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Analytical Characterization of
Biotherapeutic Products, Part I:
Quality Attributes
Anurag S. Rathore, Ira S. Krull, and Srishti Joshi
T
To ensure he potential for the presence of multiple impurities and
the reliable variants in biotherapeutic products highlights the need for
having an arsenal of orthogonal analytical techniques that
and accurate together can yield a reliable and accurate characterization of
characterization the product. Here, in the first part of this two-part series, we discuss
of the various quality attributes that are pertinent to a biotherapeutic.
In part II, we will present the current and evolving practices that are
biotherapeutics, being used for analysis of these attributes.
an arsenal of
orthogonal Since the commercialization of human insulin in 1982, the first
genetically engineered therapeutic, there has been exponential
analytical progress in the field of biotherapeutics, both in terms of the
techniques is complexity of the molecules produced as well as their characterization.
needed. Moreover, as the innovator drugs reach their patent cliff, they have
paved the way for biosimilars, which are copies of innovator drugs
that offer similar physical and chemical characteristics. However,
unlike a “generic” drug that is a chemically synthesized identical copy
FEBRUARY 2022 | LCGC 2

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ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES
of the innovator drug, the production of arrangement of single or multiple linear

biosimilars is complex because it involves the polypeptide chains, respectively.
recombinant expression of the specific protein
or peptide in a biological host (1,2). Therefore, Parallels can be drawn between the structural
the amount and type of permissible structural hierarchy of a protein and other forms of
and functional variations have to be strictly coded information. An example of a collection
controlled to match the innovator molecule. of books can be used to understand this
This requires extensive physicochemical layering of information, where a sentence,
characterization of the structure, structural
variation, and impurities that may arise from FIGURE 1: Structural complexity of a
the manufacturing process itself or the host mAb. The primary structure consists
used for the protein production (3). Typically, of a linear chain of amino acids that
these variations can be categorized into those arrange in three dimensions in a
that result in substantial changes to the safety specific manner to form the secondary
and efficacy profile of the product (product structure consisting of alpha helix, beta
impurities) and those that result in less than sheets, and random coil. The tertiary
substantial changes (product variants) (4). structure consists of functionally active
arrangements of secondary structures.
The potential for the presence of multiple In mAbs, interactions between a
impurities and variants in biotherapeutic number of the same or different tertiary
products highlights the need for having structures can occur to form the
an arsenal of orthogonal analytical quaternary structure.
techniques that together can yield a
reliable and accurate characterization of
the product. Here, we discuss the various
quality attributes that are pertinent to a
biotherapeutic. In part II of this series,
we will present the current and evolving
practices that are being used for the analysis
of these attributes.
Biotherapeutics as Proteins
Before delving deeper into the quality
attributes and their effect on product
efficacy and safety, it is important to
appreciate that biotherapeutics are
essentially proteins or protein complexes
arising from a three-dimensional (3D)

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information like the conserved functional

SPONSORED CONTENT
domains (the constant region in mAbs) or
Determination of Protein the variable region on which the antigen
Secondary Structures of binding sites unique to each antibody are
Monoclonal Antibody
located. The location of conserved cysteine
using FTIR Spectroscopy
participating in disulfide bond formation
(insulin, mAbs) are all identifiable from the
although meaningful as a stand-alone, only primary structure of a biotherapeutic (6).
imparts limited information, and can be However, it is important to remember that
used in conjunction with other sentences the sequence specificity of this translated
to create a paragraph that conveys more linear information is but a prerequisite to
meaning and context. Similarly, a chapter a protein and correct processing of higher
consisting of many paragraphs would hold orders of structure needs to take place for it
more information, and so on (FIGURE 1). The to attain proper functionality.
different levels of structural complexity of a
protein are discussed in further detail below Higher Order Structure
and illustrated (FIGURE 1) using the example of Protein higher order structure (HOS) includes
a monoclonal antibody (mAb). all orders of the structure above the primary
structure, such as secondary, tertiary, and,
Primary Structure in some cases, quaternary structure. HOS
The unique, linear amino acids sequence assessment is a key component in defining
of a polypeptide, held together by covalent a biotherapeutic’s critical quality attribute.
peptide bonds, is known as its primary Further, changes in higher order structure
structure. This sequence is a fingerprint of the can impact quality, stability, potency, and
protein and defines its structure, function, efficacy of the product with increased
and, in certain cases, even its intracellular potential for immunogenicity and loss of
localization. Because different amino acids biological function.
differ structurally by different side-chain
substituents, their unique arrangement Secondary structure relates to the 3D
confers different structural and functional arrangement of local segments of proteins.
properties to proteins. The primary structure The major types of secondary structures in a
has two ends, one with a free carboxyl group protein molecule are alpha helices, beta sheets,
called the carboxy-terminus or C-terminus, turns, and random coil. The arrangement of the
the other with a free α-amino group known as secondary structure of a protein is conserved
the amino-terminus or N-terminus (5). and is important for proper folding.
In the context of biotherapeutics, the When designing a therapeutic drug,

primary structure holds important secondary structure plays a significant role

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in determining proper functioning of the

protein of interest. An example of this is SPONSORED CONTENT
the formation of loop structures, a common Disulfide Bond Characterization
feature in proteins, that play a significant of Monoclonal Antibody (mAb)
role in downstream signaling by post- Using Q-TOF Mass Spectrometer
translational modifications or as recognition
sites for other proteins. Complementarity
determining region (CDR) loops of the heavy shown in several recent publications that
and light chain in antibodies are a prominent even innovator products show a wide range
example of this structure (7,8). of variability in their critical quality attributes
over long periods of time, possibly as a
Tertiary structure refers to the 3D result of process improvements, technology
arrangement of a single polypeptide chain transfers, scale-ups, and other such product
containing all the functional domains life-cycle associated activities (11). This
of the monomer. Different secondary variability is of particular importance to the
elements present in the protein fold into biosimilar industry because the approval of
stable globular structures through salt a biosimilar is based on the premise that the
bridges, hydrogen bonds, or disulfide bridge biosimilar product is analytically similar to
formation (9). For a lot of proteins, the the innovator product and hence a reduced
tertiary order is the highest and they are fully clinical evaluation is warranted (4).
functional after achieving this conformation
(for example, insulin, granulocyte-colony In this context, a major issue is to define
stimulating factor). However, certain how much variability is acceptable (12).
biotherapeutics, such as mAbs, attain their This step requires risk assessment and
functionality via a specific arrangement of an understanding of the types of product
the different monomers (light chain, heavy heterogeneity, its origin in the manufacturing
chain) into a quaternary structure (2,10). process, as well as its effect on product
potency and efficacy. Product associated
Quality Attributes of features that have an impact on quality and
Biotherapeutic Products under which the heterogeneity is assessed
An inherent amount of heterogeneity is and characterized are known as quality
always present in biotherapeutic products attributes (QA). Amongst the numerous QAs,
in response to variations in the biological some are critical to effective functioning of
processing of the host organism used for the drug as well as its clinical safety and are
production. Controlling manufacturing known as critical quality attributes (CQA).
processes precisely at an intracellular level Other attributes or variation that may not
such that the product at the end of each cycle directly impact the drug efficacy or its clinical
is identical is a significant task. It has been safety, but are important from a process and

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TABLE 1: A list of quality attributes of biotherapeutics and their impact on drug

product performance
Category Attributes Critically Impact References
Glycosylation Critical Potency, PD, PK (16, 17)

Disulfide modifications Critical Potency (18,19)
Glycation Noncritical None (20-22)
Methionine oxidation Noncritical Potency (23, 24)
Deamidation Potency (25)
Aggregation Critical Immunogenicity and potency (26)
Fragmentation Critical Potency (16)
Terminal modification Noncritical None (27)
Process- Host cell protein
related Critical Immunogenicity (28)
Host cell DNA
Critical Immunogenicity (29)
Raw materials/leachable
Critical Immunogenicity (30)
compounds
Other Adventitious agents Critical Immunogenicity (31)
contami-
nants Toxic by-products Critical Immunogenicity (30)
product consistency point of view are known manufacturing process. PTMs that are
as key quality attributes (KQA) (13–15). essential for function mostly occur inside the
Quality attributes and their potential impact cell (in vivo PTMs). Other PTMs might occur
on overall product performance are listed in outside the cell post lyses (in vitro PTMs), in
TABLE 1 and discussed in further detail in the response to stress, the sample handling, or
subsequent section. deliberate addition of the chemical group
to improve a certain product attribute (such
Post-Translational Modifications as PEGylation). Such PTMs are generally
Translation is the process by which ribosomes undesirable because they add to the pool of
in a cell’s cytoplasm process specific protein- protein variants in the formulation (1,32).
coding gene sequences into proteins. Any
modifications that may occur post the Glycosylation
release of the protein from the ribosomal Of the different PTMs that are known to occur
assembly come under the category of in the cell, the most studied PTM relevant
post-translational modifications (PTMs). to biotherapeutics, especially to mAbs, is
Some PTMs are essential for the protein to glycosylation. This PTM involves the addition
attain its function and are hence desirable of sugar residues to amino acids bearing amino
or even essential in the biotherapeutic or hydroxyl groups. Glycans (monosaccharides

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linked glycosidically) are highly versatile in their within the evolutionarily conserved domains
size (ranging from a few hundred up to several of the protein family, such as in human
thousand daltons) and in their arrangement immunoglobulins and cysteine synthase
(single, linear, or branched). complex in plants. For small molecules, the
probability of disulfide bond scrambling
The glycan profile of a biotherapeutic, is low because of the presence of limited
especially mAbs can be highly variable owing cysteine residues. However, with complex
to the number of possible arrangements molecules such as the monoclonal antibody
of the sugar moieties. Depending on the complex, the 16 disulfide bonds present
site of attachment to the polypeptide are crucial to the stability and hence the
backbone, glycans can be N-linked or downstream efficacy of the complex (18,19).
O-linked. Depending on the type and site
of glycosylation, the presence or absence of Glycation
glycosylation can lead to increased stability, Glycation is the nonenzymatic addition
reduced aggregation, and changes in of a monosaccharide to a protein or
potency. A popular example of the negative lipid molecule that usually occurs upon
impact of the absence of glycosylation on prolonged incubation with reduced sugars.
activity is interferon-beta (INF-b), where As it is a chemically driven modification, the
glycosylation imparts a significant increase attachment of monomeric sugar adjacent
in protein activity (33–35). In complex to a lysine molecule can also occur post
molecules such as mAbs, glycosylation of the administration of the biotherapeutic, during
Fc region has been shown to be essential its circulation time in the blood. Therefore, it
for FcyII and FcyIII receptor binding (36). is critical to ascertain its effects on induced
Inversely, an absence of fucosylation leads immunogenicity. After they are glycated,
to an increase in FcyIII receptor binding in proteins can undergo further oxidation to
mAbs (33,37). generate a complex set of end products,
collectively termed advanced glycation
Disulfide Bond Formation end (AGE) products. These products are
Another functionally important PTM with involved in a range of pathological conditions
the potential effect on protein stability and like diabetes and osteoarthritis (38,39). In
efficacy is the disulfide bond formation. biotherapeutics, studies done so far have
Disulfide bonds can form between two shown that induced glycation increases the
cysteine residues within a single polypeptide clearance rate and reduces the circulation
chain, such as in insulin, or between time of recombinant IgGs in mice (21).
two proteins to stabilize a complex like However, this might be specific to the
immunoglobulins (IgGs). Cysteine residues point of glycation on the mAb as well as
that are structurally or functionally important the mechanism of clearance, as indicated
for a class of proteins are usually present by a study done on the effect of glycation

