[mAbs 1:3, 254-267; May/June 2009]; ©2009 Landes Bioscience

Report

Potential aggregation prone regions in biotherapeutics

A survey of commercial monoclonal antibodies

Xiaoling Wang, Tapan K. Das, Satish K. Singh and Sandeep Kumar*

Pharmaceutical R & D; Global Biologics; Pfizer Global Research & Development; Chesterfield, MO USA

Abbreviations: mAb, monoclonal antibody; FDA, food and drug administration; CDR, complementarity determining region; Fab, frag-
ment, antigen binding; Fc, fragment, crystallize easily; Fv, fragment, variable; VL, variable domain of light chain; VH, variable domain of
heavy chain; scFv, single chain Fv; CL, constant domain of light chain; CH, constant domain of heavy chain; PDB, protein data bank;
Q/N, glutamine and/or asparagine; QbD, quality by design
Key words: monoclonal antibody, aggregation, antibody sequence, aggregation-prone region, aggregation prediction

Aggregation of a biotherapeutic is of significant concern and Introduction

judicious process and formulation development is required to
minimize aggregate levels in the final product. Aggregation of a Therapeutic monoclonal antibodies (mAb) are a class of medi-
protein in solution is driven by intrinsic and extrinsic factors. In cations derived from living organism and produced by means
this work we have focused on aggregation as an intrinsic property of biologic techniques such as hybridoma,1 recombinant DNA2
of the molecule. We have studied the sequences and Fab struc- and phage display.3 Monoclonal antibodies are highly specific,
tures of commercial and non-commercial antibody sequences for have high affinity as well as selectivity for binding with thera-
their vulnerability towards aggregation by using sequence based peutic targets and exhibit less degree of non-mechanism toxicity.
computational tools to identify potential aggregation-prone The antibodies can also be exogenously engineered in commer-
motifs or regions. The mAbs in our dataset contain 2 to 8 aggre- cially viable manner. The fraction of therapeutic mAbs has been
gation-prone motifs per heavy and light chain pair. Some of these increasing in the research and development portfolios of phar-
motifs are located in variable domains, primarily in CDRs. Most maceutical industry. So far, more than 20 therapeutic mAbs with
aggregation-prone motifs are rich in β branched aliphatic and various indications have been approved by US Food and Drug
aromatic residues. Hydroxyl-containing Ser/Thr residues are also Administration (FDA) and hundreds of monoclonal antibodies are
found in several aggregation-prone motifs while charged residues currently in various stage of development.4
are rare. The motifs found in light chain CDR3 are glutamine Antibodies have been studied for over a century, so the basic
(Q)/asparagine (N) rich. These motifs are similar to the reported structures and functions of antibodies are now well understood.5-7
aggregation promoting regions found in prion and amyloido- Briefly, the building units of antibodies consist of two identical
genic proteins that are also rich in Q/N, aliphatic and aromatic light and two identical heavy polypeptide chains held together by
residues. The implication is that one possible mechanism for disulfide bridges. Light chains have two isotypes, namely λ and κ,
aggregation of mAbs may be through formation of cross-β differing in sequence composition. Heavy chains have five isotypes
structures and fibrils. Mapping on the available Fab—receptor/ based on chain structure and effector function. All currently
antigen complex structures reveals that these motifs in CDRs approved therapeutic mAbs belong to the IgG class. IgG has the
might also contribute significantly towards receptor/antigen simplest form and is the major immunoglobulin type in human sera.
binding. Our analysis identifies the opportunity and tools for IgGs are further divided into four subclasses, IgG1, IgG2, IgG3 and
simultaneous optimization of the therapeutic protein sequence IgG4. The sequences of light chains and different IgG heavy chains
for potency and specificity while reducing vulnerability towards are variable in the N-terminal variable domain and conserved in
aggregation. the remaining constant domain. The variable domains, especially
the complementarity determining regions (CDRs), determine the
*Correspondence to: Sandeep Kumar; Pfizer Global Research and Development; antigen binding specificity. Each IgG subclass has characteristic
Pharmaceutical R & D; AA324; 700 Chesterfield Parkway West; Chesterfield, MO disulfide bond pattern, differing mostly in the hinge region. The
63017 USA; Tel.: 636.247.0176; Fax: 636.247.0247; Email: [Link]@ hinge is the most flexible region in a mAb. It connects the two Fab
[Link]
and the Fc domains into ‘Y’ or ‘T’ like overall structures as revealed
Submitted: 12/10/08; Accepted: 01/29/09 from the crystal structures for intact murine antibodies.8,9
Previously published online as a mAbs E-publication: Monoclonal antibodies, like other protein therapeutics, are
[Link] susceptible to degradation at all stages from production to dose

