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Kmab 11 02 1553476
Kmab 11 02 1553476
REVIEW
The discovery and development of monoclonal antibody of a developability assessment is to critically evaluate the
(mAb) therapeutics is resource demanding and technically biochemical and biophysical properties of mAb lead candi-
challenging. Specifically, challenges associated with dates and select the molecules with the lowest risks for
Chemistry, Manufacturing, and Controls (CMC) development development.
such as high aggregation, high viscosity and susceptibility to Numerous studies have shown the importance of develop-
chemical degradation and insufficient product stability have ability assessment of mAb lead candidates. For example, poor
been commonly recognized. Conventionally, only limited cri- biophysical properties resulted in mAbs with lower expression,
teria such as antigen binding and in vivo properties including instability or shorter in vivo half-life.1,2 Continuous asparagine
safety, pharmacokinetics (PK) and pharmacodynamics (PD) (Asn) deamidation in the complementarity-determining region
in animal models are used to select a mAb candidate from the (CDR) has also caused loss of potency of a mAb.3 Given the
early discovery to development stage. Without extensive char- limitations of timelines and resources at the early stage of
acterization to understand the biochemical and biophysical development, a thorough developability evaluation may not
properties of the selected candidate, issues can arise from eliminate all risks that could occur later, but it does allow the
unexpected modifications, stability or poor PK and PD, selection of lead candidates with fewer development risks.
which can result in delayed project progress or even termina- Meanwhile, the knowledge gained through thorough evaluation
tion. The development risks are often associated with the also provides a strong foundation that in turn allows for
intrinsic properties of the drug candidates. Therefore, it is a quality by design (QbD) approach for process and formula-
critical to carry out a developability assessment before enter- tion development to mitigate any remaining identified risks.
ing process development. Developability assessment is When risks are deemed critical and cannot be mitigated, an
a process used to systematically evaluate drug candidates, early decision to re-engineer the molecule is much more pre-
including structural assessment and CMC liabilities, safety, ferable than a later decision because it allows companies to save
PK and PD, as well as manufacturability (Figure 1). resources and avoid excessive delays of the timeline.
Although, many interdependent factors contribute to the suc- The biochemical and biophysical properties of mAb candi-
cessful development of an mAb therapeutic, selection of dates are evaluated based on in silico and experimental eva-
a candidate with favorable biophysical and biochemical beha- luation, and according to: 1) the general properties of the
vior help lay down a solid foundation. Thus, the primary goal 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.
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
properties of mAb molecules including posttranslational MAbs with attributes within or better than these ranges are
modifications (PTMs), stability and degradation pathways; expected to have relatively lower development risks.
and 3) drug developers’ internal knowledge from develop- Because of the highly conserved primary and similar high-
ment of similar molecules. order structures of mAbs, the commonly recognized degrada-
To date, approximately 80 mAb and Fc-fusion protein drug tion hot-spots can guide drug candidate evaluation to identify
products have been approved by the US Food and Drug potential problematic features. For example, Asn and aspar-
Administration (FDA) and the European Medicines Agency tate (Asp) residues in the flexible CDRs are susceptible to
(EMA),4,5 and more than 70 are in late-stage development.6 deamidation and isomerization,17 respectively, and thus need
Among them, some generally preferred drug properties for more careful examination. The correlation between aggrega-
mAbs have begun to emerge (Table 1). It should be noted that tion or faster clearance and exposed hydrophobic18-22 or
some attributes such as high molecular weight (HMW) species charged patches23,24 in the variable domains can also be
are dependent on the sample shelf-life and handling history. 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
Minor forms
● G0, G0F-GlcNAC <5% 10–16
∘
NANA <12% 12,14
∘
NGNA <2% Low (murine cell lines) 12,15
allow identification of potential risks based on the amino acid spots identified from steps 1 and 2, and second, reveal candi-
sequences of mAb candidates. date-specific degradation hot-spots that were not identified
Drug developers’ internal knowledge from process and from Step 1. Forced degradation studies also provide additional
formulation development also plays a significant role in devel- stability information to rank the candidates. Step 2 and Step 3
opability evaluation. Limitations of stable cell line, cell culture can be carried out simultaneously to allow a thorough evalua-
media components, and drug substance process steps are tion of mAb candidates within a short timeframe.
common elements that may exist across the pipeline and
should be part of the developability assessment consideration.