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on the Fc region of a mAb where glycation Deamidation

of 0.5 Glc/Lys did not show any significant Similar to oxidation and glycation,
difference to FcyIII receptor binding (20,21). deamidation is a nonenzymatically driven
chemical reaction that leads to conversion or
Oxidation removal of an amide functional group in the
Oxidation of a protein is caused because side chain asparagine (asn) or glutamine (Gln)
of covalent modification of a protein upon residues in a protein. Asn is converted to
exposure to reactive oxygen species (ROS) aspartic acid (Asx) or isoaspartic acid and Glu
or reaction with by-products of oxidative to α- and γ-Glu (42). Deamidation of proteins
stress. Modifications because of oxidation has been shown to be a contributing factor
lead to a range of changes in the protein to aging and several diseases such as celiac
structure and behaviors such as increased disease, urinary tract infection, cataract
side-chain hydrophilicity, side-chain and formation, cancer, and neurodegenerative
backbone fragmentation, aggregation diseases. The rate of deamidation is
via covalent cross-linking or hydrophobic affected by physiological conditions around
interactions, protein unfolding and altered the protein such as pH. The effect of
conformation, altered interactions with deamidation on protein function is not clear
biological partners, and modified turnover because this modification does not always
(40). In the context of biotherapeutics, alter the functionality of a protein. However,
oxidation of methionine and tryptophan cases where an effect on protein function
residues are known to be common have been reported include glycoprotein
degradation pathways, especially for CD4 (43), where deamidation at Asn52-
complex molecules such as mAbs. Asp53 of CD4 reduced the in vitro biological
Oxidation of conserved methionine at activity of the product. In the context of
CH2-CH3 sites (Met256 and Met432) has biotherapeutics, because of its potential
been shown to cause a loss of FcRn receptor effect on protein turnover and function,
binding resulting in a loss of efficacy (41). it is considered a CQA for biotherapeutic
Moreover, as the protein A and protein G manufacturing (42,44,45).
sites are located close to the CH2-CH3
region, oxidation of Met256 and Met432 Aggregation and Fragmentation
has also been shown to cause a decrease All self-associations of proteins into
in protein A and G binding, thus leading assemblies other than the native HOS
to a loss in manufacturing productivity come under the umbrella term “aggregates.”
(23). Because of the above reasons, it is Aggregates can have a range of species
considered a CQA for biotherapeutics depending upon size (dimer, trimer), type of
and as such is closely monitored with interaction (covalent, noncovalent), solubility
a well-defined, acceptable range for (soluble, nonsoluble), type of formation
quality tolerance. (ordered and disordered), and mode of

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formation (reversible, nonreversible) (46). chemical disruption of the peptide bond. The
Initiation of aggregation can take place in impact of fragmentation would depend on
response to various abiotic (temperature, the specific activity of the cleaved portion.
pH, shear, and pressure) and biotic stresses For example, generation of Fab fragments by
(immunological state of the host species cleavage at hinge region leads to an altered
and genetic predisposition). Because of pharmacokinetic profile. Similarly, dissociation
the multitude of causal agents, predicting of the variable region responsible for antigen
aggregation is a challenging task (33). recognition leads to disruption in downstream
signaling and activation of associated immune
In therapeutic proteins, the aggregation pathways, thus reducing drug efficacy (33).
phenomenon is of particular interest because
of its potential for varied immunological C- and N-Terminal Modifications
responses in humans. Aggregated drug Cyclization of the N-terminal (Gln) or Glu to
species can elicit formation of antidrug form pyroglutamate (pyroE) and incomplete
antibodies that bind the drug molecule, removal of leader sequence are the two
thus reducing its efficacy and also activation major types of N-terminal modifications of
downstream of immune responses in monoclonal antibodies. PyroE formation is a
patients (47). Immune- response pathways, spontaneous event that occurs even during
specific to aggregate species, still need to post-drug administration. Studies have shown
be segregated and understood in detail that the presence of PyroE does not lead to
to predict the type of immune response any changes in the antibody structure or affect
elicited by a certain aggregate. Instances its receptor binding kinetics. Another form
that have served as learning experiences for of N-terminal modification is the presence
the biotherapeutic manufacturing industry of portions of uncleaved leader peptide
include early formulations of intravenous sequence. Because of the failure of efficiency
immune globulin (IVIG) and human growth in cell cycle machinery, a percentage of the
hormone where antibody mediated adverse recombinant protein might show the presence
events were linked with the presence of of uncleaved leader sequence. Although it
aggregate species in the formulation (48). contributes to the charge variant species in
the final product, it has not been known to
The activity of a protein is a consequence of cause an immunogenic response in humans.
its sequence. In a multimeric protein such Moreover, studies have shown that the
as a mAb, different interacting partners presence of the extra amino acids does not
have different functions. Loss of any of cause any structural change or negatively
the subproteins of the assembly typically impact the binding kinetics (50,51).
leads to loss of the associated function.
Fragmentation of protein can occur both Process-Related Factors
enzymatically or nonenzymatically through Process-related impurities consist of

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contaminants generated by the host cell opposed to mammalian systems. As per the
itself (DNA and host cell proteins), as well International Conference on Harmonization
as the components of raw materials used (ICH) Q6b, an acceptable range of HCPs in
throughout the manufacturing process. the final formulation is under 100 ppm (53).
Immunogenic responses to specific host
Host Cell Impurities cell proteins are yet to be studied, but the
Host cell impurities are additions due to improvement of purification processes has
the production of the biotherapeutic in a led to a decrease in immunogenic responses
biologically active cell. Major contaminants indicating that the presence of HCPs does
in this category are host cell DNA and host have some effect in aggravating the immune
cell proteins. During cell lysis, host cell DNA system (54,55).
released can carry over into the final product
formulation. The risk associated with this Miscellaneous Components
contaminant comes from the integration of Other adventitious contaminants include
host cell DNA into the human genome. An viruses, nonhost origin bacterial population,
early study in which 108 genome equivalents fungal contamination, mycoplasma,
of DNA from a human tumor cell line was endotoxins, and impurities from
injected into nonhuman primates showed no contaminated raw materials. Sufficient viral
evidence of neoplastic disease, post 8 years clearance needs to be demonstrated for
of observation. Currently, the World Health biotherapeutics to be considered safe for
Organization (WHO) outlines 10 ng of host commercial use (56).
cell DNA per dose as the permissible limit
(29,33,52). Summary
With the advent of genetic engineering, it has
Other cell components that can carry over become possible to engineer and manufacture
into the final formulation are the host cell complex molecules. This capability has
proteins (HCPs). Given the substantial revolutionized modern medicine leading to a
proteome of hosts used in the biopharma rapidly evolving segment of biotherapeutics.
industry, especially the mammalian cell lines But as the old adage goes, “with great
such as Chinese hamster ovary cells, this is power comes great responsibility.” There
a potential cause of concern because the is the responsibility to understand the
presence of certain HCPs in the formulation quality attributes of a therapeutic protein
can cause immunogenic response upon product and its clinical efficacy. A better
administration. The susceptibility of understanding of the product allows for
immunogenic response is seen to be higher efficient and effective risk assessment. In
for bacterial proteins, probably because of this regard, implementing scientific practices
the larger phylogenetic distance between to define CQAs on a case by case basis is
the bacterial systems and primates as becoming a mandatory, regulatory exercise.

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Cano, D. Tesar, I. Nijem, D.E. Allison, P.Y. Wong,

Y.H. Kao, C. Quan, A. Joshi, R.J. Harris, and P.
Motchnik, MAbs 2(6), 613–624 (2010).
Anurag S. Rathore
51. H. Liu, G. Ponniah, H.M. Zhang, C. Nowak, A.
Professor, Department of Chemical Engineering
Neill, N. Gonzalez-Lopez, R. Patel, G. Cheng, Indian Institute of Technology
A.Z. Kita, and B. Andrien, MAbs 6(5), 1145–1154
(2014).
Ira S. Krull
52. C.E. Hogwood, D.G. Bracewell, and C.M. Smales,
Professor Emeritus, Department of
Bioengineered 4(5), 288–291 (2013). Chemistry and Chemical Biology
53. International Conference on Harmonization, Northeastern University
Expert Working Group, Specif. Test Proced. LCGC’s editorial advisory board member
Accept. Critreia Biotechnol. Prod. (ICH, Geneva,
Switzerland, 1–20, 1999). Srishti Joshi
54. A.J. Chirino and A. Mire-Sluis, Nat. Biotechnol. Post-Doctoral Research Fellow
22(11), 1383–1391 (2004). Indian Institute of Technology
55. B. Sharma, Biotechnol. Adv. 25(3), 325–331 This article first appeared in LCGC North America,
(2007). 36 (6), 376–384 (2018).
56. D.M. Strauss, T. Cano, H. Delucchi, M. Plancarte,
D. Coleman, G.S. Blank, Q. Chen, and B. Yang,
Biotechnol. Prog. 26(3), 750–755 (2010).