254 mAbs 2009; Vol. 1 Issue 3

 Aggregation prone regions in commercial mAbs

administration. Several chemical and physical pathways, such addressed. Mapping of the aggregation-prone motifs on the avail-
as deamidation, oxidation, hydrolysis/fragmentation, isomeriza- able Fab—receptor/antigen complex structures revealed that, when
tion and aggregation, are responsible for the degradation of the present in mAb CDRs, these motifs might also contribute towards
proteins.10 Among these, aggregation is the least understood degra- receptor/antigen binding. This poses an interesting challenge to
dation route for mAbs. antibody engineers: how to minimize the vulnerability of the mole-
Aggregation is widely seen in proteins and refers to self- cule towards physical (or chemical) degradation while still retaining
association of a number of protein molecules that may form visible high potency and specificity towards the cognate targets?
or invisible particles, precipitates or fibrils.11,12 Self-association We must also point out that our analysis delves into only one
may or may not be preceded by adoption of non-native conforma- part of the complicated phenomena of aggregation. Apart from
tions by the protein or parts thereof. Aggregation is an important the intrinsic factors, there are a whole host of extrinsic factors that
factor in the development of a biotherapeutic.13-15 The primary together ultimately determine the levels of aggregation seen in a
concern around aggregation is the potential to enhance immune product. Furthermore, judicious process and formulation devel-
response, which presents a risk-factor in the development of these opment diminishes the impact of intrinsic factors and attenuates
therapeutics.16-18 If aggregation in the liquid state can not be the impact of extrinsic factors, as evidenced by the number of
controlled, the product may need to be lyophilized, increasing the successful commercial mAb products that have had a significant
cost of production. Aggregation problems are further exacerbated impact on human health over the last two decades.
when the mAb needs to be formulated at high concentrations
Therapeutic Monoclonal Antibodies in the Market
for subcutaneous route of administration.19,20 Hence, scientists
engaged in product development and formulation of the biologic Since the launch of muromonab-CD3 (Orthoclone OKT3) in
drug products must discover ways to counter these problems. In 1986, therapeutic monoclonal antibodies have evolved from the
the absence of a molecular-level understanding of the aggregation early murine origin, through chimeric and humanized, to fully
mechanism, work in preventing aggregation has been mainly based human form in little over 20 years. All 22 FDA approved commer-
on experimental heuristic screening, and dependent on particulars cial therapeutic monoclonal antibodies, as well as two polyclonal
of each case. antibody fragments and a diagnostic mAb approved by FDA,
Protein misfolding and aggregation is an intrinsic property are listed in Table 1. Indications, formulations and marketing
that underlies a variety of neurodegenerative disorders such as for these antibodies are summarized in refs. 29–31. The increase
Alzheimer and Parkinson disease.21-23 A hallmark of these diseases in the number of therapeutic antibody drug products over time
is aggregation by formation of fibrillar amyloid deposits. Through mirrors the advances in antibody production techniques.32-34 The
extensive research, sequence motifs that lead to this pathogenesis first therapeutic candidates were murine antibodies produced by
have been identified. Computational tools have been developed hybridoma technique,1 but use of these mAbs was limited due
to analyze protein sequences and predict the propensity for such to their immunogenic complications in human patients. Patients
aggregate formation. Two such sequence-based prediction tools are produced human anti-mouse antibody (HAMA) response as
TANGO24 and PAGE.25 the murine antibody was 100% foreign protein.35 Due to these
We believe that the intrinsic (in)stabilities of mAbs, as governed concerns, production of murine mAbs acrituomab (CEA-scan,
by the sequence-structure-function paradigm, are also coded in [Link]), fanolesomab (Neutrospec, www.
their sequences. In this report, we have collected the sequences of [Link]), imciromab pentetate (Myoscint, [Link]/cder),
commercial mAbs and performed sequence analysis in a system- satumomab pendetide (Oncoscint, [Link]
atic manner. We used sequence-based prediction tools (TANGO edu/resources/[Link]), and nofetumomab (Verluma,
and PAGE) to identify the potential aggregation-prone regions in [Link]
commercial mAb sequences. Our analyses reveal several common was discontinued.
features among the aggregation-prone motifs found in both vari- Subsequent generations of therapeutic antibodies have been
able and constant domains. The aggregation-prone motifs in re-engineered to reduce mouse origin content in order to reduce
variable domains are primarily located in the CDR loops, the immunogenicity. In the mid-1980s, simple chimeric mouse-
regions that bind directly with the cognate receptor or antigen. human antibodies42-44 were made using recombinant DNA
Almost all aggregation-prone motifs are rich in hydrophobic or techniques.2 Chimeric antibodies consist of variable domain of
aromatic residues. Hydroxyl group containing residues, Ser and mouse antibody and constant domains of human antibody. The
Thr, are also found in several aggregation-prone motifs. However, mouse content is reduced to approximately 33% in chimeric
charged residues (Asp, Glu, Lys, Arg and His) are rare in such mAbs. Two chimeric mAbs, rituximab (Rituxan) and infliximab
regions. The aggregation-prone motifs found in light chain CDR3 (Remicade) are among the five top selling antibodies today.
regions are glutamine (Q)/asparagine (N) rich. Sequence motifs Antibodies were further humanized by grafting only murine CDR
rich in Q/N, hydrophobic or aromatic residues have been previ- regions into the human antibody framework.45,46 Humanized anti-
ously implicated in aggregation of prion or amyloidal proteins.26-28 bodies have 5–10% of mouse content. Following the approval of
We also performed identical analyses on a set of non-commercial the first humanized therapeutic antibody (daclizumab [Zenapax])
antibody sequences and obtained similar results. The importance in 1997, humanized antibodies trastuzumab (Herceptin) and
of location of aggregation-prone regions on 3D structure is also bevacizumab (Avastin) have gone on to achieve blockbuster status.