In silico evaluation
Therefore, we are proposing an overall developability assess-
ment (Figure 1), focusing on evaluation of biochemical and We propose to categorize mAb structural features into three
biophysical properties at early stage. groups (Table 2), namely, Problematic, Unnecessary and
Below is a three-step workflow for the assessment of struc- Further Considerations for in silico evaluation, based on the
tural and CMC liabilities (Figure 2), taking into consideration general properties of mAbs, including PTMs and degradation
previous proposals.3,25-29 Step 1 is in silico evaluation, including pathways.30,31 Problematic attributes have been associated
the use of computational methods, based on amino acid with known safety or efficacy concerns. Unnecessary attri-
sequence to identify the known degradation hot spots and butes have not been linked to any biological functionalities.
problematic motifs. Step 2 is to perform in-depth characteriza- Attributes for further considerations have not been consid-
tion to experimentally evaluate key biochemical and biophysi- ered traditionally for mAb lead selection, but may be consid-
cal properties, such as structure, stability, charge profiles, ered for next-generation molecules with improved properties,
PTMs, solubility, and hydrophobicity. Step 3 is to carry out such as minimal immunogenicity, enhanced stability and
limited forced degradation studies to, first, further confirm hot- extended half-life.
do not raise safety or efficacy concerns. The presence of these Host cell protein (HCP) in mAb therapeutics can also con-
attributes possesses unnecessary challenges for the manufac- tribute to immunogenicity.93,94 The type and amount of HCP in
turing of product with consistent profiles, and complicates drug substance can be dependent on the specific mAb sequence
analytical method development. or manufacturing process conditions, such as cell line, cell
Cyclization of N-terminal glutamine (Gln) to form pyro- culture and stringency of purification parameters. However,
glutamate (pyroGlu) is a major source of heterogeneity of mAbs with more general stickiness due to exposed hydrophobic
mAbs. This reaction occurs spontaneously during drug sub- or charged patches are expected to have more HCP problems.
stance production process, storage in liquid formulation and The potential for self-administration through subcutaneous
continues under physiological conditions74 and during sto- injection requires mAbs to be formulated at high concentra-
rage. This N-terminal modification has no impact on struc- tion (≥100 mg/mL) and delivered at small volumes (≤2mL).
ture, stability, or biological functions of mAbs.75 N-terminal In terms of developability, high solubility and low viscosity
Gln of human endogenous IgGs is almost completely con- are thus required. Since strong mAb self-association is often
verted to pyroGlu.76 It has been proposed to substitute the cause of low solubility and high viscosity, protein engi-
N-terminal Gln with other amino acids to eliminate this neering can be applied to reduce self-association by modifying
source of heterogeneity. the protein sequences and thus increase mAb solubility95 or
Incomplete processing of C-terminal lysine (Lys) is another decrease viscosity.26,96-99 Aside from the previously discussed
common modification. C-terminal Lys has no impact on mAb potential issues with variable domain glycosylation, the intro-
potency77,78 PK, PD or immunogenicity.78 However, removal duction of glycosylation sites near aggregation-prone regions
of C-terminal Lys showed optimal C1q binding and CDC.79 (APRs) was demonstrated to improve mAb solubility.95,100-103
C-terminal Lys is rapidly removed during circulation,80 Modulation of in vivo half-life has been explored by chan-
explaining its absence from endogenous IgGs.80 Removal of ging amino acid sequence around the FcRn binding site83,104
the codon for C-terminal Lys can eliminate this heterogeneity. 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
Further considerations binding can be addressed by altering amino acid sequences
MAbs for therapeutic purposes have evolved from murine ori- to disrupt charged or hydrophobic patches.2,26 MAbs can be
gin, to chimeric, humanized and fully human molecules to tailored to have either extended or shortened half-life to better
reduce immunogenicity.81 However, immunogenicity remains fit their therapeutic purposes and to increase patient compli-
a concern even for mAbs with full human sequences.81,82 ance, e.g., longer half-life reducing dosing frequency.