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Siarhei/stock.adobe.com
Analytical Characterization of
Biotherapeutic Products, Part II: The
Analytical Toolbox
Anurag S. Rathore, Ira S. Krull, and Srishti Joshi
I
Review of the n the first part of this series, we discussed the various quality
utility of the attributes that are pertinent to a biotherapeutic. In this issue, we
will present the current and evolving practices that are being used
traditional for analysis of these attributes.
tools and new,
orthological With increasing importance being attached to structural attributes
of biotherapeutics and subsequent biosimilars, the analytical
techniques. techniques used for characterizing these attributes have also evolved.
There is an ever-increasing interest in attaining a higher structural
resolution of these products (1,2). In view of the complexity exhibited
by biopharmaceutical products, it has become the norm to use a
multitude of orthogonal, high-resolution tools for characterization.
These tools are carefully chosen such that the platform covers all the
critical structural, physicochemical, immunochemical, and biologically
relevant characteristics of the molecule (3–5). Attributes to be covered
in biotherapeutic characterization, as per World Health Organization
(WHO) guidelines to evaluate quality, safety and efficacy (2), are

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ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX
TABLE 1: Techniques used for analytical characterization as mentioned in WHO

and ICH Q6b guidelines.
Type Attributes
WHO (2013) ICH Q6b (1999) Others
Physiochemical Amino-acid sequence,
characterization Molecular weight, extinc-
tion-coefficient, disulfide
linkage, N-terminal
methionine, signal/leader
sequence, N-/C-terminal HPLC (SEC, RP, IEX)
modifications, C-terminal Affinity), MS, SEC,
Primary processing, N-terminal HPLC, MS (ESI, SDS-PAGE,
MALS, DSC
structure pyroglutamate, deamida- MAL-DI-TOF), MS/MS IEF, UV-VIS
tion, oxidation, isomer- spectroscopy,
ization, fragmentation, Western Blot, CE
disulfide bond mismatch,
N-/O-linked oligosac-
charide, glycosylationm,
aggregation, C-terminal
lysine presence
Glycan content, glycan
Glycan structure, glycan pattern, HPLC, Electrophoresis, MS/
CE-MS
structure glycosylation site, glycan MS, UV-FLD, CE, IEF
charge pattern
X-ray crystallography NMR,
CD, FTIR, Fluorescence,
secondary structure,
Higher Order DSC, proton nuclear mag- Cryo-EM, Raman
tertiary structure, CD, NMR
Structure netic resonance (IH-NMR), spectroscopy
quarternary structure
Hydrogen-Deuterium
exchange MS
Biological Animal-based
ADCC, CDC, Apoptosis
characterization Effector function, biological assays,
assay, Fc-y receptor
complement binding and cell culture-based SPR, BLI, ITC
binding, Neonatal Fc
activation potency assays, biochemical
receptor binding
assays
Immunochemical Affinity, avidity, immuno-
characterization Product reactivity, epitope char- Binding assays,
Cell-based assays SPR, BLI, ITC
related acterization, glycosyla- western blot, ELISA
tion/PEGylation profile
Impurities, Fragmentation, amino HPLC, Electrophoresis, HPLC, SEC-HPLC,
contaminants acid modification, Higher MS, CE, SEC, FFF, AUC, SDS-PAGE, peptide
MALS, SEC, MFI
molecular weight species, Fluorescence, Light mapping, CE,
particles scattering MS, DD
CD - Circular Dichroism, DSC - Dynamic Light Scattering, FTIR - Fourier Transform Infra-Red Spectrometry, LC- Liquid Chromatography,
MS - Mass Spectrometry, NMR - Nuclear Magnetic Resonance, Cryo-EM - Cryo Electron Microscopy, IEX - Ion Exchange Chromatography,
CE - Capillary Electrophoresis, IEF - Isoelectric Focusing, RP-HPLC - Reverse Phase High Performance Liquid Chromatography, SEC -
Size Exclusion Chromatography, UV - Ultraviolet, MALS - Multi Angle Light Scattering, FFF - Field Flow Fractionation, AUC - Analytical
Ultracentrifugation, SDS-PAGE - Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis, TEM - Transmission Electron Microscopy,
ELISA - Enzyme Linked Immunosorbent Assay, SPR - Surface Plasmon Resonance, ITC - Isothermal Titration Calorimetry, BLI - Bio-Layer
Interferometry.

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listed in TABLE 1. This review focuses on the An interesting recent introduction in the
utility of the traditional characterization tools 2D platform has been of 2D-Difference Gel
and covers the new generation of analytical Electrophoresis (2D-DIGE). Proteins are
hardware that is increasingly being used directly labeled with fluorescent dyes (CyDyes),
orthogonally to the traditional toolbox. pooled and separated on a 2D-PAGE. The dye
binds covalently to ε-amino groups of lysine
Tools for Analytical Characterization residues in proteins, allowing for accurate
Cost-effectiveness and relative ease quantification of spots. The gels are scanned
of use have made Poly-Acrylamide Gel using an instrument capable of detecting
Electrophoresis (PAGE) one of the most different CyDye independently. 2D-DIGE can
commonly used techniques for protein further be coupled with mass spectrometry for
analysis. In the case of biotherapeutic protein identification (7).
characterization, PAGE is typically used to
estimate the size and isoelectric point of the A major advantage of PAGE is its versatility.
molecule. It serves both as a detecting as Gel matrix can be easily modified to achieve a
well as resolving technique, and relies on the specific resolution. Gradient gels (pH gradient)
property of charged molecules to migrate in are routinely used to ascertain the isoelectric
an electric field. Proteins are resolved on a point. Moreover, PAGE can be coupled with
polyacrylamide matrix, either in their native Mass Spectrometry for identification on
form (non-denaturing PAGE) or in reduced specific gel bands (first dimension) or spots
form (SDS-PAGE). A combination of the two (second dimension) (8, 9).
can also be employed for resolving complex
molecules such as monoclonal antibodies High resolution, varied choice of phase and
(mAbs), where separation from other robustness have made High Performance
proteins is attained in a non-denaturing Liquid Chromatography (HPLC) a cornerstone
first dimension followed by a denaturing of biopharmaceutical analysis. It is
second dimension resolution (2D-PAGE). ubiquitously used for analysis of proteins,
Iso-Electric Focusing (IEF), which resolves nucleic acids, or small molecules in complex
molecules based on their iso-electric point is mixtures. Typical separation is achieved
also a commonly used platform for 2D-PAGE by using a liquid mobile phase and a solid
and provides higher resolution, either in stationary phase. Diversity in solid phase
the first or the second dimension when chemistry and choice of mobile phase
compared with 2-D separation based only allows the analyst to fine-tune the type of
on size. Using Trastuzumab as an example, interactions allowed between the analyte and
2D-PAGE has also been shown to be a quick the stationary phase. This yields unparalleled
and easy method for qualitative evaluation selectivity between a biotherapeutic
of charge heterogeneity, stability and post- and related variants and impurities,
translational modifications (6). which otherwise may have near identical

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on their size. The stationary phase

SPONSORED CONTENT consists of a gel containing beads of a
Analysis of Modification Site of specific pore distribution. The choice
Chemically Modified Antibody Using
of pore distribution allows the user to
MALDImini™-1 Compact MALDI
Digital Ion Trap Mass Spectrometer achieve separation of species in the
desired range of size. In the case of
biotherapeutics, a popular application
physicochemical properties. Some of the is the resolution of size based
commonly used modalities of HPLC include: heterogeneities, especially aggregates
(12). Co-eluting host cell proteins can
• Ion Exchange (IEX) Chromatography also be resolved using SEC (9).
is used to separate molecules based • Hydrophobic Interaction
on their total charge. It enables the Chromatography (HIC) separates
separation of molecules based on molecules based on their
their charge. The strength of binding hydrophobicity and their charge. It is a
is determined by the affinity of the relatively gentle separation technique
proteins to the “Ion-Exchanger” as the chosen conditions are minimally
linked to the resin (stationary phase). denaturing and as a result do not
Cationic exchangers possessing significantly affect the biological activity
negative charge bind positively charged of the protein. Traditionally used as a
entities, whereas anionic exchangers polishing step in monoclonal antibody
bind negatively charged entities. purification, it has also been used
Furthermore, ion-exchangers can be to characterize drug distribution in
weak or strong depending upon the antibody-drug conjugates (ADCs) by
range of pH within which they can exploiting the hydrophobicity of the
sustain their charge. This influences the conjugated small molecule (13).
range and type (strong/weak) of binding • Reverse Phase (RP) Chromatography
that can be achieved (10). Elution is exploits reversible adsorption
typically achieved by either altering the of biomolecules based on their
pH of the mobile phase or increasing hydrophobicity under conditions
the ionic concentration of the mobile where the stationary phase is more
phase (salt gradient IEX). A commonly hydrophobic than the mobile phase.
used application involves separation of It is quite similar to HIC in principle,
charged variants, which are similar in however, the RPC medium is much
size but differ in charge, using cation more hydrophobic than in HIC and
exchange HPLC (11). elution is achieved by the use of non-
• Size Exclusion Chromatography (SEC) polar, organic solvents. This makes it a
enables separation of molecules based very desirable technique for coupling

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with MS for peptide mapping and and capillary damage due to handling.
other comparability studies. Coupled Demonstrated applications include
with mass spectrometry, the reverse glycosylation profiling of mAb directly from
phase has been extensively used in cell culture with sample volume requirement
primary structure characterization of under fifty microliters (17). The technique
of biotherapeutics as well as in shows much promise in monitoring batch to
comparability studies of biosimilars (14). batch variation in manufacturing as well as in
biocomparability exercise.
High Performance Capillary Electrophoresis
(CE) is another technique increasingly being X-ray crystallography is considered the
applied in conjunction with MS for charge gold standard for protein structural
variant characterization, isoelectric focusing studies. It works on the principle of X-ray
and biosimilarity assessment. Molecules diffraction. The angle and the intensity of
are separated across a fine capillary with the diffracted beam from a crystal are used
the internal diameter as small as 50 µM to construct a 3-dimensional image of the
via application of high voltage (~30 kV). molecular structure. However, application
This miniaturized format requires minimal of this technique for characterization and
sample with flow rates in the range of nL/ biocomparability is challenging as the
min, making it a cost-effective technique. protein of interest has to be purified and
CE coupled with nano-ESI-MS has been crystallized, which may not be possible for
shown to be particularly useful for N-Glycan biotherapeutics due to the presence of
analysis of monoclonal antibodies as well inherent heterogeneity in the form of several
as for detection of charge variants (15). The post-translational modifications (PTMs).
availability of various CE modes such as Although the data obtained is a direct
capillary zone electrophoresis, capillary gel measurement of the crystal structure, the
electrophoresis, capillary isoelectric focusing technique itself is too cumbersome to be used
and micellar electrokinetic chromatography as a routine analytical technique for biosimilar
allows for characterization of different characterization and comparability (18).
attributes such as intact mass, reduced mass,
charge variants, as well as glycosylation Nuclear Magnetic Resonance (NMR) exploits
pattern. CE coupled with MS is increasingly the magnetic properties of specific atomic
being used as a complementary platform nuclei. Although a gold standard for protein
to traditional LC-MS for biotherapeutic structural studies, its routine application
characterization (16). Innovative integration in the biotherapeutic industry has been
of CE with MS, such as in ZipChip has limited due to several factors, including the
reduced the analysis time to under large size of protein biopharmaceuticals,
three minutes and bypassed issues the relatively low sensitivity of the NMR
of individual component integration signals, and the low natural abundance of

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experiments to capture structural changes

