Modificationsshould causing be selected.
heterogeneity Modifications in this category
ifications
weight of to eacheither the Fab,
fragment F(ab’)2,kDa,
to 23–25 Fc regions,
allowinglight chain or
the measure- may also be highly
N-terminal pyroGlu dependent
formation on celland lines partial
and cell culture
removalpara- of
8,116,117
heavy
ment of chain.
monoisotopic In addition
moleculartoweight. the traditionally
107
Ultimately, used analysis
papain, meters
C-terminal suchLysasaretemperature,
two of the well pH,characterized
media composition modifications and
more
at specific
the117,118
peptide level digestion can be localize
can precisely achieved using limited
modification sites formulation.
that cause molecule heterogeneity, but have no impact on safety
107,119,120
Lys-C at intact or Ides andenzyme
subunitdigestion. The combina-
#3detected
tiontheof peptide
at IdeS digestion
level can anddetect
levels. More importantly,
reduction decreasesthat
modifications the cannot
analysis
molecular be
or efficacy. Additionally, the levels of these modifications are
Modifications
highly dependent causing
on cellheterogeneity
line and cell culture conditions.
p.6
weight ofateach
detected intact fragment
and subunit to 23–25 levels,kDa,such allowing
as Asn the measure-
deamidation, N-terminal
The level of pyroGlu
terminalformation
Gal associated and with partial removal of
the glycosylation
107
ment ofthe
where monoisotopic
molecular weight molecular weight.is about
difference Ultimately, analysis
1 Da. Because of Fc is alsoLys
C-terminal of are
notetwo dueofto theitswell characterized
sensitivity to process modifications
changes.
at chromatographic
of the peptide levelseparation, can precisely localize modification
modifications without molecu- sites that cause
The terminalmolecule Gal heterogeneity,
has no impactbuton have no impact
structure, on safety
179-181,193,194

detected
lar weightatdifferences,
intact and subunit such aslevels. More importantly,
Asp isomerization, 41-43 analysis
L-Cys to or efficacy.
stability Additionally,
184,195
or clearance. the 70,185,186,189,196,197
levels of these modifications Recent studies are
MABS
2019, VOL. 11, NO. 2, 239–264
https://doi.org/10.1080/19420862.2018.1553476

REVIEW

Structure, heterogeneity and developability assessment of therapeutic antibodies

Yingda Xua, Dongdong Wangb, Bruce Masonc, Tony Rossomandoc, Ning Lid, Dingjiang Liu e, Jason K Cheungf,
Wei Xug, Smita Raghavah, Amit Katiyari, Christine Nowakc, Tao Xiangj, Diane D. Dongj, Joanne Sunk, Alain Beckl,
and Hongcheng Liuc
a
Protein Analytics, Adimab, Lebanon, NH, USA; bAnalytical Department, Bioanalytix, Inc., Cambridge, MA, USA; cProduct Characterization, Alexion
Pharmaceuticals, Inc., New Haven, CT, USA; dAnalytical Chemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA; eFormulation
Development, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA; fPharmaceutical Sciences, MRL, Merck & Co., Inc., Kenilworth, NJ, USA;
g
Analytical Method Development, MRL, Merck & Co., Inc., Kenilworth, NJ, USA; hSterile Formulation Sciences, MRL, Merck & Co., Inc., Kenilworth, NJ,
USA; iAnalytical Development, Bristol-Myers Squibb, Pennington, NJ, USA; jManufacturing Sciences, Abbvie Bioresearch Center, Worcester, MA, USA;
k
Product development, Innovent Biologics, Suzhou Industrial Park, China; lAnalytical chemistry, NBEs, Center d’immunologie Pierre Fabre, St Julien-
en-Genevois Cedex, France

ABSTRACT ARTICLE HISTORY

Increasing attention has been paid to developability assessment with the understanding that thorough Received 2 October 2018
evaluation of monoclonal antibody lead candidates at an early stage can avoid delays during late-stage Revised 12 November 2018
development. The concept of developability is based on the knowledge gained from the successful Accepted 24 November 2018
development of approximately 80 marketed antibody and Fc-fusion protein drug products and from the KEYWORDS
lessons learned from many failed development programs over the last three decades. Here, we reviewed Developability; monoclonal
antibody quality attributes that are critical to development and traditional and state-of-the-art analytical antibody; posttranslational
methods to monitor those attributes. Based on our collective experiences, a practical workflow is modifications
proposed as a best practice for developability assessment including in silico evaluation, extended
characterization and forced degradation using appropriate analytical methods that allow characteriza-
tion with limited material consumption and fast turnaround time.

The discovery and development of monoclonal antibody
(mAb) therapeutics is resource demanding and technically
challenging. Specifically, challenges associated with
Chemistry, Manufacturing, and Controls (CMC) development
such as high aggregation, high viscosity and susceptibility to
chemical degradation and insufficient product stability have
been commonly recognized. Conventionally, only limited cri-
teria such as antigen binding and in vivo properties including
safety, pharmacokinetics (PK) and pharmacodynamics (PD)
in animal models are used to select a mAb candidate from the
early discovery to development stage. Without extensive char-
acterization to understand the biochemical and biophysical
properties of the selected candidate, issues can arise from
unexpected modifications, stability or poor PK and PD,
which can result in delayed project progress or even termina-
tion. The development risks are often associated with the
intrinsic properties of the drug candidates. Therefore, it is
critical to carry out a developability assessment before enter-
ing process development. Developability assessment is
a process used to systematically evaluate drug candidates,
including structural assessment and CMC liabilities, safety,
PK and PD, as well as manufacturability (Figure 1).
Although, many interdependent factors contribute to the suc-
cessful development of an mAb therapeutic, selection of
a candidate with favorable biophysical and biochemical beha-
vior help lay down a solid foundation. Thus, the primary goal
of a developability assessment is to critically evaluate the
biochemical and biophysical properties of mAb lead candi-
dates and select the molecules with the lowest risks for
development.
Numerous studies have shown the importance of develop-
ability assessment of mAb lead candidates. For example, poor
biophysical properties resulted in mAbs with lower expression,
instability or shorter in vivo half-life.1,2 Continuous asparagine
(Asn) deamidation in the complementarity-determining region
(CDR) has also caused loss of potency of a mAb.3 Given the
limitations of timelines and resources at the early stage of
development, a thorough developability evaluation may not
eliminate all risks that could occur later, but it does allow the
selection of lead candidates with fewer development risks.
Meanwhile, the knowledge gained through thorough evaluation
also provides a strong foundation that in turn allows for
a quality by design (QbD) approach for process and formula-
tion development to mitigate any remaining identified risks.
When risks are deemed critical and cannot be mitigated, an
early decision to re-engineer the molecule is much more pre-
ferable than a later decision because it allows companies to save
resources and avoid excessive delays of the timeline.
The biochemical and biophysical properties of mAb candi-
dates are evaluated based on in silico and experimental eva-
luation, and according to: 1) the general properties of the
approved mAbs; 2) scientific literature regarding the general

CONTACT Alain Beck alain.beck@pierre-fabre.com Analytical chemistry, NBEs, Center d’immunologie Pierre Fabre, St Julien-en-Genevois Cedex, France;
Hongcheng Liu hcliu2008@gmail.com Product Characterization, Alexion Pharmaceuticals, Inc., 100 College Street, New Haven, CT, USA
© 2018 Taylor & Francis Group, LLC
240 Y. XU ET AL.