[Link] mAbs 255

 Aggregation prone regions in commercial mAbs

Table 1 List of commercial mAbs

# Brand name Molecule name Isotype and format Source Source of sequence Approval year Company
1 Avastin Bevacizumab IgG1 κ Humanized CAS ID: 216974-75-3 2004 Genentech and
BioOncology
2 Bexxar Tositumomab and IgG2a λ Murine 2003 Corixa and GSK
I-131Tositumab Conjugated
to Iodine
3 Campath Alemtuzumab IgG1 κ Humanized DB ID: DB00087 2001 Ilex Oncology;
Millenium and Berlex
4 Cimzia Certolizumab IgG1 K; Fab; Humanized CAS ID: 428863-50-7 2008 Nektar/UCB
pegol Conjugated to
Polyethylene glycol
5 CroFab Crotailidae Fab Ovine 2002 Protherics Inc
Polyvalent
Immune Fab
6 DigiFab Digoxin Immune IgG2a κ; Fab Ovine DB ID: DB00076 2001 Protherics Inc
Fab PDB ID: 1IGJ
7 Erbitux Cetuximab IgG1 κ Chimeric DB ID: DB00002 2004 ImClone and BMS
8 Herceptin Trastuzumab IgG1 κ Humanized DB ID: DB00072 1998 Genentech
9 Humira Adalimumab IgG1 κ Human CAS ID: 331731-18-1 2002 CAT and Abbott
10 Lucentis Ranibizumab IgG1 κ; Fab Humanized CAS ID: 347396-82-1 2006 Genentech
11 Mylotarg Gemtuzumab IgG4 κ Humanized ref. 36, Fv only 2000 Celltech and Wyeth
ozogamicin Conjugated to Chimeric
Calicheamicin
12 Orthoclone Muromonab-CD3 IgG2a κ Murine DB ID: DB00075 1986 Ortho Biotech
OKT
13 ProstaScint Indium-111 IgG1 κ Murine 1996 Cytogen
Capromab Conjugated
pendetide to Indium
14 Raptiva Efalizumab IgG1 κ Humanized CAS ID: 214745-43-4 2003 Genentech
ref. 37 and Xoma
15 Remicade Infliximab IgG1 κ Chimeric ref. 38 1998 Centocor
16 ReoPro Abciximab 1gG1; Fab Chimeric 1994 Centocor/Lilly
17 Rituxan Rituximab IgG1 κ Chimeric DB ID: DB00073 1997 Genentech and IDEC
18 Simulect Basiliximab IgG1 κ Chimeric DB ID: DB00074 1998 Novartis
19 Soliris Eculizumab IgG2/4 κ Humanized CAS ID: 219685-50-5 2007 Alexion
ref. 39, 40 Pharmaceuticals
20 Synagis Palivizumab IgG1 κ Humanized PDB ID: 2HWZ ref. 41 1998 MedImmune
21 Tysabri Natalizumab IgG4 κ Humanized CAS ID: 189261-10-7 2004 Elan and
Biogen Idec
22 Vectibix Panitumumab IgG2 κ Human CAS ID: 339177-26-3 2006 Amgen
23 Xolair Omalizumab IgG1 κ Humanized CAS ID: 242138-07-4 2003 Genentech w
Novartis
24 Zenapax Daclizumab IgG1 κ Humanized CAS ID: 152923-56-3 1997 Roche
25 Zevalin Ibritumomab IgG1 κ Murine DB ID: DB00078 2002 IDEC
Conjugated
to Indium
or Yttrium

The current mAb market is dominated by chimeric and human- Most commercial antibodies are intact conventional mAbs.
ized antibodies. However, fully human antibodies generated using However, for enhanced affinity, reduced toxicity and improved
phage display technique3,47,48 and transgenic animals49-51 are tissue permeation, antibody fragments such as Fab, Fv and single
reaching the market. Adalimumab (Humira; launched in 2002) chain Fv (scFv) can be used. The Fab products are currently most
is one such example. common. Marketed Fabs (Table 1) are ranibizumab (Lucentis),

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 Aggregation prone regions in commercial mAbs

abciximab (ReoPro), certolizumab (Cimzia), digoxin immune (Simulect) and rituximab (Rituxan). Their aligned scores are close
Fab (DigiFab) and crotailidae polyvalent immune Fab (CroFab). to 90%. The murine mAb muromonab-CD3 (Orthoclone OKT)
The latest technologies for the design of high affinity antibodies is and the ovine mAb digoxin immune Fab (DigiFab) share lowest
reviewed by Wark et al.52 sequence similarities with other mAbs, as indicated by the low
aligned scores of ~60%.
Sequence Differences and Similarities Figure 2A shows the heavy chain sequence alignment for
Sequence availability for commercial mAbs. We have collected commercial mAbs. Similar to light chains, sequences in constant
the sequences of commercial mAbs in as complete a manner as regions for mAbs of the same isotype are very similar, if not iden-
feasible to perform sequence analysis. The sources of sequences tical. Variability comes from VH, especially CDRs. In the IgG1
are Drug Bank database ([Link])53 and CAS registry group, overall sequence identities of human and humanized mAbs
([Link]). Some partial sequences are collected from RSCB (adalimumab [Humira], bevacizumab [Avastin], alemtuzumab
Protein Data Bank ([Link]/pdb/home/)54 and literature. [Campath], trastuzumab [Herceptin], efalizumab [Raptiva], omal-
The Drug Bank ID, CAS ID, PDB ID, and references are listed izumab [Xolair], daclizumab [Zenapax], certolizumab [Cimzia
in Table 1. Fab] and ranibizumab [Lucentis Fab]) are greater than 85%, while
The sequences of commercial mAbs are not all publically the VH sequence aligned scores are ~67%. The same applies to the
available. For example, we could not find the sequences of tosi- chimeric IgG1 mAbs cetuximab (Erbitux), basiliximab (Simulect)
tumomab (Bexxar), abciximab (ReoPro), capromab pendetide and rituximab (Rituxan). These mAbs share 85% overall sequence
(ProstaScint) and crotailidae polyvalent immune Fab (CroFab) in identities, but 54% identities for VH sequence only. The human-
the public domain. In a few other cases, only partial sequences are ized mAb palivizumab (Synagis) appears to be an exception as
publicly available. The sequences of only the Fab and Fv regions are its aligned scores with other human or humanized IgG1 mAbs
available for palivizumab (Synagis) and gemtuzumab ozogamicin are below 70% when only Fab portions of the sequences are
(Mylotarg), respectively. Fv sequence of infliximab (Remicade) aligned (note that only Fab sequence is available for palivizumab).
has been published,38 but the CDR regions are missing. Thus, Ibritumomab (Zevalin) is the only IgG1 murine mAb in Figure
infliximab (Remicade) was not included in subsequent sequence 2A. Its sequence similarities with others are lower than 60%.
analysis. The hinge region is important for flexibility and effector func-
Sequence isotypes of the commercial mAbs. Table 1 summa- tions of the IgG molecules.55 Each IgG subclass has characteristic
rizes the brand name, generic name, isotype, source, year of first
sequence composition and disulfide bonding pattern in the hinge
FDA approval, and the manufacturer for each antibody included in
region.62,63 All the IgG1 commercial mAbs, except ibritumomab
this study. The heavy chains are dominantly IgG1 with only a few
(Zevalin), have the characteristic IgG1 hinge with three cysteine
of the IgG2 or IgG4 type. The choice of IgG isotype commonly
residues (Fig. 2A). The first cysteine is disulfide bonded with the
depends on the requirement for complement binding and effector
C-terminal cysteine in light chain. The other two cysteines form
function.55,56 IgG1 has a flexible hinge and good Fc-mediated
inter-heavy chain disulfide bridges. The constant region of eculi-
effector activity, while IgG2 has limited effector activity. IgG2 is
zumab (Soliris) is the hybrid of IgG2 (CH1 domain and hinge
commonly chosen when only the binding specificity is desired.56,57
region) and IgG4 (CH2 and CH3 domain).64 Both eculizumab
However, mAbs of IgG2 type have been reported to show disulfide
scrambling.58-60 Disulfide scrambling affects the hinge structure in (Soliris) and panitumumab (Vectibix) have the typical IgG2 hinge.
IgG2 mAbs and likely leads to product heterogeneity. However, the hinge sequence for panitumumab as published in
Most light chains are of κ type. Tositumomab (Bexxar) is the CAS registry (CAS ID: 339177-26-3) does not show an IgG2
only product with λ light chain. The light chain type for abcix- characteristic pattern. In Figure 2A, the sequence of panitumumab
imab (ReoPro) and crotailidae polyvalent immune Fab (CroFab) (Vectibix) is shown after replacement with a canonical IgG2 hinge.
is unknown from public sources. κ light chains have a high abun- Muromonab-CD3 (Orthoclone OKT) is another outlier as an
dance in mice (95%) but much lower (60%) in humans.61 IgG2 mAb with an IgG1 hinge. Natalizumab (Tysabri) has the
Sequence alignments for commercial mAbs. The multiple IgG4 hinge in which the four cysteines form inter-heavy chain
sequence alignment of light chains for commercial mAbs is disulfide bonds.
presented in Figure 1A. All the light chains shown in Figure 1A Sequence alignments for non-commercial antibodies. To
are κ chains. The degree of sequence identity is consistent with the compare our data set of commercial mAbs, we have randomly
sequence origin, i.e., source organism. The sequences of human collected 20 non-commercial antibody sequences. The crystal
and humanized mAbs have very high identities. The pair-wise structures for all of these are available in the protein data bank
aligned scores are as high as 90%. As expected, the differences (PDB).54 Three of the 20 are the sequences for intact mAbs (PDB
primarily lie in CDR regions. The aligned scores are reduced entries: 1IGT, 1IGY, 1HZH), seven are antigen-antibody (Fab)
to 80% if only VL sequences are considered. The aligned scores complexes and the remaining ten are Fabs only. We have done
between the human or humanized and the chimeric mAbs are the same analyses on these 20 antibody sequences. Their light and
75–80%. Even though ibritumomab (Zevalin) is a murine mAb, heavy chain sequence alignments are presented as Figures 1B and
its light chain is very similar to the two chimeric mAbs, basiliximab 2B to facilitate the direct comparison with the commercial mAbs.