SPONSORED CONTENT
due to biochemical reactions (19, 20)
Detect Slight Differences in
Hydrophobicity: Hydrophobic
Transmission Electron Microscopy (TEM) is
Interaction Chromatography
a microscopy technique in which a beam of
Column Shim-pack™BIO HIC
electrons is transmitted through a specimen
to form an image. Due to the much smaller
active nuclei. However, in instances where wavelength of electrons as compared to light,
biotherapeutics of small molecular size are the resolution of the image is greater by orders
being characterized, NMR might be utilized of magnitude with details up to atomic level,
to gain in-depth HOS information (18). and hence TEM finds use in the characterization
of biotherapeutic aggregates (21). Recent
It should be noted that although both X-ray developments in achieving greater resolution,
crystallography and NMR provide structural especially in Cryo-TEM, have enabled
details at a near-atomic level, the techniques researchers to observe protein complexes such
differ in their principle, information and as mAbs in their native formulation (without
bottlenecks. X-ray crystallography requires crystallization) (22). The technique is currently
the formation of the uniform crystal lattice underutilized in the field of biotherapeutics but
and utilizes very high energy X-rays for has significant potential to grow.
deflection by the surrounding electron
clouds, NMR is based on the absorption Circular Dichroism (CD) Spectroscopy
of electromagnetic radiation in the radio- is based on the difference observed in
frequency (RF) range. In NMR, proteins are the absorption of left and right-handed
analyzed in solution and the final image is circularly polarized light of a molecule in the
a compilation of a number of low energy presence of light absorbing chiral groups.
states of the protein in different orientations This property of biomolecules is frequently
as compared to single instance images of utilized in the examination of the secondary
X-ray crystallography. Both the techniques structure of the proteins and is employed
require high concentration, homogenous for assessing HOS comparability. Using
sample preparations. With respect to protein thermal denaturation, information about
size, NMR is more limiting. Proteins with molecule stability as well as folding and
a size larger than 40 kDa exhibit a slower unfolding mechanisms can be elucidated.
molecular tumbling in solution leading to An application of CD in the aggregate
spectral overlap and peak broadening. In characterization of mAbs has also been
terms of information obtained, NMR can demonstrated (23, 24).
unravel information on structural dynamics
and flexibility more readily than x-ray Fourier Transform Infrared Spectroscopy
crystallography which requires time-resolved (FTIR) is a spectroscopic technique that

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monitors the characteristic infrared frequency modes in a system, generally

absorption of molecules and translates applied to study the secondary structure
it into structural information. Similar to of proteins by studying the Amide I band
CD, it gives structural information about between 1600 and 1700 cm-1. An advantage
the secondary structure of the protein, that Raman spectroscopy offers over
mainly the alpha helices and beta sheet. It other secondary structure characterizing
serves as an orthogonal technique to CD techniques is its low susceptibility to water
in characterization and biocomparability interference as it detects scattered light
studies (25). and water is inefficient at scattering in
this part of the spectrum (28). In a recent
Although both CD and FTIR can provide study, Raman Optical Activity (ROA) was
information with regards to components of evaluated as a means to detect early thermal
the secondary structure of a molecule, it is instability in mAb samples kept at 50 °C
only through higher resolution visualization for a month. Significant structural changes
techniques such as CryoTEM, X-ray could be observed at one week of stress.
crystallography and NMR that the specific This provides an advantage over SEC in
arrangement of these components in space monitoring aggregation as ROA would
can be unraveled. provide information about subtle differences
in tertiary structure whereas Raman/ROA
Dynamic Light Scattering (DLS) is commonly spectra can elucidate on changes at the
used to determine the size distribution secondary structure level (29). Another
profile of small particles in biotherapeutic interesting recent application of Raman
formulations. The particle size profile is spectroscopy has been in drug identification,
determined by measuring the random specifically in the identification of
changes in the intensity of light scattered monoclonal antibodies by exploiting subtle
from the sample solution. It is often used differences in vibrational modes of the
in conjunction with SEC and analytical antibodies (30). It would be interesting to see
ultracentrifugation for aggregate studies. An if this application can be further modified
interesting application of High-throughput to distinguish between biosimilars and
platform in DLS (HT-DLS) has been in innovator product.
screening studies to quantify viscosity of
mAb formulations (26). Using automated Analytical Ultracentrifugation (AUC) is a
HT-DLS, effects of buffer conditions and versatile tool for quantitative analysis of
temperature on aggregate formation have macromolecules in solution. It employs
also been reported (27) the principle of centrifugal acceleration
to separate particles based on their size
Raman Spectroscopy is used to observe and mass. Two types of hydrodynamic
vibrational, rotational, and other low- analyses, namely sedimentation velocity

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and sedimentation equilibrium, are able to to Surface Plasmon Resonance (SPR) to study
make distinctions in formulation components receptor binding kinetics in biotherapeutics.
based on shape and mass or mass alone, Unlike SPR, CG-MALS would not require
respectively. Sedimentation velocity is binding of the receptor to any chip and hence
a mode of choice for aggregate analysis would be a truly label-free technique to
because it causes physical separation of quantify interactions, similar to Isothermal
molecular species of different mass or shape. Titration Calorimetry (ITC). Although the latter
This is a matrix-free technique as no column is more suited to study the thermodynamic
or gel matrix is required for size fractionation parameters of an interaction.
to occur (31). AUC-Sedimentation Velocity
(AUC-SV) is often used to quantify high Field Flow Fractionation (FFF) is a unique
molecular weight species present in separation technique used for analysis of
biopharmaceuticals (32). aggregates. Samples are pumped into a
narrow tube perpendicular to the flow and
Multiple Angle Light Scattering (MALS) separation occurs due to the difference
is typically used in line with a resolving in mobility of the species in the mixture
technique such as SEC to determine the under the field applied. Depending upon
size of the different molecular species as the properties of the species in the sample
they pass through the MALS detector. It mixture, different flows such as electrical,
adds a quantitative measure of analysis to magnetic, thermal-gradient, gravitational or
techniques like SEC. As the sample passes centrifugal can be applied. FFF serves as a
through the laser beam, light is scattered at complementary technique to DLS and MALS
multiple angles and the detector collects this for determining the presence of sub-micron
data to approximate the sample size (33). particles in the sample (36).
It has been used to monitor the formation
of soluble, HMWS so as to better quantify Fluorescence spectrometry is based on
and model non-native aggregation kinetics the principle that certain molecules called
in α chymotrypsinogen (34). Composition- fluorophores emit light upon excitation by
Gradient Multi-Angle Light Scattering (CG- an external source such as an incandescent
MALS), a variation on MALS, employs a lamp or a laser which produces a spectrum.
series of unfractionated samples of different This technique is helpful in exploring HOS of
composition or concentration in order to a protein (to an extent), mainly the tertiary
characterize a wide range of macromolecular structure via the intrinsic fluorescence of the
interactions. CG-MALS, a complementary protein. Changes in the local environment
technique to AUC and DLS, has been used to of tryptophan, the strongest intrinsic
characterize self-association in model mAb fluorophore, are reflected in the emission
molecule (35). Although not yet commonly spectra of the molecule and are a measure
used, CG-MALS could be used orthogonally of change in the protein tertiary structure

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(37). In cases where a biomolecule does are displayed as a spectrum of the relative
not have an intrinsic fluorophore, extrinsic abundance of different ions based on the
fluorophore dyes such as Thioflavin-T m/z ratio.
(ThT) or 1-anilino-8-naphthale-nesulfonate
(ANS) can be used to induce fluorescence. Analytical capability of an MS platform is
Fluorescence is routinely used in thermal defined by the kind of ionization source
stability studies of biotherapeutics, especially and the type of mass analyzer being used.
monoclonal antibodies (38). At best, it is an Examples of ionization sources include fast
indirect measure of changes in a protein’s atom bombardment (FAB), chemical ionization
tertiary structure and does not provide (CI), atmospheric-pressure chemical ionization
information about position and the specific (APCI), electrospray ionization (ESI), and
nature of these changes. matrix-assisted laser desorption/ionization
(MALDI). Examples of mass analyzers include
Micro-Flow Imaging (MFI) is an up and Time-of-flight (TOF), quadrupole mass
coming technique that combines digital filter, and ion-traps. Some of the popular
microscopy with microfluidics to capture combinations of the two are ESI-TOF, MALDI-
and quantify sub-visible particles in the TOF and ESI-Q-TOF (TABLE 2).
range of 1 to 300 µm in a solution. It
can provide information about particle MS is usually coupled with LC or CE as the
size, concentration and morphology first dimension of separation. Characterization
(39). However, rather than protein of complex molecules such as monoclonal
characterization, it is more suited for antibodies requires mapping of the different
profiling and classifying the particulate size fragmentation patterns in MS such as Electron
in a given formulation. Transfer Dissociation (ETD) and Collision-
Induced Dissociation (CID) along with the use
Mass Spectrometry (MS) is a powerful and of different proteases make it possible to map
data-intensive technique for determining disulfide links present and the different glycans
protein mass (intact, fragmented and attached to the protein (18,40).
reduced), sequence, and for probing and
quantifying protein modifications. MS Another useful application of MS, when
involves ionization of the sample fragments coupled with Hydrogen/Deuterium
followed by their separation based on their exchange (HDX) is in elucidating protein
mass to charge ratio (m/z). By accelerating conformational dynamics and protein
the ionized particles and subjecting them interactions. In the biopharmaceutical
to an electric or magnetic field, the ions industry, HDX-MS has established itself
get deflected depending on the mass and in the analysis of protein-small molecule
charge that they carry. Ions are detected interactions, characterization of bio-
by an electron multiplier and the results therapeutics/biosimilars, and epitope

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TABLE 2: Publications per technique since 2010 for biotherapeutics

(Google Scholar)
Category
Tool 2010-2017
Biopharmaceuticals Monoclonal antibodies
HPLC 7440 1600
CE 1880 1040
HPCE 109 44
ESI-TOF 2140 703
Q-TOF 559 441
MALDI-TOF 2630 835
ORBITRAP 882 603
TRIPLE-QUAD 39 251
HDX-MS 406 665
CD 1750 781
DSC 1670 847
FTIR 1720 362
DLS 4330 1080
NMR 3550 796
CRYO-TEM 144 24
TEM 3760 650
FFF 550 307
SPR 2060 997
ITC 505 156
LC - Liquid Chromatography, CE - Capillary Electrophoresis, TOF - Time of flight, Q-TOF- Quadrupole-time of flight, MALDI - Matrix
Assisted Laser Desorption ionization, Triple-Quad - Triple Quadrupole, CD - Circular Dichroism, DSC - Dynamic Light Scattering,
FTIR - Fourier Transform Infra-Red Spectrometry, NMR - Nuclear Magnetic Resonance, HDX-MS - Hydrogen/Deuterium exchange-
Mass spectrometry, CRYO-TEM - Cryo-Transmission Electron Microscopy, TEM - Transmission Electron Microscopy, FFF - Field Flow
Fractionation, SPR - Surface Plasmon Resonance, ITC - Isothermal Titration Calorimetry. The publication data were generated using
Google Scholar for papers from 2010-2018 (excluding citations and patents). Search word combination used was “technique name”
“biopharmaceutical” and “technique name” “biopharmaceutical” and “monoclonal antibody”