Structural assessment and CMC liabilities

• In silico: Hot spots, Benchmarking (approved mAbs),

literature, prior knowledge
• Extended characterization: Chromatographic and
electrophoretic profiling, mass spectrometry
• Forced degradation studies

Manufacturability
Safety/PK/PD
• Cell line stability
• Expression titer
• Pharmacokinetics (PK) • Purification recovery
• Pharmacodynamics (PD) • Scalability
• Toxicology • Stability (in-process, long-term, in use)
• Immunogenicity • Comparability
• Cost of goods

Figure 1. Major components of mAb developability assessment.

properties of mAb molecules including posttranslational
modifications (PTMs), stability and degradation pathways;
and 3) drug developers’ internal knowledge from develop-
ment of similar molecules.
To date, approximately 80 mAb and Fc-fusion protein drug
products have been approved by the US Food and Drug
Administration (FDA) and the European Medicines Agency
(EMA),4,5 and more than 70 are in late-stage development.6
Among them, some generally preferred drug properties for
mAbs have begun to emerge (Table 1). It should be noted that
some attributes such as high molecular weight (HMW) species
are dependent on the sample shelf-life and handling history.
MAbs with attributes within or better than these ranges are
expected to have relatively lower development risks.
Because of the highly conserved primary and similar high-
order structures of mAbs, the commonly recognized degrada-
tion hot-spots can guide drug candidate evaluation to identify
potential problematic features. For example, Asn and aspar-
tate (Asp) residues in the flexible CDRs are susceptible to
deamidation and isomerization,17 respectively, and thus need
more careful examination. The correlation between aggrega-
tion or faster clearance and exposed hydrophobic18-22 or
charged patches23,24 in the variable domains can also be
applied to mAb candidate evaluation. These general principles

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 <2% for most of the tested mAbs 7

(HMW) species
● Low molecular weight <0.9% 7

(LMW) species
8
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
Charge variants
● pI 6.1–9.4 9

● Acidic 18–60% (cIEF), 5–36% (WCX), 9

● Main 20–70% (cIEF), 25–92% (WCX) 9

● Basic 1–40% (cIEF), 3–40 % (WCX) 9

7
Hydrophobicity 58% tested mAbs with low to moderate hydrophobicity
33% tested mAbs with moderate to high hydrophobicity
8% tested mAbs with very high hydrophobicity
Glycosylation
Major forms
● G0F 30–85% 10–16

● G1F 10–55% 10–16

● G2F 1–15% 10–16

Minor forms
● G0, G0F-GlcNAC <5% 10–16

● High mannose <15% 10–16

● Afucosylation <20% 10–16

● Sialylation <15% (up to 35% with Fab glycan) 10,12,14–16

∘
NANA <12% 12,14

∘
NGNA <2% Low (murine cell lines) 12,15

● Alpha 1,3-gal <3% 10,12,13,15

● Bisecting <5% 10,13

● Hybrid <10% 12,13,15

 MABS 241

allow identification of potential risks based on the amino acid
sequences of mAb candidates.
Drug developers’ internal knowledge from process and
formulation development also plays a significant role in devel-
opability evaluation. Limitations of stable cell line, cell culture
media components, and drug substance process steps are
common elements that may exist across the pipeline and
should be part of the developability assessment consideration.
Therefore, we are proposing an overall developability assess-
ment (Figure 1), focusing on evaluation of biochemical and
biophysical properties at early stage.
Below is a three-step workflow for the assessment of struc-
tural and CMC liabilities (Figure 2), taking into consideration
previous proposals.3,25-29 Step 1 is in silico evaluation, including
the use of computational methods, based on amino acid
sequence to identify the known degradation hot spots and
problematic motifs. Step 2 is to perform in-depth characteriza-
tion to experimentally evaluate key biochemical and biophysi-
cal properties, such as structure, stability, charge profiles,
PTMs, solubility, and hydrophobicity. Step 3 is to carry out
limited forced degradation studies to, first, further confirm hot-
spots identified from steps 1 and 2, and second, reveal candi-
date-specific degradation hot-spots that were not identified
from Step 1. Forced degradation studies also provide additional
stability information to rank the candidates. Step 2 and Step 3
can be carried out simultaneously to allow a thorough evalua-
tion of mAb candidates within a short timeframe.
In silico evaluation
We propose to categorize mAb structural features into three
groups (Table 2), namely, Problematic, Unnecessary and
Further Considerations for in silico evaluation, based on the
general properties of mAbs, including PTMs and degradation
pathways.30,31 Problematic attributes have been associated
with known safety or efficacy concerns. Unnecessary attri-
butes have not been linked to any biological functionalities.
Attributes for further considerations have not been consid-
ered traditionally for mAb lead selection, but may be consid-
ered for next-generation molecules with improved properties,
such as minimal immunogenicity, enhanced stability and
extended half-life.

Step 2: Extended characterization

• Confirmation of amino acid sequence
• PTMs
• FcRn
• Thermal stability
Step 1: In-silico evaluation • Solubility
1. Problematic attributes • Viscosity
• Asn deamidation • Aggregation propensity
• Asp isomerization • Charge variants
• Oxidation • Hydrophobicity Lead mAb Yes
• Extra Cys • Free thiols meeting the Development
• Variable domain glycosylation • Protein-protein interactions expectation?
• Identification of non-obvious
attributes
2. Unnecessary attributes
• N-terminal Gln
Step 3: Forced degradation No
• C-terminal Lys • Degradation pathways
3. Further considerations • Stability ranking
• Immunogenicity motif Protein
• Aggregation prone motif engineering

Figure 2. Workflow of structural and CMC liabilities assessment.

Table 2. Categories of mAb attributes.

Categories
Problematic (Especially in
CDRs)
Negatively impact safety or
efficacy
Nonhuman
Degradation products
Unnecessary
Causing product heterogeneity
without
impacting safety and efficacy
Further considerations
Improving product quality
Structural features Rational
● Asn deamidation ● Decreased affinity and immunogenicity
● Asp isomerization ● Decreased affinity and immunogenicity
● 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
● N-terminal Gln ● Increased heterogeneity
● C-terminal Lys ● Increased heterogeneity
● Immunogenicity motif ● Immunogenicity
● Aggregation prone regions ● Aggregation and clearance
● Mutations to increase half-life ● Patient convenience
242 Y. XU ET AL.

Problematic attributes decreased complement-dependent cytotoxicity (CDC),50

decreased binding affinity to FcRn50,54,55 and shorter in vivo
Problematic attributes of mAb candidates mainly encompass
half-life.56 Oxidation of Trp residues in the CDRs has been
PTMs in CDRs that can negatively impact potency, immuno-
reported, which can lead to reduced potency, decreased thermal
genicity and stability. MAbs are subject to PTMs and degradation
stability and increased aggregation propensity.57-59 Trp oxida-
during cell culture, purification, storage and even after adminis-
tion has also been shown to cause a yellow coloration to the mAb
tration. Exposure to various stress conditions during manufac-
solution,60 due to the formation of kynurenine. Because higher
turing, such as elevated temperature (e.g., cell culture, process
Trp oxidation correlates with higher solvent exposure,26 Trp
hold steps), extreme pH (e.g., low pH protein A chromatography
residues in CDRs are expected to be more susceptible to oxida-
elution, or virus inactivation), agitation (e.g., cell culture, pump-
tion. Overall, Met and Trp in CDRs should be carefully evaluated
ing, mixing, filtration, or shipping), shear forces (e.g.,UF/DF)
to determine their susceptibility, and implement an appropriate
and ambient light can accelerate degradation.
control strategy during processing and formulation, if necessary.
Asn deamidation is one of the most commonly encountered
Though rare, mAbs may have an unpaired cysteine (Cys)
degradation pathways in mAbs, especially for Asn residues in
residue in CDRs. The unpaired Cys can be readily modified
CDRs. 17 Asn followed by the small and flexible glycine (Gly)
by free cysteine in cell culture medium,61,62 which has been
residue (NG motif) is highly susceptible to deamidation.3,17,32-37
shown to decrease antigen binding affinity.61 Candidates with
Additionally, protein structures can have a substantial impact on
unpaired Cys in the CDRs or other regions should be elimi-
Asn deamidation, and this has been demonstrated in cases where
nated due to the highly reactive nature of the Cys side chain.
Asn not in NG motif are susceptible to deamidation,17,37
However, the formation of a disulfide bond from two Cys
whereas, cases where Asn in NG motif are resistant to
residues in the heavy chain CDRs offers some unique epitope
deamidation.34,37 Asn deamidation in CDRs may cause
recognition properties, though no impact on stability and
a decrease in antigen binding affinity 33,35-37; therefore, deami-
solubility has been observed.63
dation in the CDRs continues to occur in circulation after
It is not uncommon for mAbs to have a consensus sequence
administrated to humans,32,33 resulting in loss of potency.3 For
for N-glycosylation (NXS/T, X cannot be P) in variable domains,
this reason, Asn followed by Gly, and to a lesser degree, Asn
in addition to the conserved N-glycosylation site in the Fc
followed by serine (Ser), threonine (Thr) in the CDRs, should be
region. The variable domain glycosylation showed variable
highlighted during in silico assessment and evaluated during
effects on antigen binding,64-69 but no impact for in vivo half-
extended characterization and forced degradation to confirm
life.67,70 Variable domain glycosylation adds another level of
deamidation liability. IgG also contains susceptible deamidation
uncertainty with regard to potency and comparability later in
sites in the so-called “PENNY” loop peptide in the Fc.38,39
development. The higher level of terminal galactose of Fab
Deamidation at this site continues to occur with mAbs in
glycosylation increases the likelihood for further galactosylation
humans and to endogenous IgG.40 Because this region is con-
by α1,3-galactose and sialylation. Fab-associated oligosacchar-
served and not associated with negative impact, deamidation at
ides with the addition of α1,3-galactose (Gal) have been shown
this site should not be a concern for developability assessment.
to cause immunogenicity.71 Sialylation could also add the immu-
Asp isomerization is another common degradation pathway
nogenic moiety of N-glycolylneuraminic acid (NGNA).12,72 It is
for mAbs. Similar to deamidation, Asp residues in CDRs are
worth mentioning that the addition of α-1,3 Gal and NGNA is
generally prone to isomerization,17 especially when followed by
highly dependent on cell line,12,13,72 and their levels should be
a Gly residue.17,37,41-46 Isomerization of Asp followed by His42
evaluated using material from the intended stable cell line.
or Ser47 have also been reported, suggesting the involvement of
In silico evaluation, especially with the use of computational
other factors such as residue flexibility, size and structure.17,26
methods, may also assist in identifying less apparent problematic
Isomerization of Asp in CDRs may also cause a decrease in
attributes. The low expression and low stability of the mAb
antigen binding affinity.37,41,42,46,48 Asp isomerization is favored
candidate was caused by uncommon amino acids identified by
around pH 5,47,49 making it challenging to formulate mAb in
statistical sequence analysis.73 Hydrophobic amino acids in
liquid formulation around this commonly used pH range. To
CDRs were responsible for precipitation and absorption of
reduce development risks, Asp followed by Gly, to a lesser
mAb to filters during manufacturing and shorter in vivo half-
degree, followed by Asp or His, should be highlighted during
life.2 Yet, another study demonstrated that asymmetric charge
in silico evaluation and further evaluated during extended
distribution, and to a lesser degree hydrophobicity, contributes
characterization and forced degradation.
significantly to the observed high viscosity.26 These hydrophobic
Oxidation occurs frequently with methionine (Met) and tryp-
or charged patches may not be obvious at the primary sequence
tophan (Trp) residues. Studies demonstrated that oxidation of
level, but visible through proper sequence analysis and structural
a Met in the heavy chain CDR2,46 as well as one in the frame-
simulation using various computational methods. Extended
work region,50 did not impact antigen binding. However,
characterization and forced degradation can confirm these non-
a negative impact may be expected with either higher levels of
obvious problematic attributes.
oxidation or at locations that are more critical to antigen bind-
ing. Two conserved Met residues close to the CH2-CH3 domain
interface and part of the neonatal Fc receptor (FcRn), Protein A, Unnecessary attributes
and Protein G binding sites have been shown to be susceptible to
oxidation.50-52 Oxidation of these Met residues results in Some sequence and structural features of mAbs cause mAb
decreased thermal stability,50-53 increased aggregation,51,53 heterogeneity, but not linked to any biological functions, and
 MABS 243