[Link] mAbs 257

 Aggregation prone regions in commercial mAbs

Figure 1. For figure legend, see page 259.

258 mAbs 2009; Vol. 1 Issue 3

 Aggregation prone regions in commercial mAbs

Figure 1. (A) Sequence alignment of light chains of commercial mAbs. The letters at the end of the mAb brand name are used to indicate its source.
‘u’ for human, ‘zu’ for humanized, ‘xi’ for chimeric, ‘o’ for mouse and ‘ov’ for sheep mAb. For molecular name of each mAb, refer to Table 1. All
cysteine amino acids are highlighted in green. The three CDRs are highlighted in yellow. The predicted aggregation-prone segments are in red letters.
(B) Sequence alignment of light chains of the twenty randomly selected non-commercial mAbs. Highlighting has the same meaning as in (A).

Figure 2A. For figure legend, see page 260.

[Link] mAbs 259

 Aggregation prone regions in commercial mAbs

Figure 2A. Sequence alignment of heavy chains of commercial mAbs. ‘G1’ or ‘G2’ after the mAb brand name are for IgG1 and IgG2, respectively.
For molecular name of each mAb, refer to Table 1. Highlighting scheme is same as Figure 1. In addition, the generic hinge region is highlighted in
blue. Cysteine amino acids in hinge are highlighted in red. Note the hinge region of Vectibix is shown with a canonical IgG2 hinge. The mAbs except
Remicade are grouped according to isotype and ordered based on degree of humanization within each group.

Figure 2B. Sequence alignment of light chains of the twenty randomly selected non-commercial mAbs. Highlighting has the same meaning as in (A).

Prediction of potential aggregation-prone regions proteins, especially ones with amyloidogenic properties, often
contain specific sequence or structural motifs that tend to promote
Commercial mAbs are subjected to numerous stress factors, such aggregation.26,65 Knowledge of the aggregation-prone regions in
as pH changes, temperature excursions, chemical modification, proteins may help alleviate aggregation concerns in biotherapeutics
shear and freeze/thaw, at various stages of production, shipping via rational design and/or selection of robust yet potent candidates,
and storage. These stress factors may trigger self-association thus, bringing Quality by Design (QbD) approaches to the early
of the mAb molecules leading to aggregation. Currently, the stages of drug discovery and development.
process by which protein molecules self associate to seed aggre- Several molecular seeding conformations may give rise to aggre-
gation and the total number of variables involved are not gates. One of the best-studied conformational seed is the cross-β
well-understood. Does molecular association require a confor- motif seen in amyloid-like fibrils.66,67 While the presence of β-
mational change from the native state? If so, what degree of strands is not essential to form this motif, several β-rich proteins
conformational change is required? Which regions of the molecule (e.g., β2-microglobulin, transthyretin, immunoglobulin light chain
are more conformationally labile than others? Are these confor- especially of type λ, immunoglobulin heavy chain, concavalin A)
mationally labile regions more likely to drive self-association and form amyloids using the cross-β motif as seed.68-73 In addition,
promote aggregation? Accumulating experimental data shows that Siepen et al. have shown that the exposed edge β-strands in