Various tools including in silico calculation, in vitro assays or
in vivo animal models are designed to identify amino acid
Experimental evaluation by extended
sequences causing immunogenicities.81,83,84 Additional immu-
characterization
nogenic components such as alpha 1,3 galactose can be elimi-
nated with the removal of variable domain glycosylation Following in silico evaluation, lead candidates are usually eval-
sites.81,83 uated experimentally through extended characterization.
Aggregation of mAb therapeutics is another contributor to Quality attributes that are highly relevant to mAb developability
immunogenicity, as well as to processing difficulties.85 Attempts assessment are summarized in Table 3. Usually, the initial
have been made to identify regions in mAbs that mediate aggre- material available for testing are produced by transient transfec-
gation through computational algorithms,18,19,86-90 or experi- tion (e.g., HEK293 cells). Properties that are independent of the
mental screening (e.g., phage display),91 and then to improve expression hosts such as primary sequence, hydrophobicity,
mAb stability by mutagenesis.2,19,83,88,91,92 solubility, thermal stability, and antigen binding affinity, can
be evaluated without any observable differences. On the other be on those modifications that correspond to the potentially
hand, most PTMs are highly dependent on cell line and cell problematic attributes. For those PTMs that are highly depen-
culture conditions, such as pH, temperature, and cell culture dent on cell lines, re-evaluation using materials from the
duration. Those cell line-dependent attributes should be evalu- stable cell line throughout the development is necessary.
ated using materials produced from the stable cell lines that will Comparison of the different candidates is based on the nature
be used for clinical and/or commercial manufacturing. of modifications and their relative percentages.
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
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
Table 4. (Continued).
Ave. Site/
Mono. mass mass Composition location Modifications Recommendation/Rationale Ref.
−33.9877 −34.08 H(−2)S(−1) C Dehydroalanine Not highly relevant because it of low 173
frequency
−43.0534 −43.07 H(−5)N(−3) R Oxidative carbonylation to glutamic Not highly relevant because it of low 159
O(−2) P
−203.0793 −203.20 H(−13)C(−8) N-linked Loss of N-acetylglucosamine Select candidates with low amount because 10,177-184
Since the level of terminal galactosylation varies with different issues during purification, sterile filtration, fill and finish, ship-
cell lines and cell culture conditions, it may have to be re- ping, storage214 and more importantly, can adversely affect
evaluated later in the development process. 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
FcRn affinity the chromatographic steps. During the final ultrafiltration/dia-
FcRn binding is one of the most critical factors affecting mAb filtration (UF/DF) step, mAbs are buffer exchanged into for-
half-life.104 The FcRn binding affinities of mAb candidates are mulation buffers at concentrations above the targeted drug
usually measured by Biacore, but alternative assays using biolayer product, thus requiring much higher solubility.
interferometry (BLI) or FcRn affinity chromatography have been Lower solubility is usually caused by strong mAb self- asso-
established. In a study evaluating mAbs, it was found that delayed ciation with exposed hydrophobic or charged patches. Naturally,
elution of mAbs from an FcRn affinity column at neutral pH the bivalent nature of mAbs amplifies their self-association
correlated with poor pK.24,199 Compared to Biacore and affinity tendencies.216 Colloidal instability caused by conformational
chromatography, much higher throughput can be obtained using changes or chemical modifications can also contribute to the
BLI.200 In general, mAbs with stronger FcRn binding at acidic pH, poor solubility of a mAb. Additionally, mAb solubility is often
but fast dissociation at neutral pH, show longer in vivo half-life.104 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
Thermal stability based on the amino acid sequences, therefore solubility of
mAbs should be studied experimentally. However, studying
Thermal stability is the ability of a protein to maintain its struc-
mAb solubility directly requires large amounts of protein,
tural and functional integrity under different temperature envir-
typically several hundred milligrams, and it is often not prac-
onment, and is an intrinsic property of mAbs that can influence
tical to produce all candidates at such large quantities for
product stability, such as aggregation, during manufacturing and
solubility study. Given the limited sample quantities available
storage. High thermal stability of a mAb candidate indicates
for developability assessment studies, indirect measurement
a well-packed structure that requires more energy to unfold.