do not raise safety or efficacy concerns. The presence of these
attributes possesses unnecessary challenges for the manufac-
turing of product with consistent profiles, and complicates
analytical method development.
Cyclization of N-terminal glutamine (Gln) to form pyro-
glutamate (pyroGlu) is a major source of heterogeneity of
mAbs. This reaction occurs spontaneously during drug sub-
stance production process, storage in liquid formulation and
continues under physiological conditions74 and during sto-
rage. This N-terminal modification has no impact on struc-
ture, stability, or biological functions of mAbs.75 N-terminal
Gln of human endogenous IgGs is almost completely con-
verted to pyroGlu.76 It has been proposed to substitute
N-terminal Gln with other amino acids to eliminate this
source of heterogeneity.
Incomplete processing of C-terminal lysine (Lys) is another
common modification. C-terminal Lys has no impact on mAb
potency77,78 PK, PD or immunogenicity.78 However, removal
of C-terminal Lys showed optimal C1q binding and CDC.79
C-terminal Lys is rapidly removed during circulation,80
explaining its absence from endogenous IgGs.80 Removal of
the codon for C-terminal Lys can eliminate this heterogeneity.
Further considerations
MAbs for therapeutic purposes have evolved from murine ori-
gin, to chimeric, humanized and fully human molecules to
reduce immunogenicity.81 However, immunogenicity remains
a concern even for mAbs with full human sequences.81,82
Various tools including in silico calculation, in vitro assays or
in vivo animal models are designed to identify amino acid
sequences causing immunogenicities.81,83,84 Additional immu-
nogenic components such as alpha 1,3 galactose can be elimi-
nated with the removal of variable domain glycosylation
sites.81,83
Aggregation of mAb therapeutics is another contributor to
immunogenicity, as well as to processing difficulties.85 Attempts
have been made to identify regions in mAbs that mediate aggre-
gation through computational algorithms,18,19,86-90 or experi-
mental screening (e.g., phage display),91 and then to improve
mAb stability by mutagenesis.2,19,83,88,91,92
Host cell protein (HCP) in mAb therapeutics can also con-
tribute to immunogenicity.93,94 The type and amount of HCP in
drug substance can be dependent on the specific mAb sequence
or manufacturing process conditions, such as cell line, cell
culture and stringency of purification parameters. However,
mAbs with more general stickiness due to exposed hydrophobic
or charged patches are expected to have more HCP problems.
The potential for self-administration through subcutaneous
injection requires mAbs to be formulated at high concentra-
tion (≥100 mg/mL) and delivered at small volumes (≤2mL).
In terms of developability, high solubility and low viscosity
are thus required. Since strong mAb self-association is often
the cause of low solubility and high viscosity, protein engi-
neering can be applied to reduce self-association by modifying
the protein sequences and thus increase mAb solubility95 or
decrease viscosity.26,96-99 Aside from the previously discussed
potential issues with variable domain glycosylation, the intro-
duction of glycosylation sites near aggregation-prone regions
(APRs) was demonstrated to improve mAb solubility.95,100-103
Modulation of in vivo half-life has been explored by chan-
ging amino acid sequence around the FcRn binding site83,104
or in CDRs by introduction of a pH switch using histidine
(His)105 or disruption of CDR positively charged patches106
Additionally, mAbs with rapid clearance due to off-target
binding can be addressed by altering amino acid sequences
to disrupt charged or hydrophobic patches.2,26 MAbs can be
tailored to have either extended or shortened half-life to better
fit their therapeutic purposes and to increase patient compli-
ance, e.g., longer half-life reducing dosing frequency.
Experimental evaluation by extended
characterization
Following in silico evaluation, lead candidates are usually eval-
uated experimentally through extended characterization.
Quality attributes that are highly relevant to mAb developability
assessment are summarized in Table 3. Usually, the initial
material available for testing are produced by transient transfec-
tion (e.g., HEK293 cells). Properties that are independent of the
expression hosts such as primary sequence, hydrophobicity,
solubility, thermal stability, and antigen binding affinity, can

Table 3. mAb quality attributes evaluated during developability assessment.