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mapping of biotherapeutics. The technique However, with high specificity techniques

relies on isotope labeling to probe the such as MS (detection) and Surface plasmon
rate at which protein backbone amide resonance (interaction) becoming affordable
hydrogens undergo exchange. MS is then and routine, ELISA can only be used for a
used to monitor the mass-shift as a result of precursor technique for quick estimation with
incorporation of deuterium in the protein. limited confidence prior to employing tools
The rate of exchange provides information with much higher specificity.
regarding the conformational mobility,
hydrogen bonding strength, and solvent Surface Plasmon Resonance (SPR) (interaction)
accessibility in protein structure (41,42) is a label-free technique that allows for real-
time detection of biomolecular interactions.
Differential Scanning Calorimetry (DSC) The SPR phenomenon occurs when polarized
is a versatile technique that measures the light strikes an electrically conducting surface
quantity of heat radiated or absorbed by at the interface between two media. In
the sample on the basis of a temperature response, plasmons are produced, which are
difference between the sample and the electron charged density waves. These waves
reference material. It is used to determine reduce the intensity of reflected light at a
equilibrium thermodynamic stability specific angle known as the resonance angle,
and folding mechanism of proteins and in proportion to the mass on a sensor surface.
finds routine use in thermal stability SPR allows for real-time monitoring of both
characterization of biotherapeutics (43). association and dissociation of an interaction,
generating reproducible kinetic data. Due to
Tools for Functional Characterization: its sensitivity, ease of use and automated data
Enzyme-linked Immunosorbent Assay (ELISA) analysis, it is widely used to study the ligand-
is a popular diagnostic technique used to receptor kinetics of monoclonal antibodies (14)
assess the immunogenicity of a therapeutic
product. In a typical assay, a ligand, typically Bio-Layer Interferometry (BLI) is a label-free,
an antigen, is non-specifically or specifically optical analytical technique that analyzes the
bound to the polystyrene well of a 96 well interference pattern of white light reflected
microtiter plate. Enzyme-linked antibodies are from two surfaces: a layer of immobilized
used for colorimetric detection of a positive protein on the biosensor tip and an internal
interaction upon addition of the enzyme reference layer. Changes occurring in the
substrate. ELISA has several applications in number of molecules bound to the biosensor
biotherapeutic characterization. A common tip is reflected as a shift in the interference
application of this assay format is in Host Cell pattern. Similar to SPR, binding kinetics
Protein analysis where polyclonal antibodies can be determined by this technique.
raised to the host cell are used to detect Because of its robustness and ease of
the presence of HCPs in the sample (44). implementation, BLI is gaining application

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as a complementary technique to SPR for Our understanding of the relevance of the

studying and comparing biotherapeutic different measured attributes with respect
binding kinetics (44). to the safety and efficacy of a biotherapeutic
has been improving with time and experience.
Isothermal Titration Calorimetry (ITC) is a Some recently evolving techniques such as
physical technique used to determine the CG-MALS for macromolecular interactions
thermodynamic parameters of interactions in and intrinsic Förster resonance energy
a solution. It measures the heat released or transfer (iFRET) for in vivo target protein
absorbed by mixing the two interactants via detection are yet to find their place in the
titration. It is widely considered an absolute biotherapeutic characterization toolbox while
and direct measurement of interaction. other well-established techniques such as
Thermodynamic parameters of an interaction Raman spectroscopy are being increasingly
can be assessed via this technique and it is used for novel applications such as drug
fast gaining importance as an orthogonal identification. A desirable direction for
technique to SPR for ligand binding studies technological advancements would be the
in biocomparability (45). increasing throughput of techniques such as LC
by parallelization (9). Short analysis time and
Summary micro- and nanoplatforms with minimal sample
With the increase in complexity of the consumption would help cut down the cost of
biotherapeutics undergoing manufacturing, analysis associated with limited and expensive
the need for in-depth analytical samples such as monoclonal antibodies.
characterization has also increased. As
protein molecules, even minute changes in Despite these advancements, we are yet
the structure of the biotherapeutic can confer to reach a point where a product such as a
altered functionality, sometimes leading to complex biotherapeutic, can be completely
immunogenic reactions in the patients. In fingerprinted in a manner similar to a
view of our dependency for production of pharmaceutical (small molecule) product.
these molecules on biological machinery, the Moreover, each technique comes with its
formation of numerous altered conformations unique limitations and pitfalls. This ensures
of the molecule is unavoidable. However, that the topic of analytical characterization
significant advancements have been made of biotherapeutics will continue to be an area
in analytical methodology increasing our of research in the time to come.
ability to characterize a biotherapeutic. This
is highlighted by the increase in the number References
of technique rich publications on analysis 1. ICH Expert Working Group, Specif. Test Proced.
of biotherapeutics since 2010 and the Accept. Criteria Biotechnol. Prod. (March), 1–20
introduction of the US-FDA BPCI Act 2009 (1999).
(TABLE II). 2. WHO, Guidelines on the Quality, Safety and Efficacy

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of Biotherapeutic Protein Products Prepared by 13–34 (Springer International Publishing, Cham,

Recombinant DNA Technology, WHO Technical 2016). doi:10.1007/978-3-319-46240-0_3.
Report Series 814, 91 (2013) 17. S.A. Berkowitz, J.R. Engen, J.R. Mazzeo, and G.B.
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Anurag S. Rathore
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Professor, Department of Chemical Engineering
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Expert Q&A: Analysis and Characterization

of Monoclonal Antibodies in the
Biopharmaceutical Industry
Interview with Ratnesh Dharamchandra Jain, Ph.D.
L
New research CGC sat down with Ratnesh Dharamchandra Jain, Ph.D., UGC
assistant professor of engineering sciences at the Institute of
on mAb Chemical Technology (ICT) in Mumbai, India, to discuss the
development. advantages of monoclonal antibody (mAb)-based medicine,
techniques for characterizing proteins and antibodies, the challenges
and advantages of mass spectrometry (MS) in the biopharmaceutical
industry, and more.
LCGC: Can you introduce the research work undertaken in your lab at
ICT and your involvement in the Indian biopharmaceutical industry?
JAIN: Our laboratory in ICT in Mumbai is primarily working on
proteins and peptides, specifically mAb therapies, which are
presently divided into originator biologics and biosimilars; we
primarily handle biosimilars in our laboratories. We also try to
understand the structure of these therapies, specifically in their
native form, and different formulation development and its
interaction with the formulation excipient.
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EXPERT Q&A: ANALYSIS AND CHARACTERIZATION IN THE BIOPHARMACEUTICAL INDUSTRY
We are also investigating structural Recently, we investigated nutrient roles in

changes that are typically experienced by antibody production, specifically when cell
these molecules, specifically mAbs during media and cell feed are given to a clone for
different processing steps. We also deal target antibody production when looking for
with upstream bioprocessing, downstream a link between the nutrient and the critical
bioprocessing, and the changes by different quality attribute of an antibody. This is also very
analytical methodologies. important because the quality and structure of
an antibody are linked to the media and feed.
LCGC: What are the advantages of mAb-
based medicine? Feed isn’t static during production—it is in
JAIN: mAbs are a highly specialized class of a bioreactor, so there is always a dynamic
drugs. One of their biggest advantages is feed optimization. Our research, which will
they are very effective against the specific be published shortly, demonstrates the
disease condition for which they are being consumption profile and how the major
manufactured, so the treatment’s chance of nutrients are linked to antibody production.
success is very high. We developed a new theory, in collaboration
with ICT Shimadzu of Advanced Mass
LCGC: What are some typical disease Spectrometry—on feed optimization and
conditions that mAbs are used for? upstream process development. We utilized
JAIN: mAbs play a major role in cancers and mass spectrometry for nutrient profiling as
autoimmune diseases. When we talk about well as understanding the critical quality
disease conditions and different types of attributes of the antibody in question.
cancers, the majority of the antibodies
developed were for cancer treatment, LCGC: Can you describe the importance of
and eventually, they were developed cultivation conditions such as media and
for autoimmune diseases. In fact, one of feed in mAb production? How will it impact
the largest antibodies is for autoimmune the final mAb drug quality?
disorders like rheumatoid arthritis (RA) and JAIN: These antibodies, which are typically
many other similar disorders. produced in Chinese Hamster Ovary (CHO)
clones, are biosynthesized in these cells
LCGC: Can you elaborate on your recent using a cellular process called transcription
research project in the field of mAb
development?
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JAIN: With respect to antibodies, we try to
Development of cell culture
secure collaborations with different commercial
supernatant analysis using
partners for clones. We have also developed LC-MS/MS and their application
our own clones for antibody production to for Chinese hamster ovary cell
understand the structural basis of antibodies.

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“The major issues with At the beginning of cell culture, you need
nutrients and micronutrients a higher amount of nutrients because all
the cells will grow. During the later stage,
are the high number of these cells achieve stability—they are in
nutrients metabolized and the plateau phase, so the requirements are
the low availability in cell- more modest. Feed and media also must
be dynamic to accommodate the growth
culture media.” pattern of these cells.
and translation. Using this process, The metabolic consumption of each

each protein is synthesized in the clone changes—each clone responsible
mammalian cell—the same way mAbs for antibody production is genetically
are biosynthesized in CHO cells. But the engineered, and whenever a clone is
final structure of this particular antibody, genetically engineered, its overall
which is biosynthesized in CHO cells, is also demands and requirements change.
dependent on a specific part called post- It’s similar to human beings: we are
translational modification (PTMs). PTMs are engineered in the same way, but we have
responsible for the biological functionality of different food demands. This demand is
an antibody. different for each clone, and the process
of feed and media must be optimized. We
During the intracellular biosynthesis have approximately 300 nutrients that are
process, a short peptide is made, and given to cells and studying them together
multiple peptides are put together to make at any given time is a very challenging
a protein. These antibodies are large—more investigation. During this time, we utilize
than 150 kDa, which is a very high molecule mass spectrometry in greater detail and
weight of antibodies. utilize its capabilities to understand this
research work.
The raw materials used to manufacture these
antibodies within the cell are amino acids, LCGC: What are the major challenges
sugars, vitamins, fatty acids, multiple growth in performing analysis for cell-culture
factors, and other micronutrients that are components of spent media?
responsible for the entire metabolic and
biosynthetic strategies. When we culture
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these cells, there is a great need for nutrients
and micronutrients in the cell-culture process LC/MS/MS Method
development—at any given time. Cells are Package for Cell
either exponentially growing or they are in a Culture Profiling Ver. 2
plateau phase.