260 mAbs 2009; Vol. 1 Issue 3

 Aggregation prone regions in commercial mAbs

proteins can initiate amyloidogenesis by forming the cross

β-motifs.74 For example, the recognition between exposed
edge β-strands is an important step in amyloid fibrilogen-
esis for transthyretin and β2-microglobulin.74 In another
example, Pande et al. has proposed that aggregation by
P23T mutant of human γD-Crystallin may be via this
mechansim.75 Moreover, work from Dobson’s group has
shown that fibrilogenesis is a general feature of proteins
under stress or denaturating conditions.76 Monoclonal
antibodies are also β-sheet rich proteins. Hence, we were
curious to explore if mAbs can also potentially aggregate
via the cross β-motif conformational seed. If so, the
sequence motifs prone to this type of aggregation should
be found in both the commercial and other mAbs. The
paper by Maas et al.17 is particularly relevant in this
regard. Using the amyloid-marker dyes Congo Red and
Thioflavin T, they detected formation of cross-β struc-
tures and amyloid-like fibrils in many biopharmaceuticals,
including two antibodies which nevertheless had among
the lowest signal levels. Besides this, we also have evidence
for Thioflavin T dye binding in aggregated mAb samples Figure 3. The TANGO and PAGE profiles for trastuzumab (Herceptin) mAb. (A)
Light chain profile. X-axis shows sequence number. Left y-axis and blue curve are
from our own work in progress. This work is our first for PAGE Z score. Right y-axis and black curve are for TANGO aggregation per-
attempt at identifying aggregation-prone motifs in thera- centage. The red horizontal line indicates Z score = 1.96. (B) Heavy chain profile.
peutic mAb sequences by taking advantage of the tools Axes and line styles have same meaning as in (A).
developed for amyloidogenic proteins. The presence of just
one aggregation-prone motif could drive cross-β motif type aggre- peptides from 16 proteins including α-synuclein, apolioprotein,
gation. However, the occurrence of aggregation-prone sequences in APP, IAPP, prion, Sup35 and tau.25
mAbs does not guarantee that aggregation shall necessarily proceed We deliberately chose to use at least two different aggregation-
by this mechanism. This may or may not be the significant mode prone region prediction tools. This approach has helped us avoid
of aggregation in therapeutic mAbs. potential biases due to the training sets, parameterization and
Prediction methods. A number of computational approaches, peculiarities of methods in a given tool. Even though the authors
such as TANGO,24 PAGE25 and Zyggregator,77 have been devel- of TANGO suggest a region is potentially aggregation-prone if
oped to predict the aggregation-prone regions in amyloidogenic the TANGO scores of more than five consecutive residues are
proteins. We have chosen two prediction tools, viz. TANGO and above 5%,24 we use a much more stringent criteria of ≥50%. The
PAGE, to identify potential aggregation-prone regions in commer- aggregation propensity (lnp) from PAGE is converted to Z score
cial mAb sequences. TANGO and PAGE have been parameterized in order to identify the regions with statistically high aggregation
based on the experimental data on amyloidogenic peptides and propensity. The Z score of residue i is calculated as follows,
proteins. However, the two tools use different algorithms.
(1)
TANGO and PAGE, developed by Serrano’s and Caflisch’ group
respectively, use the primary amino acid sequence only and make
predictions based on the physicochemical properties of amino
acids. The TANGO algorithm relies on physicochemical rules where, is the average aggregation propensity of the sequence,
behind β-sheet formation and assumes that the core of an aggregate std(lnπ) is the standard deviation. A region with Z score >1.96 is
is completely desolvated. TANGO calculates relative probabilities considered as aggregation-prone. We identify a region as aggrega-
of peptide segments to exist in different conformational states such tion prone if it is strongly predicted by at least one program. Use of
as a-helix, b-strand, turn, random coil and b-aggregate. The 24 stringent criteria ensures that our predicted regions have a greater
TANGO algorithm was tested against 179 peptides derived from probability of being truly aggregation-prone. However, we must
24
21 different proteins as published. TANGO yielded a success rate note that our choice is rather arbitrary, and additional aggregation-
of 92% for aggregation tendency of 5% or greater. prone motifs could have been identified with less stringent criteria.
PAGE computes the aggregation propensity (ln�) and absolute The real test lies in experimental confirmation. Experiments on
aggregation rate (lnR) for a given amino acid sequence by sliding a polypeptides containing the predicted aggregation-prone regions
small window of 5–9 residues along the sequence. The aggregation are planned.
propensity is calculated based on aromaticity, b-strand propensity, We have performed TANGO and PAGE analysis on all the
charge, solubility, average polar/non-polar/accessible surface area collected commercial mAbs and the 20 non-commercial anti-
25
of each residue in the given widow. PAGE was tested against body sequences. The TANGO and PAGE profiles of trastuzumab