Attributes Objectives and objectives
Primary structure and Determine the primary structure and PTMs. The intended primary structure needs to be confirmed. PTMs may impact efficacy and safety
PTMs
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 Rank ordering mAb candidates based on their aggregation propensity. Aggregates are the common product-related impurity potentially
propensity 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 Rank ordering mAb candidates based on self- or unspecific interactions. Strong self-interaction correlates with higher aggregation
interactions propensity, lower solubility and higher viscosity. Strong non-specific interactions may cause shorter half-life.
244 Y. XU ET AL.
be evaluated without any observable differences. On the other
hand, most PTMs are highly dependent on cell line and cell
culture conditions, such as pH, temperature, and cell culture
duration. Those cell line-dependent attributes should be evalu-
ated using materials produced from the stable cell lines that will
be used for clinical and/or commercial manufacturing.
Primary structure confirmation and sequence variants
The confirmation of the intended amino acid sequence is
a prerequisite for further analysis and development of
a mAb lead candidate. Modern mass spectrometry (MS) has
the capability of accurately measuring the molecular weight of
an IgG at approximately 150 kDa with the accuracy of ≤2 Da.
The ability to obtain and confirm the monoisotopic molecular
weights of mAbs at the subunit level provides strong evidence
for confirming the primary structure.107 Ultimately, the full
primary sequence can be confirmed by liquid chromatogra-
phy (LC)-MS and MS/MS peptide mapping.
Several studies have shown the presence of low abundance
sequence variants.108-112 Detection and identification of low
levels of sequence variants are made possible by using LC-MS
/MS in combination with database searches.113-115 The pre-
sence of very low abundance sequence variants is likely caused
by the naturally occurring low frequency errors during tran-
scription and translation. Selection of mAb candidates and
clones with minimal sequence variants is made possible by
extensive characterization.
Posttranslational modifications
LC-MS plays an essential role for developability assessment
because of its high sensitivity, fast turnaround time and, most
importantly, the ability to obtain an in-depth level of informa-
tion. PTMs and degradation of mAb lead candidates can be
obtained from LC-MS analysis at intact, subunit or peptide
levels. LC-MS analysis at the intact level enables detection of
modifications above its resolution and detection limit, such as
glycoforms, N-terminal pyroGlu, C-terminal Lys, C-terminal
amidation, and glycation. LC-MS analysis at the subunit level
or after reduction into light and heavy chains localizes mod-
ifications to either the Fab, F(ab’)2, Fc regions, light chain or
heavy chain.8,116,117 In addition to the traditionally used papain,
more specific digestion can be achieved using limited
Lys-C117,118 or Ides enzyme digestion.107,119,120 The combina-
tion of IdeS digestion and reduction decreases the molecular
weight of each fragment to 23–25 kDa, allowing the measure-
ment of monoisotopic molecular weight.107 Ultimately, analysis
at the peptide level can precisely localize modification sites
detected at intact and subunit levels. More importantly, analysis
at the peptide level can detect modifications that cannot be
detected at intact and subunit levels, such as Asn deamidation,
where the molecular weight difference is about 1 Da. Because
of chromatographic separation, modifications without molecu-
lar weight differences, such as Asp isomerization,41-43 L-Cys to
D-Cys,121,122 and Ser racemization123 can also be detected by
LC-MS.
All the reported modifications (to our best knowledge) for
mAbs detected by LC-MS are listed in Table 4. Focus should
be on those modifications that correspond to the potentially
problematic attributes. For those PTMs that are highly depen-
dent on cell lines, re-evaluation using materials from the
stable cell line throughout the development is necessary.
Comparison of the different candidates is based on the nature
of modifications and their relative percentages.
Modifications with safety or efficacy concerns
This group of modifications are known to be linked with
safety and efficacy issues. These modifications correspond to
Problematic attributes listed in Table 2, which includes dea-
midation, isomerization, Met and Trp oxidation, unpaired
cysteine and additional glycosylation in the variable domains.
This group of modifications should be carefully examined
during extended characterization and forced degradation
studies.
There are three types of oligosaccharides, α1,3-Gal, NGNA
and high mannose, that should be evaluated carefully. As dis-
cussed previously, α1,3-Gal and NGNA are immunogenic. High
mannose has been shown to cause shorter in vivo half-life185-191
and enhanced antibody-dependent cell-meditated cytotoxicity
(ADCC) due to the lack of core-fucose.187,190,191 Additionally,
the afucosylation level must be monitored because of its corre-
lation with enhanced ADCC,141 which could be either beneficial
or harmful depending on the mAb’s mechanism of action
(MOA).192 Higher levels of afucosylation are beneficial for
mAbs targeting cell surface antigen and initiating cell killing,
while it is harmful for mAbs that only block cell surface anti-
gens. The levels of those oligosaccharides should be re-evaluated
using material generated from the stable cell lines that will be
used for clinical and commercial production.
Modifications corresponding to degradation
This group of modifications has not been reported to impact
product safety or efficacy, but could potentially cause immuno-
genicity because these modifications are not present in either
human endogenous IgG or degradation products. This group of
modifications includes the presence of partial leader sequence,
trisulfide bond, thioether, and glycation. To minimize this risk,
mAb lead candidates with the lowest levels of these types of
modification should be selected. Modifications in this category
may also be highly dependent on cell lines and cell culture para-
meters such as temperature, pH, media composition and
formulation.
Modifications causing heterogeneity
N-terminal pyroGlu formation and partial removal of
C-terminal Lys are two of the well characterized modifications
that cause molecule heterogeneity, but have no impact on safety
or efficacy. Additionally, the levels of these modifications are
highly dependent on cell line and cell culture conditions.
The level of terminal Gal associated with the glycosylation
of Fc is also of note due to its sensitivity to process changes.
The terminal Gal has no impact on structure,179-181,193,194
stability 184,195 or clearance.70,185,186,189,196,197 Recent studies
have demonstrated that the terminal galactose may have
minimal impact on ADCC, but substantial influence on
CDC.139,198 Therefore, the level of terminal galactose should
be considered for mAb candidates with MOA involving CDC.
 MABS 245

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

Ave. Site/
Mono. mass mass Composition location Modifications Recommendation/Rationale Ref.
116,124-127
+ various + various Various N-terminal Partial leader sequence Select candidates without or with low level
amino acids of leader sequence because of unknown risks
12,72
307.0903 307.26 H(17)C(11) N-linked Sialylation by Neu5Gc Select candidates with low amounts because
NO(9) glycan of potential immunogenicity
128
305.0681 305.31 H(15)C(10)N Cys Glutathionylation Select candidates without or with low
(3)O(6)S amount because it indicates free cysteine/
broken disulfides
12
291.0954 291.26 H(17)C(11) N-linked Sialylation by Neu5Ac Not highly relevant because of low
NO(8) glycan abundance, however, high levels could
indicate variable domain glycosylation
129
174.0164 174.11 C(6)H(6)O(6) N-terminal Citric acid modification Not highly relevant because it only occurs
primary after long-term storage in formulation
amine containing citric acid
130-138
162.0528 162.14 H(10)C(6)O Lys and glycation Select candidates with low levels of glycation
(5) N-terminal because it is a modification product between
primary protein amines and reducing sugars
amine
12,13,71
H(10)C(6)O N-linked Alpha 1,3-galactose Select candidates without or with low
(5) glycan amounts because of potential
immunogenicity
10,12,139
H(10)C(6)O N-linked Galactosylation Not highly relevant to manufacturability, but
(5) glycan high levels could be due to glycosylation in
variable domain. High level in Fc may
correlate with higher CDC
129
156.0059 156.09 H(4)C(6)O(5) N-terminal Citric acid modification Not highly relevant to manufacturability
primary because it occurs after long-term storage in
amine formulation containing citric acid
148.0372 148.12 H(8)C(5)O(5) N-terminal; Modification by xylosone – Selects candidates with low amounts 140

K degradation product of ascorbate – because of uncertainty towards safety and

to form hemiaminal product efficacy
141-144
146.0579 146.14 H(10)C(6)O N-linked N-linked core fucosylation Low fucose results in high ADCC. Highly
(4) glycan 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
145
146.0579 146.14 H(10)C(6)O S O-linked fucosylation Select candidates without O-linked
(4) fucosylation because of uncertainty towards
safety and efficacy
138
144.042 144.13 H(10)C(6)O R Advanced Glycation end product Select candidates with low amount because
(4) 3-deoxyglucosane the presence of this indicate susceptible Arg
hydroxyimidazolone towards glycation
146
140.022 140.10 H(4)C(5)N(2) N-terminal; Modification by maleuric acid Not highly relevant because its only present
O(3) K in mAb expressed from transgenic goats
130.0266 130.10 H(6)C(5)O(4) N-terminal; Modification by xylosone – Not highly relevant due to low frequency 140

K degradation product of ascorbate –

to form Schiff base product
147,148
128.0950 128.17 H(12)C(6)N C-terminal Addition of Lys Mutate C-terminal Lys out to decrease
(2)O K heterogeneity;
Not very relevant because of lack of
biological functions
61,62,149,150
119.0041 119.14 H(5)C(3)NO C Cysteinylation Avoid candidates with extra Cys in general
(2)S 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
151
79.9568 80.06 O(3)S Y Sulfation Not highly relevant because of low frequency
of occurrence
138
72.0211 72.06 H(4)C(3)O(2) K Advanced glycation end product, Select candidates with lower amounts
carboxyethyllysine (CEL) because its presence indicates susceptible
Arg towards glycation
138,152
72.0211 72.06 H(4)C(3)O(2) R Glycation degradation to Select candidates with low amounts because
dihydroxyimidazolidine Or direct its presence indicates susceptible Arg
modification by methylglyoxal in cell towards glycation
culture
138
58.0055 58.04 H(2)C(2)O(2) K Advanced glycation product, Selects candidates with low amounts
Carboxymethyllysine (CML) because its presence indicates susceptible
Lys towards glycation, advanced glycation
end product
153
56.0262 56.06 H(4)C(3)O N-terminal; Modified by citric acid photo Not highly relevant because it occurs under
K degradation product harsh conditions in formulation buffers
containing citric acid
(Continued )
246 Y. XU ET AL.