methods are commonly used. For example, addition of poly-
Therefore, higher thermal stability of a mAb generally correlates
ethylene glycol (PEG) to mAb solutions causes precipitation
with a lower tendency towards partial unfolding and thus
at much lower concentrations, and thus can be used to deter-
aggregation.19,73,201-206 Besides aggregation, mAbs with lower
mine the apparent solubility of mAbs through extrapolation
thermal stability have been shown to have lower expression.73,207
to zero PEG concentration.212,220-222 This approach can be
Thermal stability has been commonly measured by differ-
implemented in a high-throughput manner for mAb candi-
ential scanning calorimetry (DSC). 19,73,201-206 An alternative
date selection. However, PEG-induced precipitation may not
method using differential scanning fluorimetry (DSF) allows
truly reflect the mechanisms of the poor solubility of
high throughput thermal stability screening of 96 or 384
mAbs,218,223 and thus orthogonal methods or direct evalua-
samples.202,206,208-210 Multiple candidates analyzed under the
tion of solubility at high concentration should be considered
same conditions can be ranked based on the obtained thermo-
to confirm the predicted solubility or validate the rank order.
dynamic parameters such as the midpoint temperature (Tm) of
Prediction of the high concentration behavior of a mAb
unfolding or the onset of unfolding temperature (Tonset).
using low concentration sample may also be done through the
measurement of the osmotic second virial coefficient B22,
Solubility a thermodynamic parameter related to intermolecular inter-
actions. Positive and negative B22 values indicate repulsive or
Solubility is an important developability parameter for attractive forces, respectively. Parameters affecting B22 include
mAbs,211,212 especially in consideration of the industry trend electrostatic interactions, van der Waals force, excluded
towards higher concentration formulations (100 mg/mL and volumes, hydration forces, and hydrophobic effects.224,225
above).213 MAbs must remain soluble throughout processing, Among many different ways to obtain B22 values, such as self-
storage and administration.97,211 Low solubility can lead to interaction chromatography (SIC),226 membrane osmometry
248 Y. XU ET AL.
(MO)227 and analytical ultracentrifugation (AUC),228 the allow the drug formulation to be delivered with manual injec-
most common method is through static light scattering tion. The viscosity target is typically developed based on each
(SLS).225,229,230 Recently this value was used to determine company’s internal development experience, in particular for
a universal solubility line, the “liquidus” line, as part of developing SC product with prefilled syringes and auto-
a phase diagram for a mAb.231,232 injector devices. For example, it was proposed that the visc-
Cross-interaction chromatography (CIC) assay, introduced osity can be grouped into 3 categories: 1) “preferred” viscosity
by Jacob et al.233 takes advantage of the accumulative effect on of 10 cP or lower; 2) “acceptable” viscosity between 10 to 20
a column to capture weak binding between testing mAb and cP; and 3) “unacceptable’ viscosity of > 20 cP. This can be
a large quantity (30 mgs) of immobilized human serum IgGs. used as a starting point for defining the viscosity target with
MAbs with late elution by CIC assay correlate with poor additional considerations of the internal experience of process
solubility, due to exposed sticky (hydrophobic or charge) sur- and product development and product knowledge of delivery
faces. The other method worth mentioning is self-interaction devices, such as autoinjector.
nanoparticle spectroscopy, which uses gold nanoparticles to
concentrate mAb molecule to a high local concentration to
Aggregation propensity
amplify weak self-interaction.216,234 This method can also be
applied to high throughput screening of mAb candidates.234 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-
Viscosity
opability assessment. In addition to utilizing predictive tools,
High concentration drug products administrated via subcuta- aggregation propensity can be directly measured during
neous (SC) injection require a formulation with manageable extended characterization and forced degradation studies.