[Link] mAbs 261

 Aggregation prone regions in commercial mAbs

Table 2 Summary of aggregation-prone motifs in commercial mAbs

# General location and Aggregation- Occurrence # General location and Aggregation- Occurrence
sequence positiona prone motifs in mAbsb sequence positiona prone motifs in mAbs
1Precede and partly LLIYSASFLY 7 9 Right after CDR H2 VTMLV 1
in CDR L2 LLIYAASYL- Residue 72–76
Residue 51–60 LLIYGA----
LLIYAA----
VLIYF-----
2 Right before CDR L3 LGIYF 1 10 Precede CDR H3 IYYCV 1
Residue 88–92 Residue 97–101
3Precede and mostly YCQQNNN 8 11 In CDR H3 VVYYSNSYWYF--- 5
in CDR L3 YCQQHNE Residue 104–118 -
Residue 92–98 YCQQYS- IFYFYGTTY-F---
YCQQYN- -
YCQQHY- -----DDHYC----
YCLQYD- -
YCQQS-- ---------
FAVWG
4 Beginning of SVFIFP 17 12 In CH2 SVFLFPP 15
CL domain SVFIF- Residue 254–259 SVFLFP-
Residue 119–124 TVFIFP SVFIFP-
5 In CL domain VVCLL 19 13 In CH2 VLMISL 1
Residue 137–141 VVCFL Residue 265–270
6 In CDR H1 YIFSNYWIQWV 3 14 In CH2 IVTCVVV 1
Residue 27–39 -IFTDF----- Residue 273–279
-----YYWTWI
7Precede and partly IGAIY 6 15 End of CH2 VVSVLTVL 14
in CDR H2 LGVIW Residue 317–324 VVSVLTVV
Residue 50–54 IGYIS
IGYIY
8 In CDR H2 TTEYN- 3 16 In CH3 GSFFLYS 14
Residue 61–66 -TEYNQ Residue 417–423 GSFFLY-
-TNYNQ -SFFLYS
-SFFLY-
aContinuous numbering in the aligned sequences as shown in Figures 1 and 2. is used for residue position. bThis column records the occurrences of the corresponding motifs in the mAbs in Figures 1 and 2.

(Herceptin) are shown in Figure 3 as an example. The aggregation- contains 2–8 aggregation-prone motifs per light and heavy chain
prone regions obtained from this analysis are highlighted in the pair. Moreover, the locations of all the aggregation-prone motifs
multiple sequence alignments shown in Figures 1 and 2. The are consistent across the alignment (Figs. 1A, 2A and Table 2).
location and composition of aggregation-prone regions for these The aggregation-prone motifs in the constant regions are nearly
non-commercial antibodies are similar to those from commercial identical due to greater conservation in these regions (Table 2).
mAbs. Table 2 provides a list of aggregation-prone motifs found in The aggregation-prone regions in variable domains are primarily
commercial mAbs sorted according to their location. located in CDRs and in the adjoining framework β-strands. The
To further validate our approach, we have also performed individual motifs in the variable domains show greater sequence
TANGO and PAGE analysis on the biopharmaceutical proteins variation (Table 2). At this time, however, it is unknown if the
used in the work of Maas et al.17 The criteria used here were the number of aggregation-prone motifs in a mAb is directly correlated
same as those for the commercial mAbs and non-commercial with the rate or degree of its aggregation. It is also not clear if any
antibodies. Out of the 11 biopharmaceuticals studied by Maas of the above mentioned motifs is a stronger aggregation driver than
et al. (Table 1 in ref. 17), the amino acid sequences for nine the others.
are available in the public domain ([Link] or CAS The degree of humanization in these commercial mAbs may
registry). Consistent with the experimental results,17 TANGO not be related to fewer aggregation prone motifs. Apart from alem-
and PAGE analyses have identified aggregation-prone motifs in all tuzumab (Campath) and daclizumab (Zenapax), the heavy chains
biopharmaceuticals (Table 3). Furthermore, the aggregation-prone of human or humanized IgG1 mAbs do not contain aggregation-
motif 14-ALYLV-18 (Table 3) coincides with the experimentally prone motifs in their variable regions (Fig. 2A). The constant
proven fibril-forming segment (12-VEALYL-17) of insulin.78 regions of heavy chains as well as the variable and constant regions
Figures 1A and 2A indicate that aggregation-prone motifs are in light chains (Fig. 1A) do not show fewer aggregation-prone
found in all domains of the antibodies except CH1. Each antibody motifs in fully human or humanized IgG1 mAbs. This is not

262 mAbs 2009; Vol. 1 Issue 3

 Aggregation prone regions in commercial mAbs

Table 3 Summary of aggregation-prone motifs in biopharmaceuticals

Therapeutic proteina Source of sequenceb Aggregation-prone motifc

Albumin DB00096 21 ALVLIAFA 153 ELLFFAK 325 VFLGMFLY
341 YSVVLLL 551 FAAFV
Somatropin DB00052 78 ISLLLIQ 101 LVYGA 161 GLLYC
Insulin aspart CAS 116094-23-6 14 ALYLV
Factor VIII DB00025 50 LFVEF 58 LFNIA 84 VVITL
136 YVWQVL 172 LIGALLV 195 FILFAVF
269 IFLFG 303 LGQFLLFC 460 TLLIIFK
572 NVILFSVF 634 VAYWYILFIG 648 FLSVFFSG
790 LLMLL 1226 NLFLLS 1445 LAILT
1698 YFIAAV 1756 LGLLG 1771 IMVTF
1875 FALFFTIF 2012 TLFLVY 2096 YISQFIIMY
2123 LMVFFGN 2271 VTLFF
Epoetin Alfa DB00016 65 GLALL 78 ALLVN
Etanercept DB00005 259 SVFLFP 322 VVSVLTVL 422 GSFFL
Glucagon DB00040 22 FVQWLM
aListof proteins is from Table 1 of ref. 17. bSequences are obtained from either Drugbank ([Link]) or CAS registry. Listed are entry ID. cNumber before each motif is the position of the first residue of
the motif.