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JAIN: The major issues with nutrients and With regards to the characterization of
micronutrients are the high number of nutrients antibodies, specifically critical quality
metabolized and the low availability in cell- attributes, you have to perform multiple
culture media. The higher the quantity, the better assays because it’s unlikely one or two
analysis, but the lower the quantity, the analytical methods will give you any information. Any
methodology may not function properly. industry working with biologics or biosimilars
Nonetheless, our research has overcome these has to go through multiple approvals, which
challenges using a simple processing strategy. are done by multiple assays—400+ different
characterization assays. High-resolution MS
LCGC: What is the best technique for is one of the best ways to understand these
characterizing biopharmaceuticals? assays, among other techniques.
JAIN: When looking at cell culture media
components analysis, we use a Liquid LCGC: Are there any specific features of the
Chromatograph (LC) coupled with a triple MS system that are particularly useful for
quadrupole MS. When it comes to quality characterization?
attribute analysis of mAbs, we use a HRMS JAIN: HRMS can be used for the
instrument like an LC coupled with a Q-TOF. characterization of mAbs by a top-down
In our collaboration with Shimadzu, we used approach, which can give information
CCP packages (LC/MS/MS Method Package about its accurate molecular weight and
for Cell Culture Profiling), which gave us a heterogeneity due to the presence of
valuable set of information about different glycoforms/proteoforms. A second approach
media components such as amino acids, is called bottom-up characterization, which
vitamins, sugars, nucleic acids etc. in a single offers information of primary subsequence
run. This standardized method package contains and any PTM.
information such as sample pre-treatment info
to the ready-to-use analytical method that Key features/requirements for HRMS are
can be set on Shimadzu’s LC-MS/MS triple mass accuracy and stability. PTMs are
quadrupole system. This has saved my students complex to characterize, and sometimes
a lot of time and has worked wonderfully for the run time of peptide mapping/ PTMs
their research work. experiments can be very long. Hence, a
mass spectrometer that can offer stable
“A second approach is called mass accuracies for a long period of time
bottom-up characterization, without frequent need of mass calibration
is beneficial. A HRMS instrument that can
which offers information of acquire an MS/MS spectra over a wide range
primary subsequence and any of collision energies is extremely useful in
post-translational modification.” PTM analysis.

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LCGC: What improvements or additional liquid chromatography (HPLC). It is used to

features are needed to improve the understand aggregation by size-exclusion
accuracy of LC-MS and other techniques in chromatography (SEC) and to characterize
the biopharmaceutical industry? proteins and antibodies. If a product needs
JAIN: There are challenges related to LC-MS to go to market quickly, it offers fast quality
attributes. Many attributes require different control. In fact, many regulatory guidelines
methodologies and processing. For example, indicate analysis by HPLC. It has also been
if I am looking for aggregation, oxidation, used for charge variant analysis by ion-
or another possible degradation, the target exchange chromatography (IEX). We use
method and process are different each time. HPLC with a fluorescence detector for
One of the biggest challenges is getting released glycan analysis and attach other
more information using a single analysis detectors for stability-related analysis.
with multiple sets of data. Data analysis and
automated sample processing are needed.
There is a lot of manual work involved with Ratnesh Dharamchandra Jain, Ph.D.
LC-MS. Automating these steps would UGC Assistant Professor in Engineering Sciences,
Department of Chemical Engineering
provide a significant amount of accuracy in Institute of Chemical Technology (ICT)
data sample analysis and data processing.
rd.jain@ictmumbai.edu.in
But LC-MS is not utilized to its full www.nano-medicine.co.in, www.ictmumbai.edu.in
potential—LC-MS analysis has increased the
understanding of antibodies and ultimately
reduced the cost of clinical development, so
even with these challenges, I still think the
technique is very promising.
LCGC: Apart from LC-MS, are there any other

analytical techniques that are widely used
by the biopharmaceutical industry?
JAIN: For the characterization of recombinant
protein, there are more than 100 assays.
So yes, many methodologies are needed,
but one method that can be used apart
from LC-MS or HRMS is high performance

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majcot/stock.adobe.com
Characterization of Control and Stress Induced

Samples of Trastuzumab Biosimilar using
LCMS-9030 by Bottom-Up Approach
Amita Puranik, Deepti Bhandarkar, Prajakta Dandekar, Pratap Rasam, Tian hua Wang, and
Ratnesh Jain
In-depth Introduction
Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals
peptide covering a large panel of diseases, from cancer to asthma,
mapping and including central nervous system disorders, infectious diseases and
PTM analysis cardiovascular diseases (1). Throughout manufacturing, storage,
transportation, and administration, mAbs are subjected to biophysical
methodology and biochemical stress from multiple sources, which may lead to
is described for their degradation via aggregation, fragmentation, and chemical
the analysis modifications, such as oxidation, deamidation, or isomerization
(2). Among these undesirable degradation products, oxidation and
of control and deamidation are the most commonly observed post-translational
stress induced modifications (PTMs). Early identification of these prone sites enables
antibody engineering to eliminate the liability of leading candidates to
samples of such modifications while maintaining binding activity.
mAb.
The bottom-up approach, which is essentially peptide mapping,
helps in the determination of primary amino acid sequence and

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CHARACTERIZATION OF CONTROL AND STRESS INDUCED SAMPLES OF TRASTUZUMAB BIOSIMILAR USING
LCMS-9030 BY BOTTOM-UP APPROACH
FIGURE 1: LCMSTM-9030 Quadrupole the solutions. Finally, trypsin was added at a

Time of Flight Mass Spectrometer 1:50 ratio and overnight digestion was carried
out. Reaction was quenched with diluted
formic acid and desalting was carried out
using solid phase extraction (SPE) cartridges.
Eluent obtained at the end of SPE clean-up
was evaporated using vacuum centrifugation
and reconstituted in a water: formic acid:
acetonitrile (100:1:2 v/v) solution and analyzed
with the LCMS-9030 (shown in FIGURE 1) for
peptide mapping analysis. A mobile phase
consisting of 0.1% formic acid in water and %
formic acid in acetonitrile was used with the
identification of site-specific PTMs. In this Shim-pack™ Arata Peptide C18 column for
application note, a methodology for the the chromatographic separation. Analysis was
identification of sites susceptible to oxidation performed in Data Dependent Acquisition
and deamidation is described by analyzing the (DDA) mode in positive polarity using an
control and chemically stress-induced (forced Electro Spray Ionization (ESI) interface (3).
degradation) samples of mAb. DDA data acquisition was controlled by the
LabSolutions™ LCMS software. A mass range
Experimental of 200-2500 m/z was used for a MS1 TOF
A trastuzumab biosimilar sample (2 mg/mL) survey scan. A base peak chromatogram
was incubated in water with a pH 9 (adjusted intensity threshold of more than 1000 was
using tris base solution) for 7 days at 37 °C to used to trigger the MS/MS fragmentation
induce deamidation. Similarly, 10 µL of 30% with a collision energy spread of 18-52 V. The
H2O2 solution was added to 2 mg/mL of the use of a collision energy spread allowed the
trastuzumab sample and incubated for 1 hour at acquisition of a comprehensive fragmentation
37 °C to induce oxidation. Oxidation reaction pattern for any given precursor ion. Seven
was quenched by adding a pinch of methionine. dependent (MS/MS) events were set to allow
sufficient MS/MS data collection. A mass
100 µg of trastuzumab control and stress- range of 100 to 2800 m/z was used to obtain
induced samples were incubated with a an MS/MS spectra. Ion exclusion and inclusion
reduction buffer containing 8 M urea and settings are available in the LabSolutions LCMS
5 mM dithiothreitol at 37 °C for 60 mins. software to automatically exclude background
Solutions were then alkylated with 20 mM ions and include ions of interest, respectively.
of iodoacetamide at room temperature for
30 mins. At the end of the incubation period, All data acquisition was performed
50 mM Tris-HCl buffer (pH 8) was added to with single external TOF calibration. No

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TABLE 1: Details of analytical intermediate TOF calibration/lock masses

conditions for bottom-up approach were used during the data acquisition/
processing. Details of analytical conditions
HPLC system Nexera™ X2
are given in TABLE 1.
Shim-pack™ Arata Peptide C18
Column (100 mm × 2.0 mmI.D., 2.2 μm) The LCMS-9030 quadrupole time-of-flight
(P/N: 227-32806-02) (Q-TOF) mass spectrometer is a powerful
Column oven 40 °C instrument that integrates the world’s
A: 0.1 % formic acid in water fastest and the most sensitive quadrupole
Mobile phases
B: 0.1 % formic acid in acetonitrile technology with TOF capabilities for
accurate mass measurement. Patented
Flow rate 0.3 mL/min
technologies of LCMS-9030, UF-FlightTube™
and iRefTOF™, ensure excellent mass
measurement accuracy (MMA) with stability,
which helps in the identification of different
0-3 min → 1 (%); 3-45 min →
1-30 (%); 45-46 min → 30 (%); peptides and PTMs present in the sample.
Gradient program
(8%)
46-46.1 min→ 30-90 (%); 46.1-50 UFaccumulation™ and UFgrating™ offer
min →90 (%); 50-50.1 min→ 1 (%);
superior sensitivity, which helps in detecting
60 min → stop
low abundant PTMs present in the samples.
LCMS system LCMS-9030 DDA data acquired by the LCMS-9030 was

Interface Heated ESI processed using the “Protein Metrics” software
Polarity suite (4). Settings of precursor mass tolerance
Positive
of “6 ppm” and fragment mass tolerance of
Acquisition mode DDA
“20 ppm”; maximum 2 missed cleavages;
Mass range for TOF
200-2500 m/z and fully specific trypsin digestion efficiency
survey scan
were used for peptide/PTMs identification.
Mass range for
precursor ion
220-2000 m/z Carbamidomethyl modification was
Mass range for MS/ considered as fixed. Other PTMs like oxidation,
100-2800 m/z
MS scan deamidation, Gln->pyro-Glu, Glu->pyro-
Collision energy
18-52 V Glu, ammonia-loss/succinamide formation,
spread
dioxidation, dethiomethylation were considered
Temperatures Interface: 300 °C
as variable. N-glycan 52 common biantennary
Desolvation line: 200 °C
database present in the software was used to
Heater block: 400 °C
obtain information about glycosylation.
Gas flow rates Heating gas: 15 L/min
Nebulizing gas: 3 L/min
Results and Discussion
Drying gas: 15 L/min
Therapeutic proteins may undergo a

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FIGURE 2: Overlay of extracted ion chromatograms of TOF survey scan (MS1) for
trastuzumab biosimilar control sample.
FIGURE 3: Summary of peptide coverage, fragmentation coverage and PTMs for

trastuzumab biosimilar control sample.
series of modifications throughout their modifications accurately. Moreover, it is also

cellular production, upstream/downstream important to find out the sites that are prone
processing, and storage. These modifications to undergo such modifications.
can include the addition or replacement
of functional groups, or structural changes Analysis of the control and artificially
such as folding/unfolding, cleavage, and stress-induced mAb samples can provide
racemization. The presence of these an understanding of such susceptible
modifications can affect biological activity, sites. The bottom-up approach for mAb
half-life and immunogenicity (5). Hence, it characterization is typically referred to as
is of utmost importance to identify these “peptide mapping.” Peptide mapping analysis