Table 4. (Continued).
Ave. Site/
Mono. mass mass Composition location Modifications Recommendation/Rationale Ref.
138,152
54.0106 54.05 H(2)C(3)O R Advanced end Glycation degradation Select candidates with low amounts because
product-methyl glyoxal its presence indicates susceptible Arg
hydroimidazolone or direct towards glycation
modification by methylglyoxal in cell
culture
150
47.9847 48.00 O(3) C Oxidation of Cys to form sulfonic acid Not highly relevant because of low frequency
of occurrence
153
38.0156 38.048 H2C3 N-terminal Modified by citric acid Not highly relevant because it occurs under
photodegradation product harsh conditions in formulation buffers
containing citric acid
138
37.979 38.00 C(2)H(−2)O R Arg modification to form glyoxal Select candidates with low amounts because
(1) hydroxyimidazolone its presence indicates susceptible Arg
towards glycation
138
31.9898 32.00 O(2) M Double oxidation to form methionine Not highly relevant because of low frequency
sulfone of occurrence under very harsh condition
60
O(2) W Oxidation to N-formylkynurenine Select candidates with low amounts because
presence of this modification indicates Trp
susceptibility
154,155
31.9721 32.06 S C Trisulfide Select candidates with low amounts because
its degradation product with unknown risks
150
O(2) C Oxidation of Cys to form sulfinic acid Not highly relevant because of low frequency
of occurrence
60
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
50,54,55,138,156
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
157
O K hydroxylysine Not highly relevant because of low frequency
57,59,60,138,158
O W Oxidized to hydroxytryptophan Select candidates with low amounts because
the presence of this modification indicates
Trp susceptibility
159
O P Oxidative carbonylation to glutamic Not highly relevant because it of low
semialdehyde frequency
160
13.9792 13.98 H(−2)O H His-His cross-linking Not highly relevant unless the candidates are
extremely sensitive to light
60,138
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
161–163
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.
33,36-
0.9840 0.98 H(−1)N(−1) N deamidation Select candidates with low amount of this
39,49,75,164,165
O modification because deamidation decreases
potency when in CDRs and in general causes
potential immunogenicity heterogeneity
41-43
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
121,122
- C Racemization Not very relevant because of low frequency
123
- S Racemization Not very relevant because of low frequency
−1.0316 −1.03 H(−3)N(−1) K Oxidative carbonylation to Not highly relevant because of low frequency 159

O aminoadipic semialdehyde
−2.0157 −2.02 H(−2) W Trp oxidation product Not very relevant because of low frequency 59
159
H(−2) T Oxidative carbonylation to keto- Not very relevant because of low frequency
threonine
−17.0265 −17.03 H(−3)N(−1) Q Pyro-Glu Mutate N-terminal Gln to lower 74,75,162,166

heterogeneity. If present, it has no impact

except causing heterogeneity
49,167
H(−3)N(−1) N Succinimide as Asn deamidation Select candidates without or with low
intermediate amount of succinimide because it indicates
the presence of susceptible deamidation
sites
−18.0106 18.02 H(−2)O(−1) E Pyro-E Not highly relevant because of lower rate of 168,169

conversion
41,43,47,48,170-
H(−2)O(−1) D Succinimide as Asp isomerization Select candidates without or with low
172
intermediate amount of succinimide because it indicates
the presence of susceptible deamidation
sites
−31.9721 −32.06 S(−1) C Thioether Select candidates with low amount of 173,174

thioether because its presence indicates

disulfide bond degradation
(Continued )

Table 4. (Continued).
Ave. Site/
Mono. mass mass Composition location Modifications
−33.9877 −34.08 H(−2)S(−1) C Dehydroalanine
−43.0534 −43.07 H(−5)N(−3) R Oxidative carbonylation to glutamic
C(−1)O semialdehyde
−58.0055 −58.04 H(−2)C(−2) C-terminal C-terminal amidation
O(−2) P
−203.0793 −203.20 H(−13)C(−8) N-linked Loss of N-acetylglucosamine
N(−1)O(−5) glycan
Since the level of terminal galactosylation varies with different
cell lines and cell culture conditions, it may have to be re-
evaluated later in the development process.
FcRn affinity
FcRn binding is one of the most critical factors affecting mAb
half-life.104 The FcRn binding affinities of mAb candidates are
usually measured by Biacore, but alternative assays using biolayer
interferometry (BLI) or FcRn affinity chromatography have been
established. In a study evaluating mAbs, it was found that delayed
elution of mAbs from an FcRn affinity column at neutral pH
correlated with poor pK.24,199 Compared to Biacore and affinity
chromatography, much higher throughput can be obtained using
BLI.200 In general, mAbs with stronger FcRn binding at acidic pH,
but fast dissociation at neutral pH, show longer in vivo half-life.104
Thermal stability
Thermal stability is the ability of a protein to maintain its struc-
tural and functional integrity under different temperature envir-
onment, and is an intrinsic property of mAbs that can influence
product stability, such as aggregation, during manufacturing and
storage. High thermal stability of a mAb candidate indicates
a well-packed structure that requires more energy to unfold.
Therefore, higher thermal stability of a mAb generally correlates
with a lower tendency towards partial unfolding and thus
aggregation.19,73,201-206 Besides aggregation, mAbs with lower
thermal stability have been shown to have lower expression.73,207
Thermal stability has been commonly measured by differ-
ential scanning calorimetry (DSC). 19,73,201-206 An alternative
method using differential scanning fluorimetry (DSF) allows
high throughput thermal stability screening of 96 or 384
samples.202,206,208-210 Multiple candidates analyzed under the
same conditions can be ranked based on the obtained thermo-
dynamic parameters such as the midpoint temperature (Tm) of
unfolding or the onset of unfolding temperature (Tonset).
Solubility
Solubility is an important developability parameter for
mAbs,211,212 especially in consideration of the industry trend
towards higher concentration formulations (100 mg/mL and
above).213 MAbs must remain soluble throughout processing,
storage and administration.97,211 Low solubility can lead to
MABS 247
Recommendation/Rationale Ref.
Not highly relevant because it of low 173
frequency
Not highly relevant because it of low 159
frequency
Not highly relevant because of low frequency 175,176
Select candidates with low amount because 10,177-184
it indicates incomplete glycan processing or
degradation
issues during purification, sterile filtration, fill and finish, ship-
ping, storage214 and more importantly, can adversely affect
activity, bioavailability, and immunogenicity.212,215 From the
process perspective, a minimum solubility (e.g., 20–30 mg/
mL) in the buffers used for bioprocessing is necessary for all
the chromatographic steps. During the final ultrafiltration/dia-
filtration (UF/DF) step, mAbs are buffer exchanged into for-
mulation buffers at concentrations above the targeted drug
product, thus requiring much higher solubility.
Lower solubility is usually caused by strong mAb self- asso-
ciation with exposed hydrophobic or charged patches. Naturally,
the bivalent nature of mAbs amplifies their self-association
tendencies.216 Colloidal instability caused by conformational
changes or chemical modifications can also contribute to the
poor solubility of a mAb. Additionally, mAb solubility is often
influenced by solution properties, such as buffer composition,
ionic strength, pH, and temperature.214,217-219
It is challenging to predict solubility of mAb candidates
based on the amino acid sequences, therefore solubility of
mAbs should be studied experimentally. However, studying
mAb solubility directly requires large amounts of protein,
typically several hundred milligrams, and it is often not prac-
tical to produce all candidates at such large quantities for
solubility study. Given the limited sample quantities available
for developability assessment studies, indirect measurement
methods are commonly used. For example, addition of poly-
ethylene glycol (PEG) to mAb solutions causes precipitation
at much lower concentrations, and thus can be used to deter-
mine the apparent solubility of mAbs through extrapolation
to zero PEG concentration.212,220-222 This approach can be
implemented in a high-throughput manner for mAb candi-
date selection. However, PEG-induced precipitation may not
truly reflect the mechanisms of the poor solubility of
mAbs,218,223 and thus orthogonal methods or direct evalua-
tion of solubility at high concentration should be considered
to confirm the predicted solubility or validate the rank order.
Prediction of the high concentration behavior of a mAb
using low concentration sample may also be done through the
measurement of the osmotic second virial coefficient B22,
a thermodynamic parameter related to intermolecular inter-
actions. Positive and negative B22 values indicate repulsive or
attractive forces, respectively. Parameters affecting B22 include
electrostatic interactions, van der Waals force, excluded
volumes, hydration forces, and hydrophobic effects.224,225
Among many different ways to obtain B22 values, such as self-
interaction chromatography (SIC),226 membrane osmometry
248 Y. XU ET AL.
(MO)227 and analytical ultracentrifugation (AUC),228 the
most common method is through static light scattering
(SLS).225,229,230 Recently this value was used to determine
a universal solubility line, the “liquidus” line, as part of
a phase diagram for a mAb.231,232
Cross-interaction chromatography (CIC) assay, introduced
by Jacob et al.233 takes advantage of the accumulative effect on
a column to capture weak binding between testing mAb and
a large quantity (30 mgs) of immobilized human serum IgGs.
MAbs with late elution by CIC assay correlate with poor
solubility, due to exposed sticky (hydrophobic or charge) sur-
faces. The other method worth mentioning is self-interaction
nanoparticle spectroscopy, which uses gold nanoparticles to
concentrate mAb molecule to a high local concentration to
amplify weak self-interaction.216,234 This method can also be
applied to high throughput screening of mAb candidates.234
Viscosity
High concentration drug products administrated via subcuta-
neous (SC) injection require a formulation with manageable
viscosity, making it another critical factor for evaluation at an
early stage to ease developability concerns.235 High viscosity
can pose challenges to the final UF/DF step 28,236,237 and fill/
finish operation.238,239 Viscous drug product can lead to diffi-
culties in delivery causing low patient compliance.240 Viscous
samples can also pose sampling challenges for analytical
method development and instrumentation.241
High viscosity has been shown to be caused by strong mAb
self-association through electrostatic 26,98,242-246 or hydropho-
bic interactions,26,242 or the combination of both.26,242,247
Although, a number of formulation parameters, including
pH, salts, sugars, and various small molecule excipients and
detergents can be explored to lower viscocity,97,248-251 selec-
tion of mAb lead candidates with minimal inherent problems
such as exposure of hydrophobic, or charged patches is one of
the most efficient means to minimize high viscosity risk.242,252
A variety of methods have been used to measure viscosity,
including Cannon-Fenske Routine viscometer, Taylor Cone
plate method, and various rheometers. Most of the conven-
tional techniques for measuring viscosity require a large
amount of materials. To overcome this challenge, in particular
for developability assessment, a high throughput DLS method
has been developed based on measurement of apparent poly-
styrene bead radii in high concentration mAb solutions to
back calculate the viscosity of a mAb solution.253 This method
can only be used for mAbs without interaction with the beads,
otherwise the apparent bead radii cannot be reliably mea-
sured. High throughput diffusion interaction parameters
derived from DLS measurement have also been shown to
correlate with viscosity.254 In recent years, the instruments
allowing viscosity measurement using ≤100 µL and with auto-
mated sample handling have become commercially available,
and these are suitable for measuring viscosity during devel-
opability assessment.
Because of the significance of viscosity for process and
product development, having a carefully defined viscosity
target for developability assessment is important. At mini-
mum, the formulation viscosity should be low enough to
allow the drug formulation to be delivered with manual injec-
tion. The viscosity target is typically developed based on each
company’s internal development experience, in particular for
developing SC product with prefilled syringes and auto-
injector devices. For example, it was proposed that the visc-
osity can be grouped into 3 categories: 1) “preferred” viscosity
of 10 cP or lower; 2) “acceptable” viscosity between 10 to 20
cP; and 3) “unacceptable’ viscosity of > 20 cP. This can be
used as a starting point for defining the viscosity target with
additional considerations of the internal experience of process
and product development and product knowledge of delivery
devices, such as autoinjector.
Aggregation propensity
Aggregates are the most commonly observed product-related
impurities, requiring close monitoring due to immunogenicity
concerns.85,255 Therefore, it is a critical component of devel-
opability assessment. In addition to utilizing predictive tools,
aggregation propensity can be directly measured during
extended characterization and forced degradation studies.
Though typically only low concentration data is available
due to material limitation, it is important to evaluate the
colloidal stability and aggregation propensity of a mAb at
medium to high (50–100 mg/mL) concentration ranges.
Routinely, sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) or capillary electrophoresis (CE-
SDS) are used to determine mAb monomer, fragments and
covalent aggregates under denaturing conditions with or
without reduction. A variety of other methods are available
to measure mAb soluble aggregates such as dimer, oligomer
or subvisible particles under native conditions.255-258 Size-
exclusion chromatography (SEC) is the most commonly
used method to determine mAb HMW species (e.g., dimer,
trimer or oligomers) and LMW species. SEC is typically
included for product release and often available as a generic
or platform method, therefore it is suitable for evaluation of
aggregates during extended characterization and forced degra-
dation. It is worth mentioning that SEC can sometimes reveal
properties other than the percentage of monomer, aggregates
and fragments. Abnormal SEC behaviors of a mAb, such as
peak tailing, could indicate non-ideal biophysical properties.1
Longer retention time and asymmetric peak shape can suggest
nonspecific interactions between a mAb and the SEC
column.2 Studies showed that SEC can even separate mAb
variants containing succinimide intermediate from those with
Asn deamidation (17Da) or Asp isomerization (18Da).167,259
SEC has also shown that a mAb variant containing oxidized
Trp eluting earlier than the main peak.158 These examples
suggest that interpretation of SEC data should be done cau-
tiously because earlier and later peaks may not always repre-
sent HMW or LMW species.
For large aggregates, light scattering can be used to char-
acterize particles in the range of <1 nm to 1–10 µm. The DLS
method can be used to determine the hydrodynamic diameters
of mAbs242 and interaction parameters, such as KD,260,261
which can be run in high throughput mode with low sample
consumption. Methods that measure turbidity, such as optical
density at visible wavelength and nephelometry, can also be
 MABS 249