surprising because the goal of humanization is to reduce immuno- tracts is associated with at least nine different neurodegenerative
genicity due to foreign origin. diseases, including Huntington’s disease.84-86 This high aggregation
In the antibody structure, CDRs form binding sites and are ­propensity of Q/N rich sequences is explained by the ‘polar-zipper’
exposed on the surface of the Fab globules to facilitate antigen/ model proposed by Perutz and coworkers based on their structural
receptor/hapten recognition. In absence of the cognate partners, studies.87 A number of computational studies have also provided
the CDRs may promote self-assembly due to their conformational insights on the aggregation mechanism of Q/N rich sequences.88-90
flexibility and solvent accessibility. However, it is not yet clear to us The crystal structures of two short peptides NNQQNY and
if the mAb self-association involves Fab: Fab or Fab: Fc association. GNNQQNY have been solved.66 These structures elucidated a
In theory, both of these association modes are possible. ‘steric zipper’ motif implicated in fibril formation. This work led
In general, the aggregation-prone motifs avoid charged residues to development of a computational method to detect similar short
(Asp, Glu, Lys, Arg and His). This feature is consistent with the peptide sequences.78 This work and other computational model of
fact that charged residues contribute favorably towards protein amyloid structures were also analyzed by Zhang et al.67 They have
solubility.79 The net charge of an unfolded polypeptide chain outlined three consensus features of amyloid organization, namely,
has been reported to be inversely correlated with its aggregation (1) polar or non-polar VDW zippers; (2) in-register β-strands; (3)
propensity.80 Surprisingly, the hydrophilic hydroxyl-containing twisted β-sheets.
Ser/Thr residues are found in several motifs (Table 2), probably Interestingly, several motifs predicted to be aggregation-prone
due to their high propensity to form β-strand and low propensity are conserved in the constant domains of almost all mAbs. SVFIFP
to form α-helix.81 More interestingly, almost all aggregation-prone in CL, SVFLFP in CH2 and GSFFLYS in CH3 are phenylalanine
motifs are rich in aromatic and hydrophobic β branched residues. rich segments (motif 4, 12 and 16 in Table 2). Phenylalanine,
The aggregation-prone motifs found in several light chain CDR3 an aromatic amino acid, is found to play an important role in
regions are rich in Gln and Asn residues. Those motifs are worth directing molecular recognition and self-assembly.28 Many experi-
further discussion as they have also been implicated in driving mentally studied β-aggregating segments contain phenylalanine
aggregation of proteins involved in neurodegenerative diseases. residues. Some examples are LVFFA in amyloid precursor protein
The aggregation-prone motifs in the third CDR of light chains (APP) and RGFFY in islet amyloid polypeptide (IAPP).25 Tyrosine
(YCQQNNN in cetuximab [Erbitux], YCQQHNE in efalizumab and tryptophan also have effects similar to phenylalanine. Jones
[Raptiva]; [motif 3 in Table 2]) are rich in Gln and Asn. These et al. have found that the aromatic residue rich peptides from
motifs are similar to the aggregation-seeding motifs YQQYN, β2-Microglobulin readily form fibrils in vitro.68 The motifs 6
YQNYQ and YQQQY found in the well-known yeast prion (YIFSNYWIQWV and YYWTWI in Heavy Chain CDR 1)
proteins sup35,27 and Ure2p,82 where the Q/N rich segments seed and 11 (VVYYSNSYWYF and IFYFYGTTYF in Heavy chain
self-association. High Q/N content domains have strong propen- CDR 3) are rich in aromatic residues (Table 2). The role
sity to form amyloid fibrils.83 The high aggregation propensity of of aromatic residues in promoting aggregation is through the
Q/N rich sequences is also manifested in the mutational expan- favorable π-stacking interactions; Phe especially is found to
sion of CAG codons which leads to the elongation of the encoded form significant homostacking.88 These interactions lead to
polyglutamine sequence. Aggregation of expanded polyglutamine aggregates with the specific ordered pattern seen in fibrils.25,68

[Link] mAbs 263

 Aggregation prone regions in commercial mAbs

However, in their study of amyloid

aggregation of several mutants of human
muscle acylphosphatase, Bemporad et al.
have suggested that the high aggregation
propensity of aromatic residues is attrib-
uted to their hydrophobicity and intrinsic
β-sheet forming propensity rather than
aromaticity.91
Besides being the driving force for
protein folding, the hydrophobic effect
also plays an important role in protein
aggregation.92,93 Most of the aggrega-
tion-prone motifs in commercial mAbs
(Table 2) are rich in hydrophobic residues,
especially β-branched aliphatic residues.
Ile, Val and Leu are among the top
aggregation-prone residues reported by de
Groot et al.26 Commercial mAbs contain
VVCLL in CL and VVSVLTVL in CH2
(motif 5 and 15 in Table 2). These are two
representative motifs with high hydropho- Figure 4. Cartoon representation of the superimposed Fab structures of bevacizumab (Avastin),
bicity. Stretches of hydrophobic residues, alemtuzumab (Campath), cetuximab (Erbitux), trastuzumab (Herceptin), basiliximab (Simulect) and
palivizumab (Synagis). All light chains are in red and heavy chains in blue.
such as IAALL in Transthyretin have been
shown to be aggregation promoting.71 The
high aggregation propensity of hydrophobic stretches could come 3D Structures of Commercialized mAbs
from two potential sources, i.e., the specific interactions mediated
by the side chains of hydrophobic residues and the non-specific IgG mAbs have very similar structure, which is in overall ‘Y’ or
92
hydrophobicity per se. Kim et al. have tried to distinguish these ‘T’ shape with Fab and Fc domains adopting the immunoglobulin
two potential sources through systematic mutation in a study fold. Currently, it is difficult to obtain crystal structures for intact
92
of Aβ42 peptide. Their findings suggest that the non-specific antibodies. So far only three crystal structures of intact antibodies
hydrophobicity is more important for aggregation than the specific have been solved with moderate resolution.8,9,95 However, the
side chain interactions. Fab or Fc domains of antibodies are relatively easy to crystallize
Amyloid fibrils and β helices show similar conformations. and many structures have been deposited in the Protein Data
Tsai et al.88 have performed a systematic analysis of β helices and Bank. The Fab structures of some commercial mAbs are available,
revealed their sequence and structure characteristics. Some relevant namely, bevacizumab (Avastin; PDB ID: 1BJ1),96 alemtuzumab
characteristics are: (1) Charged residues except Asp disfavor the β (Campath; 1CE1),97 cetuximab (Erbitux; 1YY8 and 1YY9),98 tras-
sheet regions; (2) Asn and Phe show strong amide stacking inter- tuzumab (Herceptin; 1N8Z),99 muromonab-CD3 (Orthoclone
actions which stabilize β helices; (3) Ile and Val have the highest OKT; 1SY6),100 basiliximab (Simulect; 1MIM),101 palivizumab
propensity to be in β strands. These features are also found in the (Synagis; 2HWZ) and digoxin immune Fab (DigiFab; 1IGJ). The
aggregation-prone motifs identified in this study. Fc structure of rituximab (Rituxan; 1L6X)102 is available as well.
In order to promote aggregation, a region in a protein should Due to their high sequence identities, the Fab structures are strik-
satisfy the following criteria: (1) the amino acid residues in the ingly similar. These Fab structures are aligned and shown in Figure
region should have high intrinsic aggregation propensity; (2) the 4. The root mean square distance (RMSD) values between these
region should be conformationally unstable or flexible; and (3) the structures are less than 5 Å.
region should be exposed or become exposed upon conformational We have mapped the aggregation-prone regions predicted
transition, to facilitate intermolecular interactions.77,94 In the case from amino acid sequence on to the 3D structure of Fabs. The
of commercial mAbs, the above mentioned motifs may well be aggregation-prone motifs in the CDRs are co-localized in the
buried in the native structure of mAb and, hence, blocked from structures to yield characteristic structural motifs in the antigen
aggregation. However, in case of conformational fluctuations, binding site. These structural motifs may be involved in recogni-
these motifs may become exposed. Therefore, the location of tion of the cognate receptors or antigens. As an example, Figure 5
aggregation-prone segments on the 3-D structure and the confor- shows the predicted aggregation-prone regions mapped on the Fab
mational flexibility of mAb molecule are also important factors structure of trastuzumab (Herceptin) in complex with extracellular
for predicting aggregation-prone regions with high accuracy. We domain of human epidermal growth factor 2 (HER2).99 HER2
are currently performing molecular dynamic simulation studies plays an important role in breast cancer. Trastuzumab (Herceptin)
to further refine our predictions. The mapping of our sequence is designed to selectively bind with high affinity to the extracel-
motifs on to the structures is given below. lular region of HER2. Two of the four aggregation-prone segments