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TABLE 2: Representative results of mass accuracies for peptide sequences

of different chain length for control and stress induced samples of
Trastuzumab biosimilar
not only provides information about the chain lengths. Representative data
primary sequence of mAbs, but it is also demonstrating the mass accuracy (less
useful in identifying sites that are susceptible than 2 ppm) obtained for peptides with
to oxidation, deamidation etc. a chain length as short as 4 amino acids
and as long as 63 amino acids is shown in
Hence, control and stress-induced samples TABLE 2. Obtaining such mass accuracies and
of trastuzumab biosimilar were subjected stability for a longer duration is of the utmost
to peptide mapping and PTMs analysis. An importance to accurately identifying PTMs.
overlay of extracted ion chromatograms
from TOF survey scan (MS1) for trastuzumab Generally, a mass shift in precursor ion m/z
control sample is shown in FIGURE 2. More for the modified peptide and retention time
than 92% of peptide sequence coverage was changes are considered to identify signs
obtained for both heavy and light chains of of PTMs. Furthermore, acquiring a good
the trastuzumab control sample even with quality MS/MS spectra is equally important
single enzyme digestion (shown in FIGURE 3). to confidently assign the location of the
Some of the short peptide chains (around 3 modification on a given peptide. The collision
to 4 amino acid) are found to not be covered; energy spread function of the LCMS-9030
however, the use of multiple enzymes for the helps acquire an MS/MS fragmentation
digestion can improve the sequence coverage. pattern over a range of collision energies
(18-52 V, in this case) instead of obtaining an
LCMS-9030 offered excellent mass MS/MS spectra at a single or few selected
accuracies for the peptides with different collision energies. Thus, comprehensive
MS/MS fragmentation pattern can be
obtained, which in turn, helps with confident
SPONSORED CONTENT site-specific PTM assignment. Examples
Understand the rationale behind of identified modifications like oxidation,
the Q-TOF LC/MS that stably deamidation etc. are discussed herein.
sustains sub-ppm mass accuracy Oxidation of biotherapeutic proteins can alter
their physical and biological properties, affecting

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FIGURE 4: Top left: Chromatograms of oxidized and unmodified “DTLMISR.”

Top right: MS1 spectra of oxidized and unmodified “DTLMISR.” Bottom: MS/MS
fragmentation spectra for oxidized and unmodified “DTLMISR.”
their potency and stability characteristics. fragmentation pattern. Identification of

The most commonly oxidized amino acid is oxidation modification is illustrated with
methionine (Met). Oxidized peptides are easily “DTLMISR” peptide and shown in FIGURE 4.
identified using MS since the addition of one The oxidized peptide “DTLMISR” elutes earlier
oxygen atom to the Met side-chain upon as compared to its unmodified counterpart
conversion to Met sulfoxide increases the mass as “oxidation” imparts hydrophilicity to the
of the affected residue by +16 Da (5). peptide. A mass shift of +8 Da (for “+2” charge
state) for an oxidized peptide precursor ion m/z
Location of oxidation modification can can be seen from the MS1 spectra. The MS/
be easily identified from an MS/MS MS fragmentation spectra revealed that the

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TABLE 3: Relative abundance summary of PTMs for control and stress induced
samples of trastuzumab biosimilar
y3 fragment is the same for both the peptide A deamidation modification

versions, however a mass shift in fragment ions example is illustrated with the
can be seen from the y4 fragment onwards “ASQDVNTAVAWYQQKPGK” peptide (refer
indicating the location of a modification as y4 to FIGURE 5). A mass shift of “+ 0.329 Da” (for
methionine. Met sulfoxide-containing peptides +3 charge state) is observed in the MS1 scan
readily lose methane sulfenic acid (CH3SOH) spectra of a modified peptide which is very
upon CID fragmentation and are thus easily close to the first 13C isotope m/z of unmodified
identified by a characteristic loss of 64 Da from precursor ion. Hence, careful evaluation of
the fragment mass which can be seen in the precursor ion mass shifts, MS/MS patterns and
MS/MS spectra of a modified peptide shown changes in the retention time is essential before
in FIGURE 4. assigning deamidation modification.
A deamidation of asparagine (Asn) and The deamidated peptide is observed eluting

glutamine (Gln) has an important role in later than its unmodified counterpart. The
regulating the heterogeneity and stability of MS/MS fragmentation pattern revealed that
recombinant mAbs. Deamidation is one of product ions m/z up to y12++ are the same
the most challenging PTMs to characterize for modified as well as unmodified peptides.
using MS-based techniques. Deamidation However, y13++ fragment ion showed a
results in conversion of –NH2 to difference of 0.49 Da (for “+2” charge state)
–OH (+0.984 Da), which has a very similar as seen in FIGURE 5 confirming the presence of
mass shift as the first 13C isotope peak of the deamidation modification and its
native peptide (+1.0034 Da) (5). location on the given peptide. A relative

Charge Variant
UHPLC
“Excellent mass accuracy with

good stability, comprehensive SPONSORED CONTENT
fragmentation pattern and Shim-pack Arata LC Columns -

Unprecedented Resolution and
sensitivity offered by the Peak Shape of Basic Compounds
LCMS-9030 helps in reliable

and in-depth characterization deamidation. Some of these modifications
of mAbs and also has helped could be sample preparation artifacts
introduced due to elevated pH used during
in the identification of sites sample preparation. Nevertheless, sites LC-30
susceptible to oxidation and and HC-318 have shown elevated levels of
deamidation modifications.” deamidation modification in the deamidation
stress-induced sample as compared to the
abundance summary of PTMs observed control and oxidative stress-induced samples
for control and stress induced samples of indicating potential susceptibility of these sites.
trastuzumab biosimilar is given in TABLE 3.
It can be observed that three sites, viz., Conclusion
HC-361, HC-107 and HC-431 have shown
methionine oxidation only in oxidative stress- • Complete peptide mapping and PTMs
induced samples and are absent in control as analysis workflow for control and
well as deamidation stress-induced samples, stress-induced mAb samples
which shows susceptibility of these sites to is described.
oxidation stress. Site HC-255 has shown • A sequence coverage of more than 92%
methionine-oxidation in all three samples, was obtained for both heavy and light
however, relative abundance of modified chains of the mAb even with single
peptides is higher (77.5%) in oxidative stress- enzyme digestion.
induced samples as compared to control and • Excellent mass accuracy with good
deamidation stress-induced samples (6.35% stability, comprehensive fragmentation
and 7.29%). This site appears to be the most pattern and sensitivity offered by the
susceptible to oxidative stress. Site HC-83 has LCMS-9030 helps in reliable and in-
shown minor levels of methionine oxidation depth characterization of mAbs and
in all three samples with almost comparable also has helped in the identification
relative abundance, which indicates that this of sites susceptible to oxidation
site may not be susceptible to oxidative stress. and deamidation modifications.
Such studies can help to deepen
Similarly, sites LC-30, HC-289, HC-387, HC- understanding of Critical Quality
392, HC-55, HC-84 and HC-318 have shown Attributes (CQA) of the product and

Charge Variant
UHPLC
to further develop Multiple-Attribute

Method (MAM).
Amita Puranik
References
Institute of Chemical Technology, Mumbai
1. Physicochemical Stability of Monoclonal
Antibodies: A Review, Journal of Pharmaceutical
Sciences, Volume-190, Issue-1, 169- 190,2020. Deepti Bhandarkar
2. Rapid assessment of oxidation via middle-down Shimadzu Analytical (India) Pvt. Ltd.
LCMS correlates with methionine side-chain
solvent-accessible surface area for 121 clinical Prajakta Dandekar
stage monoclonal antibodies, Mabs, Volume 9 (4), Department of Pharmaceutical Science and
646-653, 2017. Technology
3. Data-Dependent Analysis Approach in LC/HRMS: Institute of Chemical Technology, Mumbai
Annotation of Natural Product Components,
04-JMST-208-EN. Pratap Rasam
4. Monoclonal Antibody Workflows on the Shimadzu Analytical (India) Pvt. Ltd.
Shimadzu Q-TOF LCMS-9030 Using the Protein
Metrics Software Suite, SSI- LCMS-103. Tian hua Wang
5. Structural Elucidation of Post-Translational Shimadzu (Asia Pacific) Pte. Ltd.
Modifications in Monoclonal Antibodies, ACS
Symposium Series, ISBN13: 9780841230293,
Ratnesh Jain
Chapter 3, Volume 1201, 119-183, 2015.
Institute of Chemical Technology, Mumbai
LCMS, Nexera, LabSolutions, UF-FlightTube, iRefTOF,
UFaccumulation, UFgrating and Shim-pack are
trademarks of Shimadzu Corporation in Japan and/or
other countries.
For Research Use Only. Not for use in diagnostic

procedures.

Charge Variant
UHPLC
Alexander Raths/stock.adobe.com
Charge Variant Analysis of mAb

Biotherapeutics by Nexera XS with a
Shim-pack Bio IEX Column
Ashutosh Shelar, Bhaumik Trivedi, Navin Devadiga, and Krutika Padhye
A robust Introduction
Overview
platform for Monoclonal antibodies (mAbs) are the most approved
charge variant biopharmaceuticals used to treat severe and chronic diseases such
analysis and as cancer, autoimmune, cardiovascular, respiratory, hematology,
and several infections. They have an enormous therapeutic and
columns for commercial value which makes them among the top 10 best sellers
excellent of pharmaceuticals for several past years. In recent years, the
reproducibility use of mAbs has been expanded due to significant advances in
design thus decreasing immunogenicity in humans, improving their
and resolution bioavailability, and specific affinity for antigen-binding.
between charge
The complexity of mAb with about 150 kDa molecular weight
variants of implements the use of quality by design (QbD) as an unavoidable
mAbs strategy for the development and manufacturing of these molecules.
QbD defines the critical quality attributes and a control strategy to
ensure stable and consistent quality during the manufacturing process.