considered for detection of submicron/sub visible particles.
These techniques may be developed with high throughout
and low volume consumption, and thus can be used for devel-
opability assessment. Light obscuration (e.g., HIAC) and flow
imaging methods (e.g., micro-flow imaging (MFI)) and
FlowCam) can be used for sub-visible particle characterization
and quantification for sizes greater than 2 µm. A visual inspec-
tion method is used for detecting the protein particles in the
visible range, typically > 70 to 100 µm.
In addition to directly measuring the level of aggregates,
aggregation propensity can be ranked based on hydrophobi-
city or protein interactions as they are the main driving forces
for aggregation. Fluorescence dyes such as 1-anilino-
naphthalenesulfonate (ANS) and thioflavin can be used to
probe exposed hydrophobic patches in a high throughput
manner with minimal sample requirement.262 Affinity capture
self-interaction nanoparticle spectroscopy (AC-SINS), which
provides coarse-grained information about interactions and
aggregation propensity in different solution conditions, is
useful to leverage during developability evaluations.234
Charge variants
Charge variation of mAbs reflects the sum of various PTMs.
Variants of mAbs need to be closely monitored throughout the
development process to ensure consistent peak profiles. Because
of its sensitivity to process changes, charge variation is one of the
quality attributes that could be challenging for demonstrating
comparability, when process changes are introduced.
A typical mAb charge variant profile characterized by
charged-based methods such as ion exchange chromatogra-
phy and isoelectric focusing usually contains one major peak
and several smaller acidic and basic peaks. Reported modifi-
cations resulting in the formation of acidic or basic species are
shown in Table 5. It is worth mentioning that several
Table 5. Modifications that form either acidic or basic species.
Modifications References
Acidic
● Deamidation 33,36,37,75,116,126,264,267-270
● Glycation 126,130
modifications may impact chromatographic separations of
mAb variants by modulating mAb structures. For examples,
mAbs with smaller oligosaccharides can contribute to the
formation of basic species,263 while the oxidized Met may
contribute to the formation of either acidic172,264 or
basic265,266 species. Similarly, the presence of the incompletely
formed disulfide bond in the heavy chain variable domain can
either contribute to acidic163 or basic162 species.
Isoelectric focusing gel electrophoresis (IEF) was tradition-
ally used to analyze mAb charge variants.37,267,268,276 This
semi-quantitative, labor-intensive method relies on dye stain-
ing for detection. It also suffers from low throughput, lack of
automation and poor reproducibility. Capillary IEF (cIEF)
overcame most of the IEF limitations and offered additional
advantages, including high sensitivity, automation, and low
sample consumption.276-278 Moreover, imaged cIEF (icIEF)
has gained popularity for the analysis of mAb charge
variants279-281 because the whole capillary imaging eliminates
the troublesome mobilization step used by cIEF.282
Capillary zone electrophoresis (CZE) separates mAb charge
variants based on both charge and hydrodynamic radius. This
method can be readily platformed with relatively high through-
put compared to cIEF.277,278,283-286 CZE can also be coupled on-
line with a mass spectrometer. CZE-MS has been used to profile
N-linked glycans from tryptic peptides16 and analyze the sites of
deamidation and isomerization.287 A single CE-MS run has been
shown to confirm 100% of the primary structure and reveal
several PTMs, including glycosylation, N-terminal Gln cycliza-
tion, deamidation and isomerization.288
Ion exchange chromatography (IEX), including cation
exchange36,37,75,166,264,267,268,271,272 and anion exchange,116,155,276
has been widely used to monitor mAb charge variants. IEX allows
fraction collection for further characterization. Multiple mAbs can
be analyzed when a pH gradient is used,289 implying the potential
for establishment of a platform method. Strong cation exchange
(SCX) chromatography allows a relatively higher throughput
compared to weak cation exchange chromatography.290,291
When comparing the overall charge profiles, IEF usually shows
comparable results with either cation37or anion276 exchange chro-
matography. However, different profiles have been observed due
to differences in the separation mechanisms.267,268