viscosity, making it another critical factor for evaluation at an Though typically only low concentration data is available
early stage to ease developability concerns.235 High viscosity due to material limitation, it is important to evaluate the
can pose challenges to the final UF/DF step 28,236,237 and fill/ colloidal stability and aggregation propensity of a mAb at
finish operation.238,239 Viscous drug product can lead to diffi- medium to high (50–100 mg/mL) concentration ranges.
culties in delivery causing low patient compliance.240 Viscous Routinely, sodium dodecyl sulfate-polyacrylamide gel elec-
samples can also pose sampling challenges for analytical trophoresis (SDS-PAGE) or capillary electrophoresis (CE-
method development and instrumentation.241 SDS) are used to determine mAb monomer, fragments and
High viscosity has been shown to be caused by strong mAb covalent aggregates under denaturing conditions with or
self-association through electrostatic 26,98,242-246 or hydropho- without reduction. A variety of other methods are available
bic interactions,26,242 or the combination of both.26,242,247 to measure mAb soluble aggregates such as dimer, oligomer
Although, a number of formulation parameters, including or subvisible particles under native conditions.255-258 Size-
pH, salts, sugars, and various small molecule excipients and exclusion chromatography (SEC) is the most commonly
detergents can be explored to lower viscocity,97,248-251 selec- used method to determine mAb HMW species (e.g., dimer,
tion of mAb lead candidates with minimal inherent problems trimer or oligomers) and LMW species. SEC is typically
such as exposure of hydrophobic, or charged patches is one of included for product release and often available as a generic
the most efficient means to minimize high viscosity risk.242,252 or platform method, therefore it is suitable for evaluation of
A variety of methods have been used to measure viscosity, aggregates during extended characterization and forced degra-
including Cannon-Fenske Routine viscometer, Taylor Cone dation. It is worth mentioning that SEC can sometimes reveal
plate method, and various rheometers. Most of the conven- properties other than the percentage of monomer, aggregates
tional techniques for measuring viscosity require a large and fragments. Abnormal SEC behaviors of a mAb, such as
amount of materials. To overcome this challenge, in particular peak tailing, could indicate non-ideal biophysical properties.1
for developability assessment, a high throughput DLS method Longer retention time and asymmetric peak shape can suggest
has been developed based on measurement of apparent poly- nonspecific interactions between a mAb and the SEC
styrene bead radii in high concentration mAb solutions to column.2 Studies showed that SEC can even separate mAb
back calculate the viscosity of a mAb solution.253 This method variants containing succinimide intermediate from those with
can only be used for mAbs without interaction with the beads, Asn deamidation (17Da) or Asp isomerization (18Da).167,259
otherwise the apparent bead radii cannot be reliably mea- SEC has also shown that a mAb variant containing oxidized
sured. High throughput diffusion interaction parameters Trp eluting earlier than the main peak.158 These examples
derived from DLS measurement have also been shown to suggest that interpretation of SEC data should be done cau-
correlate with viscosity.254 In recent years, the instruments tiously because earlier and later peaks may not always repre-
allowing viscosity measurement using ≤100 µL and with auto- sent HMW or LMW species.
mated sample handling have become commercially available, For large aggregates, light scattering can be used to char-
and these are suitable for measuring viscosity during devel- acterize particles in the range of <1 nm to 1–10 µm. The DLS
opability assessment. method can be used to determine the hydrodynamic diameters
Because of the significance of viscosity for process and of mAbs242 and interaction parameters, such as KD,260,261
product development, having a carefully defined viscosity which can be run in high throughput mode with low sample
target for developability assessment is important. At mini- consumption. Methods that measure turbidity, such as optical
mum, the formulation viscosity should be low enough to density at visible wavelength and nephelometry, can also be
MABS 249
considered for detection of submicron/sub visible particles. modifications may impact chromatographic separations of
These techniques may be developed with high throughout mAb variants by modulating mAb structures. For examples,
and low volume consumption, and thus can be used for devel- mAbs with smaller oligosaccharides can contribute to the
opability assessment. Light obscuration (e.g., HIAC) and flow formation of basic species,263 while the oxidized Met may
imaging methods (e.g., micro-flow imaging (MFI)) and contribute to the formation of either acidic172,264 or
FlowCam) can be used for sub-visible particle characterization basic265,266 species. Similarly, the presence of the incompletely
and quantification for sizes greater than 2 µm. A visual inspec- formed disulfide bond in the heavy chain variable domain can
tion method is used for detecting the protein particles in the either contribute to acidic163 or basic162 species.