Charge Variant
UHPLC
CHARGE VARIANT ANALYSIS OF mAb BIOTHERAPEUTICS BY NEXERA XS WITH A SHIM-PACK BIO IEX COLUMN
“The most common methods the characterization of mAbs charge

for charge variant analysis still heterogeneity and is routinely used as
a fingerprint of the distribution of post-
use a salt elution mechanism. translational modifications present on
Because of this, we investigated the mAb. Also, CEX analysis is necessary
the robustness of this method.” for mAbs quality control analysis and is
requested by regulatory agencies.
Different post-translational modifications There are two major mechanisms involved

during the upstream process such as in CEX of mAbs charge variants namely salt
amino or carboxy-terminal processing and gradient and pH gradient. In salt gradient
glycosylation or during downstream processes elution, mAbs charge variants are pushed down
or storage, such as deamidation, oxidation, the column from exchange site by competing
and fragmentation could result in different with the salt ions in the mobile phase. With
charge variants that could affect the safety, pH gradient elution, mAbs charge variants will
quality, and efficacy of mAbs. Therefore, elute from the column when the pH reaches
characterization and quantification of mAbs the point where they have little to no charge.
charge variants are required for assessing
consistent product quality. The most common methods for charge
variant analysis still use a salt elution
Methods of mAb charge variant analysis mechanism. Because of this, we investigated
Analytical methods with the capability to the robustness of this method. The control
separate differently charged molecules of pH during chromatography is of high
are used to characterize the variants importance to keep the retention times
of mAbs. Charge-based variants have stable and the method robust. The ion
been categorized as acidic, main, and exchange column itself will have an inherent
basic species. Chromatographic and buffering capacity which controls the pH
electrophoretic tools include ion-exchange during analysis. The degree of buffering
chromatography (IEX) and isoelectric a column is related to the exact column
focusing (IEF), which are the most common capacity and the type of charged group
and simple analytical methods for the bound to the resin.
analysis of mAbs charge heterogeneities.
IEF may be used for visualizing the charge
SPONSORED CONTENT
isoforms, but chromatographic tools are
more appropriate for precise quantification. Nexera series-Ultra
High Performance Liquid
Cation exchange chromatography Chromatograph
(CEX) has been a standard method for

Charge Variant
UHPLC
In this study, we describe a salt-gradient FIGURE 1: NexeraTM XS system

method having a common gradient program
for separating the charge variants of all the
four mAbs namely trastuzumab, omalizumab,
rituximab and cetuximab using Shimadzu
Nexera XS (UHPLC system shown in
FIGURE 1) and a Shim-pack Bio IEX column.
NexeraTM series
Key features
• Automated support functions utilizing
digital technology such as machine-
to-machine communication (M2M),
Internet of things (IoT), and Artificial
Intelligence (AI) enables higher The SP groups attached to the stationary
productivity and maximum reliability. phase are strong cation exchangers. These
• Allows a system to monitor and are less prone to ionization changes with
diagnose itself, handle any issues during different pH during salt gradient elution and
data acquisition without user input, maintains consistency in the retention times
and automatically behave as if it were of charge variants during analysis.
operated by an expert.
• Supports the acquisition of high-quality, Experimental
reproducible data regardless of an The mAbs solutions namely trastuzumab,
operator’s skill level for both routine omalizumab, rituximab and cetuximab were
and demanding applications. prepared in tris buffer with a concentration
of 5 mg/mL.
Shim-pack Bio IEX: Ion exchange
chromatography columns The samples were directly injected and
Shim-pack Bio IEX Columns are available analyzed with the Nexera XS with a UV
in Q (quaternary ammonium) and SP detector. The elution was monitored at
(sulfopropyl) chemistries and are based on
porous (Q and SP columns) and non-porous
(Q-NP and SP-NP columns) hydrophilic SPONSORED CONTENT
polymers with low nonspecific adsorption. Shim-pack Bio IEX Ion

The columns offer excellent binding Exchange Chromatography
capacity with exceptionally high efficiency Columns
and resolution.

Charge Variant
UHPLC
TABLE 1: LC analytical conditions FIGURE 2: The charge variant profile of

trastuzumab
Column Shim packTM Bio IEX SP-NP, (100
mm × 4.6 mmI.D., 3 µm
(P/N: 227-31005-03)
Oven temperature 25°C
Mobile phase A: 20 mmol/L (sodi-

um) phosphate buffer, pH-6 Mo-
Mobile phase bile phase B: 20 mmol/L (sodium)
phosphate buffer with 250 mmol/L
NaCl, pH-6
0-1.50 min (0%); 1.50-15.00
Gradient
min (0-100%); 15.00-18.00 min
program (B %)
(100%);18.10-20.00 (0%)
Flow rate 0.5 mL/min
Total run time 20.0 min FIGURE 3: The charge variant profile of
Injection volume 2 µL omalizumab
Autosampler
15°C
temperature
Detector
280 nm
wavelength
280 nm. The relative peak area (%) was used

to quantify the charge variants of all four
mAbs. The gradient program was optimized to
separate the charge variants of all four mAbs.
TABLE 1 shows the analytical conditions for

salt-gradient IEX.
FIGURE 4: The charge variant profile
Results and Discussion of rituximab
In this study, salt-gradient IEX methods were
performed for trastuzumab, omalizumab,
rituximab and cetuximab charge variant
analysis. FIGURES 2–5 show the charge variant
profile of all four mAbs on a Shim-pack Bio
IEX SP-NP column with a salt-gradient elution
program, demonstrating good separation of
charge variants in 20 minutes. The tallest peak
is the main component. The early- and late-

Charge Variant
UHPLC
FIGURE 5: The charge variant profile

FIGURE 7: Overlay chromatograms of
of cetuximab
six replicate injections for omalizumab
eluting peaks were called acidic variants and

FIGURE 6: Overlay chromatograms of
basic variants, respectively.
six replicate injections for trastuzumab
The relative peak area percent of charge
variants and main component for all four
mAbs based on six consecutive analyses is
summarized in TABLE 2.
The injection-to-injection repeatability

of the UHPLC-UV system was evaluated
based on the six consecutive analyses. As
shown in FIGURES 6–9, the overlay of six
TABLE 2: Relative area percent of main component and charge variants (n=6)
Relative Area
Trastuzumab Omalizumab Rituximab Cetuximab
percent (%)
Acidic variant 1 8.94 5.65 2.46 1.97
Acidic variant 2 NA 14.26 7.36 13.02

Acidic variant 3 NA NA 17.46 20.99
Main component 77.41 73.17 69.86 42.38
Basic variant 1 9.91 6.89 2.85 13.64
Basic variant 2 1.30 NA NA 2.64
Notes: NA = not applicable. The charge variants were not observed

Charge Variant
UHPLC
TABLE 3: Relative area percent of main component and charge variants (n=6)
Relative Area
Trastuzumab Omalizumab Rituximab Cetuximab
percent (%)
Acidic variant 1 0.83 1.97 4.87 4.80
Acidic variant 2 NA 1.81 4.11 0.93

Acidic variant 3 NA NA 1.68 4.07
Main component 2.60 1.65 0.55 1.29
Basic variant 1 1.29 2.97 3.96 3.05
Notes: NA = not applicable. The charge variants were not observed
replicates indicates an excellent Nexera and Shim-pack are trademarks of Shimadzu

separation reproducibility. Corporation in Japan and/or other countries.
Variations in the area counts of main For Research Use Only. Not for use in
component peak as well as charge variant diagnostic procedure.
peaks of all 4 mAbs for six replicate
injections were found to be less than
5 %RSD as shown in the TABLE 3.
Ashutosh Shelar
Shimadzu Analytical (India) Pvt. Ltd.
Conclusion
• Shimadzu Nexera XS system provides
Bhaumik Trivedi
a robust platform for charge variant
Shimadzu Analytical (India) Pvt. Ltd.
analysis of mAbs.
• Method development and optimization
have been performed on the Shim-pack Navin Devadiga
Bio IEX SP-NP cation exchange column. Shimadzu Analytical (India) Pvt. Ltd.
• The salt gradient method for charge
variant analysis of four mAbs gave Krutika Padhye
consistent results. Spinco Biotech Pvt. Ltd.
• The repeatability (n=6) results in terms
of peak area counts for all the acidic
and basic charge variants as well as the
main component of four mAbs were
found to be less than 5 %RSD.

Ultra High Performance Liquid Chromatograph
Nexera XS inert
Standard UHPLC (stainless steel-based)

mAU
EXPERIENCE 30
20
ADP
AMP
NEWFOUND CLARITY
10
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
Metal-sensitive Poor peak shape

compounds
Adsorption to internal surface Peak tailing
Stainless
steel tubing
Unconstrained Recovery and Sensitivity mAU
Nexera XS inert
ADP AMP
30
ATP
Clear Resolution without Restrictions 20
10
Assured Reliability and Reproducibility 0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
Material that inhibits Sharp peaks

adsorption Excellent
separation
www.shimadzu.com Learn more about Nexera XS inert

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FEBRUARY 2022

Solutions for Increasing the

Analytical Expert Q&A: Analysis Characterization of Control

FEBRUARY 2022 | LCGC 2

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

of the innovator drug, the production of arrangement of single or multiple linear

FEBRUARY 2022 | LCGC 3

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

information like the conserved functional

In the context of biotherapeutics, the When designing a therapeutic drug,

FEBRUARY 2022 | LCGC 4

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

in determining proper functioning of the

FEBRUARY 2022 | LCGC 5

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

TABLE 1: A list of quality attributes of biotherapeutics and their impact on drug

Glycosylation Critical Potency, PD, PK (16, 17)

FEBRUARY 2022 | LCGC 6

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

FEBRUARY 2022 | LCGC 7

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

on the Fc region of a mAb where glycation Deamidation

FEBRUARY 2022 | LCGC 8

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

FEBRUARY 2022 | LCGC 9

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

FEBRUARY 2022 | LCGC 10

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

FEBRUARY 2022 | LCGC 11

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

22. S. Fischer, J. Hoernschemeyer, and H.-C. Mahler, A. Whitty, K. Kimball, M. Brickelmaier, C.

FEBRUARY 2022 | LCGC 12

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART I: QUALITY ATTRIBUTES

Cano, D. Tesar, I. Nijem, D.E. Allison, P.Y. Wong,

FEBRUARY 2022 | LCGC 13

FEBRUARY 2022 | LCGC 15

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

TABLE 1: Techniques used for analytical characterization as mentioned in WHO

FEBRUARY 2022 | LCGC 16

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

FEBRUARY 2022 | LCGC 17

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

on their size. The stationary phase

FEBRUARY 2022 | LCGC 18

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

FEBRUARY 2022 | LCGC 19

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

experiments to capture structural changes

FEBRUARY 2022 | LCGC 20

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

monitors the characteristic infrared frequency modes in a system, generally

FEBRUARY 2022 | LCGC 21

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

FEBRUARY 2022 | LCGC 22

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

FEBRUARY 2022 | LCGC 23

ANALYTICAL CHARACTERIZATION OF BIOTHERAPEUTIC PRODUCTS, PART II: THE ANALYTICAL TOOLBOX

TABLE 2: Publications per technique since 2010 for biotherapeutics

HPLC 7440 1600

MALDI-TOF 2630 835

ORBITRAP 882 603

HDX-MS 406 665

DSC 1670 847

“Excellent mass accuracy with

“The most common methods the characterization of mAbs charge