● Sialic acid 75,126,271-273

● Incomplete disulfide bond 163

● Tyrosine sulfation 151 Hydrophobicity and related heterogeneity

● Trisulfide bond 155

● IgG2A/B and IgG2B 256 Hydrophobicity can impact mAb aggregation, solubility and
● O-fucosylation 145
viscosity.292,293 Higher hydrophobicity correlates with higher pro-
● Modification by maleuric acid 146

● Modification by methylglyoxal 152 pensity towards aggregation18,19,21,22 and precipitation.293,294

● Cysteinylation 61
Hydrophobic patches in CDRs can lead to a higher degree of inter-
● Methionine oxidation 172,264
molecule interaction, higher viscosity and shorter in vivo half-
● Asp isomerization intermediate succinimide 274

● Leader sequence 275 life.2,26

● Asp isomerization 116
Hydrophobic interaction chromatography (HIC) has been
Basic
● C-terminal Lys 75,77,126,127,166 used to measure the relative hydrophobicity of different
● N-terminal remaining Gln 75,127,162,166
mAbs7,25,292 or separate variants of the same mAb caused by
● Succinimide intermediate from Asp 37,43,47,49

● C-teminal amidation 116,124,175 PTMs or degradation.37,41,43,161 Reported modifications caus-

● Asp isomerization 37,274
ing HIC retention time shift, as compared to the main peak,
● Leader sequence 75,116,126,127
are listed in Table 6. Some modifications such as Asp isomer-
● Incomplete disulfide bond 162

● Met oxidation 265,266 ization and deamidation can shift HIC retention times both
● Smaller oligosaccharides 263
ways, suggesting the involvement of other factors impacting
● Cysteinylation 275
chromatographic behavior.
250 Y. XU ET AL.
Table 6. Modifications causing HIC retention time shift.
Modifications
Early shift
● C-terminal Lys
● Deamidation
● Asp isomerization
● Met oxidation 156,161,172,296
● Trp oxidation
● Fragments
● O-fucosylation
● Fab N-glycosylation
● Isomerization succinimide
● Cysteinylation
Late shift
● Asp isomerization
● Variable domain incomplete disulfide bond
● Succinimide as isomerization and deamidation 37,41,43,44,161,167,172
intermediate
● Aggregates
Alternative methods for measuring mAb relative hydro-
phobicity of mAbs have also been reported, such as the use
of gold nanoparticle via salt gradient screening.295 In this
method, the testing mAb is loaded onto gold nanoparticles,
followed by salt gradient stress to strip water molecules from
hydrophobic patches on the surface of a mAb molecule. The
results demonstrated a good correlation with HIC retention
times for tested mAbs.
Free thiols
The presence of significant levels of free Cys negatively
impacts mAb stability and potency. The level of free Cys
and free Cys-related modifications and degradations are
highly dependent on mAb sequence, as well as environmental
factors during cell culture and purification.
MAbs contain low levels of free thiols at each Cys
residue.298-300 Free thiols have been shown to lower thermal
stability298 and increase formation of reducible covalent
aggregates.301,302 These mAb-associated free cysteines can
react with free cysteine present in cell culture media to form
cysteinylated or other covalent adducts.61,62,128,149,150 In a few
cases, relatively higher levels of free cysteine were detected,
mainly due to incomplete formation of heavy chain variable
domain disulfide bonds,161-163 which could result in reduced
potency.161
Protein-protein interactions
Protein-protein interaction has drawn increasing attention
during developability evaluation due to its impact on solubi-
lity, viscosity, and aggregation propensity96,254,294,303-305 In
addition, non-specific off-target binding in vivo resulted in
fast clearance and poor PK23,306
A variety of techniques have been developed to study
protein-protein interactions for mAbs, including self-
interactions and non-specific interactions with other mole-
cules. Among these techniques, Biacore,307 bio-layer interfero-
metry (BLI),308 and self-interaction nanoparticle spectroscopy
(SINS).216,234,309,310 have been used to study self-interaction.
On the other hand, cross-interaction chromatography (CIC)
can be used to study non-specific interaction when different
proteins were immobilized.233,293,311 Positive correlation
References between delayed retention between CIC and HIC suggests
that hydrophobic interaction being a major contributing fac-
41,172
37,270 tor to the general stickiness (non-specific interaction) of these
37,172 mAbs.293 Other assays including the polyspecificity reagent
binding assay,312 and binding to heparin,313 HEK293 cells,106
296
172 baculovirus particles,314 chaperone proteins,315 and yeast316
145
have also been used to study non-specific interactions.
20
43
150
41,43,44,161
Experimental evaluation by forced degradation
43,44,161,297
Forced degradation studies are playing an ever-increasing role
172 providing critical information to support mAb drug
development.31 Forced degradation studies can be predictive
of in vivo degradation3,33,35,40 and can also be used to validate
liable spots identified from in silico evaluation, reveal degra-
dation hot-spots that are not obvious from in-silico and
provide a ranking order of mAb candidates based on stability.
The commonly used forced degradation conditions and major
degradation pathways, and recommendations to support
developability are shown in Table 7.
Forced degradation studies are important for
confirmation3 or rejection 34 of the predicted degradation hot-
spots from in silico assessment. More importantly, forced
degradation studies can reveal the hidden degradation hot-
spots that are not generally recognized or specific to indivi-
dual mAb lead candidates. The relatively harsh conditions
used in forced degradation studies can increase the detect-
ability of degradation products that are normally present at
low levels during extended characterization. For example,
susceptibility of Asp to isomerization and peptide bond
hydrolysis in an Asp-Asp motif in the CDR315 or susceptibil-
ity of a Ser-Ser motif in the CDR to peptide bond hydrolysis
316
can only be detected during forced degradation.
Forced degradation studies are valuable for defining pro-
cess parameters and for prediction of long-term stability.317
Low pH is a common stress during protein
A chromatography elution and virus inactivation steps used
for a typical mAb purification process. MAb candidates with
poor low pH stability will require extensive efforts using
alternative purification and virus inactivation processes.
MAbs could also be transiently exposed to high pH conditions
during anion exchange chromatography elution and pH neu-
tralization after low pH exposure. MAbs that are sensitive to
agitation, light, freeze/thaw cycle can be a challenge to the
establishment of a robust process due to limited design space.
Forced degradation results for any given mAb are highly
dependent on the selected conditions. It is well known that
the first and second Asn residues in the “PENNY” peptide are
highly susceptible to deamidation.38,39 However, the Asn resi-
due within the amino acid sequence VSNK was found to have
an even higher level of deamidation compared to the
“PENNY” peptides when stored at pH 5.2.169 Specific gui-
dance regarding photostability is provided in ICH Q1B, but
the recommended conditions are different from room light
conditions.349 From the developability assessment perspective,
forced degradation conditions may be best selected based on
their relevance to process, stability and in vivo conditions,
Table 7. Forced degradation conditions, degradation pathways, and recommendations for developability evaluation.
Degradation pathways and
Conditions Objective impact Whether or not to include in developability assessment
Thermal To accelerate degradation and increase detectability of degradants Aggregation132,164,317327 Include
Fragmentation164,317,320,324- Thermal stress is the most relevant condition to product development because high temperature is the
326,328-333
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
3,33,49,164,324,334,335

Cyclization of N-terminal
Glu164
Met oxidation156,324
Asp isomerization41,47,48,170,171
Disulfide bond degradation
through β-elimination
322,323,333