visible range, typically > 70 to 100 µm. Isoelectric focusing gel electrophoresis (IEF) was tradition-
In addition to directly measuring the level of aggregates, ally used to analyze mAb charge variants.37,267,268,276 This
aggregation propensity can be ranked based on hydrophobi- semi-quantitative, labor-intensive method relies on dye stain-
city or protein interactions as they are the main driving forces ing for detection. It also suffers from low throughput, lack of
for aggregation. Fluorescence dyes such as 1-anilino- automation and poor reproducibility. Capillary IEF (cIEF)
naphthalenesulfonate (ANS) and thioflavin can be used to overcame most of the IEF limitations and offered additional
probe exposed hydrophobic patches in a high throughput advantages, including high sensitivity, automation, and low
manner with minimal sample requirement.262 Affinity capture sample consumption.276-278 Moreover, imaged cIEF (icIEF)
self-interaction nanoparticle spectroscopy (AC-SINS), which has gained popularity for the analysis of mAb charge
provides coarse-grained information about interactions and variants279-281 because the whole capillary imaging eliminates
aggregation propensity in different solution conditions, is the troublesome mobilization step used by cIEF.282
useful to leverage during developability evaluations.234 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-
Charge variants 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
Charge variation of mAbs reflects the sum of various PTMs. N-linked glycans from tryptic peptides16 and analyze the sites of
Variants of mAbs need to be closely monitored throughout the deamidation and isomerization.287 A single CE-MS run has been
development process to ensure consistent peak profiles. Because shown to confirm 100% of the primary structure and reveal
of its sensitivity to process changes, charge variation is one of the several PTMs, including glycosylation, N-terminal Gln cycliza-
quality attributes that could be challenging for demonstrating tion, deamidation and isomerization.288
comparability, when process changes are introduced. Ion exchange chromatography (IEX), including cation
A typical mAb charge variant profile characterized by exchange36,37,75,166,264,267,268,271,272 and anion exchange,116,155,276
charged-based methods such as ion exchange chromatogra- has been widely used to monitor mAb charge variants. IEX allows
phy and isoelectric focusing usually contains one major peak fraction collection for further characterization. Multiple mAbs can
and several smaller acidic and basic peaks. Reported modifi- be analyzed when a pH gradient is used,289 implying the potential
cations resulting in the formation of acidic or basic species are for establishment of a platform method. Strong cation exchange
shown in Table 5. It is worth mentioning that several (SCX) chromatography allows a relatively higher throughput
compared to weak cation exchange chromatography.290,291
Table 5. Modifications that form either acidic or basic species. When comparing the overall charge profiles, IEF usually shows
Modifications References comparable results with either cation37or anion276 exchange chro-
Acidic matography. However, different profiles have been observed due
● Deamidation 33,36,37,75,116,126,264,267-270
to differences in the separation mechanisms.267,268
● Glycation 126,130
● 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
● 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. proteins were immobilized.233,293,311 Positive correlation
Modifications References between delayed retention between CIC and HIC suggests
Early shift that hydrophobic interaction being a major contributing fac-
● C-terminal Lys 41,172
● Isomerization succinimide 43
● Cysteinylation 150
Late shift
● Asp isomerization 41,43,44,161
Experimental evaluation by forced degradation
● Variable domain incomplete disulfide bond 43,44,161,297
Cyclization of N-terminal
Glu164
Met oxidation156,324
Asp isomerization41,47,48,170,171
Disulfide bond degradation
through β-elimination
322,323,333
Asn deamidation58
Covalent cross-linking160,346
251
252 Y. XU ET AL.
while taking into consideration the intrinsic properties of the rank mAb candidates to lower the potential immunogenicity
mAb candidates. risk.
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)
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