264 mAbs 2009; Vol. 1 Issue 3

 Aggregation prone regions in commercial mAbs

on the Fab of trastuzumab (Herceptin)

are located at CDR L2 and CDR L3,
respectively. Up to 45% of the surface
area of the two segments is buried after
binding to HER2. This suggests that the
two motifs are major participants in the
binding sites. While we present trastu-
zumab (Herceptin) HER2 extra cellular
domain as an example, our observations
for the other Fab structures, except palivi-
zumab (Synagis), are similar (data not
shown).
Our observations indicate that the
sequence/structural motifs in mAb CDRs
that bind the cognate receptors/antigens
may also be the aggregation-prone regions.
If this is true, then an interesting challenge
is posed to the mAb engineers: how should
potent and specific mAb therapeutics with
low vulnerability towards aggregation be
designed? This is a significant challenge
because therapeutic mAb drug products
are stored in absence of their cognate Figure 5. Structure of trastuzumab (Herceptin) Fab in complex with extracellular domain of HER2
partners, leaving these motifs potentially is shown in cartoon representation. Green chain is HER2. Light and heavy chains of trastuzumab
(Herceptin) are shown in gray and blue, respectively. The predicted aggregation-prone motifs are
vulnerable to aggregation. Structure based highlighted with different colors. The magenta segment is LLIYSASFLY in CDR L2 and the red is
biologic drug design as part of QbD YCQQHY in CDR L3. The two motifs SVFIFP and VVCLL in L are in cyan and green, respectively.
C
approaches can help address such issues.
The other two aggregation-prone
segments in trastuzumab (Herceptin) are the two closing β-strands locations such as CDR loops; (2) it is possible to successfully use
in CL domains of the Fabs (Fig. 5). One of the strands (SVFIFP) is the methods developed for amyloidogenic peptide and proteins to
at the edge of the 4-stranded β-sheet of CL which packs against the predict aggregation in biotherapeutics. Sequences/motifs predicted
4-stranded β-sheet of CH1 domain. The edge β-strands can readily to be involved in aggregation are seen to be important for function
form edge-to-edge aggregates.74,103 The amyloid fibril formation also. Our approach is validated by the finding that TANGO and
of a number of proteins, such as transthyretin (TTR), has been PAGE also identified aggregation-prone motifs in non-mAb biop-
linked to the edge β-strand interactions.74 harmaceuticals with available experimental aggregation data (Table
3).17 The tools and the analyses utilized in this work can thus be
Summary and Conclusions applied to mutational studies to optimize activity while reducing
The technologies driving advances in antibody development aggregation propensity at the discovery stage. The next step in
have focused on increasing the efficacy and potency of mAbs while prediction of aggregation-prone regions in biologic drugs shall be
reducing immunogenic potential. Aggregation issues with candi- incorporation of the 3D structural information.
date mAbs are usually addressed at the formulation stage during Acknowledgements
drug development. However, consideration of aggregation issues We thank Drs. James Carroll, Kevin King, Sandeep Nema,
at early stages of biologic drug discovery may save both time and Sa V. Ho, Graeme Bainbridge and Bernard Violand for several
resources while reducing risk at the later stages of drug formulation helpful discussions. The authors are grateful to Pfizer IP committee
and clinical development. QbD approaches including structure members for review of this manuscript and for several useful
based biologic drug design can be useful in addressing these chal- comments. Drs. Arindam Bose and Thomas Warren are thanked
lenges. for their interest and support for our work. A postdoctoral
The aggregation-prone regions in commercial and non-commer- fellowship for Xiaoling Wang via predictive stability initiative at
cial antibodies, predicted by TANGO and PAGE, have sequence Pharmaceutical Research and Development, Global Biologics, in
motifs reminiscent of those found in amyloidogenic proteins. Pfizer is gratefully acknowledged. Two anonymous referees are
As such, this is not surprising because TANGO and PAGE have acknowledged for their critical review of the manuscript.
been parameterized using data from amyloidogenic peptides and
proteins. However, the novel findings of our study are as follows:
(1) aggregation-prone motifs in commercial and non-commercial
antibodies are consistent and present at functionally important

[Link] mAbs 265

 Aggregation prone regions in commercial mAbs

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[Link] mAbs 267