High pH To probe for liable deamidation sites Asn deamidation267,276,322 Include

Aggregation322,323 Deamidation is one of the most commonly observed modifications that can cause loss of potency and
potentially immunogenicity
Fragmentation322,328,332,336,337
Disulfide bonds
degradation322,337
Oxidation To probe for liable oxidation sites Met oxidation8,54-56,324 Optional
Trp oxidation60 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.
Aggregation323
Low pH To determine low pH susceptibility towards aggregation, Aggregation322,336,338,339 Optional
fragmentation, and increase detectability of succinimide from Asp
isomerization
Fragmentation330,332,333,337 Low pH conditions can be encountered during purification such as protein A chromatography elution and
low pH virus inactivation.
Accumulation of succinimide
intermediate41-43,170,171,259
Agitation To determine susceptibility towards aggregation Aggregation301,319,323,336,340- Do not include
344
324
Met oxidation 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 oxidation324
Freeze/ To determine feasibility of freezing Aggregation and Do not include
thaw precipitation321,325,327,336,345
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 Trp oxidation58,59,346,347 Do not include
cross-linking
Met oxidation58,59,156,346,348 Generally, light sensitivity is not a common concern unless there are Trp residues in CDRs
Histidine (His) oxidation58
Aggregation58,346
Fragmentation58,346
MABS

Asn deamidation58
Covalent cross-linking160,346
251
252 Y. XU ET AL.
while taking into consideration the intrinsic properties of the
mAb candidates.
Computational tools
Computational approaches are attractive for mAb developabil-
ity assessment as the amino acid sequence is the only necessary
input. Thus, they can be applied to in-silico evaluation for in-
depth evaluation, in addition to identifying the obvious degra-
dation hot-spots. Potential problematic attributes from known
motifs or the presence of less frequently observed amino acid at
certain positions can be easily highlighted.73,84,90 Several com-
putational tools, based on machine learning, are available to
calculate the solvent-accessible surface area (SASA).350 SASA
showed good predictability of the chemical liability, such as
oxidation of Met8 and Trp26 or Asp isomerization,26 as well as
HIC retention times.350
Computational tools are also available to rank viscosity of
mAb lead candidates based on the known amino acid
sequences. The variable domain net charge, asymmetric
charge distribution and, to a lesser degree hydrophobicity,
were found to contribute to higher viscosity.26 The spatial
charge map was established based on amino acid sequence
and homology modeling to predict viscosity.351 Calculation of
biochemical and biophysical properties based on homology
modeling of variable regions of low and high viscosity mAbs
revealed that net negative charge, zeta-potential and variable
domain isoelectric point are the critical parameters impacting
viscosity.245 Homology modeling has identified surface nega-
tively charged patches causing high viscosity.99 Interestingly,
homology modeling also identified positively charged patches
in the variable domain of a mAb responsible for its shorter
half-life, which was related to decreased dissociation from
FcRn at neutral pH.24 Coarse-grained modeling has also
been used to understand inter-molecule interactions, and
has revealed that domain-level electrostatic interactions play
an important role.349,352,353
Various computational tools have also been developed to
identify structural features that could trigger aggregation.86,87
Spatial-aggregation-propensity (SAP), based on molecular
simulation, can be used to identify aggregation-prone motifs,
due to formation of hydrophobic patches in the tertiary
structure.18,19,89,294 Mutation of the identified hydrophobic
residues to hydrophilic ones has been shown to increase
stability.18,19,294 SAP identified APRs in the constant
domains,18 as well as in variable domains,90 highlighting the
need to balance the affinity and aggregation propensity. Lauer
et al proposed the concept of a developability index, to rank
mAbs based on aggregation propensity calculated from the
net charge and SAP of the CDRs.27 Based on experimental
data from over 500 mAbs, a model was built using statistical
modeling and machine learning to categorize mAbs to high or
low risk towards aggregation.354
Computational tools have also been developed to identify
unwanted in vivo behaviors such as poor PK29 and
immunogeneicity.355 Studies have shown that faster mAb
clearance correlated with exposed hydrophobic or charged
patches in the variable domains.26,356 T-cell epitopes can be
identified by computational tools,355 which can be used to
rank mAb candidates to lower the potential immunogenicity
risk.
Emerging analytical methods
Emerging analytical methods may have the potential to gather
information faster with less material consumption, and thus
can be applied to mAb developability assessment. Hydrophilic
interaction liquid chromatography (HILIC) has been applied
to mAb characterization.357,358 With a polar stationary phase
and an organic mobile phase, HILIC is fully compatible with
MS and offers a complementary retention mechanism com-
pared to reversed-phase high-performance liquid chromato-
graphy (RP-HPLC). This chromatographic method was
mostly used for released glycan profiling and glycopeptide
separations. It can also be used as an orthogonal method to
RP-HPLC at the subunit level following IdeS digestion.
Two-dimensional LC (2D-LC) with MS and other detec-
tion methods (e.g., UV) clearly facilitate a deep structural
understanding of mAbs.359-361 The additional chromato-
graphic selectivity and resolution of 2D-LC compared to the
conventional 1D-LC methods enables the direct and efficient
identification of different variants present in these materials.
2D-LC with various combinations, such as SEC×RP–MS,
CEX×RP–MS, and HIC×RP–MS, have the potential to pro-
vide extensive characterization of mAbs with automation and
low material consumption to support developability
evaluation.
Recommendations
Acceleration of development activities to bring mAb drug
candidates into first-in-human clinical studies, and then to
market is the ultimate goal of drug developers. There is a fine
balance between minimizing the risks at early stages and
accelerating the later stages of development to provide the
essential therapies to patients. It is not practical, nor neces-
sary, to apply all of the discussed methods and studies for
a developability evaluation.
We propose a workflow as outlined in Figure 3, which
categorizes studies and testing as “Essential”, which must be
included, or “Non-essential”, which are optional. The goal of
this workflow is to gather scientific information and experi-
mental data within a reasonable timeframe for candidate
ranking and selection.
It is worth mentioning that ex vivo and in vivo serum
stability has gained popularity because of the relevance to
physiological conditions. Those studies can be considered
under specific occasions in order to ease the remaining con-
cerns of the selected mAb candidates. For example, in the case
of incomplete heavy chain variable domain disulfide bond
formation, additional studies demonstrated that they can be
formed rapidly under ex vivo and in vivo conditions, thus
eliminating the concern of reduced potency.166 In contrast, in
the case of CDR region deamidation, although the rate of
deamidation may be controlled by appropriate process con-
trols and formulation, the risk of continuous deamidation and
loss of potency in vivo means that this risk cannot be
mitigated.3
 MABS 253

Essential Non-Essential
• Problematic attributes by in-silico evaluation
• Amino acid sequence
• PTMs • Unnecessary attributes by in-silico evaluation
• Thermal unfolding • Uncommon amino acids and aggregation prone
• FcRn regions by computational tools
• Solubility • Free thiols
• Viscosity • Protein-protein interactions
• Aggregation propensity • Forced degradation (other than thermal stress)
• Charge profile
• Hydrophobicity
• Protein-protein interactions
• Forced degradation (Thermal stress)

Figure 3. Recommended attributes for developability assessment.

Conclusions LMW Low molecular weight

IEX Ion exchange chromatography
Developability assessment has been recognized as a critical Lys Lysine
step that should occur early in the process of selecting a drug mAb Monoclonal antibody
Met Methionine
candidate for development. The candidate is selected based on MFI Micro-flow imaging
a thorough evaluation using in silico assessment with the aid MO Membrane osmometry
of computational approaches and experimental data from MOA Mechanism of action
extended characterization and forced degradation. NGNA N-glycolylneuraminic acid
Candidates with the most favorable biochemical and biophy- PD Pharmacodynamics
PD Pharmacokinetics
sical properties, and thus lower inherent risks, are selected for PEG Polyethylene glycol
further development. Knowledge gained from developability PTMs Post-translational modifications
assessments will also help guide process and product devel- QbD Quality by design
opment to reduce timelines and resource consumption, thus SAP Spatial-aggregation propensity
providing affordable and high-quality mAb therapeutics to SC Subcutaneous
SCX Strong cation exchange
patients. SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEC Size exclusion chromatography
SIC Self-interaction chromatography
Abbreviations SLS Static light scattering
Trp Tryptophan
AC-SINS Affinity capture self-interaction nanoparticle spectroscopy Tyr Tyrosine
ADCC Antibody-dependent cell-mediated cytotoxicity UF/DF Ultrafiltration/Diafiltration
ANS 1-anilino-naphthale-nesulfonate WCX Weak cation exchange
Asn Asparagine
Asp Aspartate
AUC Analytical ultracentrifugation
BLI Biolayer interferometry
Disclosure statement
C1q Component of complement No potential conflict of interest was reported by the authors.
CDC Complement-dependent cytotoxicity
CDR Complementarity-determining region
CE Capillary electrophoresis ORCID
CIC Cross-interaction chromatography
cIEF Capillary isoelectric focusing Dingjiang Liu http://orcid.org/0000-0002-8665-2426
CMC Chemistry, Manufacturing, and Controls
Cys Cysteine
CZE Capillary zone electrophoresis References
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