Deamidation as a

Source of
Heterogeneity in
MAbs
By: Paul Brogee, Julia Manalil, Vikshit Girish Patel, Leila Emami
Taba, and Arpit Dharambhai Thumar
March 30, 2016
ChE 660
M. Aucoin

Introduction

Monoclonal antibodies (MAbs) are functionally identical immune-response

proteins. They are derived from a single clonally propagated parent cell. Mabs
are monospecific; each antibody in a collection of MAbs binds to the same
antigen or epitope.
MAb-derived therapeutics have become popular for various reasons:
High antigen specificity reduces potential for side-effects
Conjugation with other therapeutic or diagnostic agents provides expanded
functionality
Advances in humanization of MAbs has greatly decreased immunogenicity
concerns

Deamidation of Proteins

2,3

Deamidation is a major protein degradation

pathway. It results in the amide group of

asparagine or, to a lesser extent, glutamine, being
removed from the chemical compound.
In an antibody, this reaction is seen when the amide
group in asparagine is spontaneously replaced by a
carboxylic acid. This occurs due to a nucleophilic
attack by the N+1 nitrogen against the adjacent amide
group, resulting in a succinimide intermediate which
is subsequently hydrolyzed to form aspartic acid or
isoaspartic acid.
Figure 1: Deamidation of L-Asparagine

Consequences of MAb
4,5,6
Deamidation
Deamidation of Asn in MAbs could have detrimental effects on the stability of
the therapeutic antibody, potentially reducing half-life significantly. Effects of
deamidation are associated with the location of Asn conversion.
Location

Effect

Complementarity
Determining Region

Changes in antigen specificity

Constant Region

Changes in immunogenicity

Figure 2: Effects of deamidation at different locations on a mAb

Factors Contributing to
2,3,6,7
Fact Contribution
Deamidation
or
pH

Asn-X motifs (most

common) are most
susceptible to
deamidation under
alkaline conditions.
Conversely, C-terminal
Asn residues are
deamidated most
frequently at low pH.

Temp Elevated temperatures

eratur promote deamidation.
e
Changes in
temperature will also

Sites of Deamidation

3,8

Not all Asn residues of monoclonal antibodies are prone to deamidation. The
reaction is dependant on the 3-dimensional structure of the protein and the
primary amino acid sequence.
The conserved region of the human IgG contains 25 Asn residues yet only 4 are
prone to deamidation. These positions are adjacent to residues that are
typically less of a steric hinderance.
When the antibody was digested with trypsin into peptides, the deamidation
occurred at a more frequent rate than in the native protein which is more rigid,
but only at the 4 mentioned positions.
It was found that the occurrence of isoaspartate as the deamidation product over
aspartate was 3:1, and was dependant on the rigidity of the structure.

Quantification &
6,9,10,11(IEC)
Ion Exchange Chromatography
Characterization
IEC is a simple and rapid analytical method that can
separate analytes by net charge. By cleaving with papain
and then utilizing IEC, the deamidation rates of Fc
(crystallizable fragment) and Fab (antigen-binding
fragments) can be independently determined.

Figure 3: Ion Exchange

Chromatography

Isoelectric Focusing (IEF)

Due to the negatively ionizing
Asp, deamidation of Asn
reduces the pI of an
antibody. IEF can be used to
determine whether a change
in pI has taken place.

Figure 4: Isoelectric Focusing

Quantification &
3,12
Characterization
Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC)

RP-HPLC is a technique used to separate particles based on their retention time in a column
containing a hydrophobic solid phase. The fragments of a trypsinized antibody submerged in a
liquid phase can be separated in this way. This technique is also useful in determining which of
the two residues, aspartic acid or isoaspartic acid (which have the same molecular mass), are
contained in a peptide since the latter elutes earlier.

Mass Spectrometry (MS)

MS is an analytical technique that sorts ions based on their mass to charge ratio. MS can further
separate peptides that coelute on RP-HPLC and reduce chemical background based on their
masses leading to more accurate measurements. To measure the deamidation percentage of a
given peptide by MS, an extracted ion chromatogram (EIC) is used to represent the amount of
peptide ions detected by MS.

Amino Acid Sequence

Optimization 3,13
Deamidation can be mitigated by
Original

Substitution

A, L

NG, NN,
NS, NH

NT, NA, NL,

NF, NY, NW

making a posteriori modifications to the

antibodys primary structure at
deamidation hotspots. This may entail
substituting Asn itself (in locations
where Asn is not critical), or adjacent
residues that enable deamidation. NG
and NN motifs were found to be
particularly susceptible.

Cell Line Development

Select Cell line

Cell Lines

Transfect with plasmid(s)

containing genes for
antibody light and heavy
chains
96-well ELISA assays used
to eliminate non- and lowproducers
Cell growth and productivity
assessed and top 12-24
clones chosen

14,15

Chinese hamster ovary (CHO) cells

predominant mAb production host
PER.C6 human embryonic retina cells
Relatively new technology that can be grown indefinitely in serumfree media
NS0 murine myeloma cells
Used less often due to potential immunogenicity concerns related
to the production of galactose- alpha-1,3-galactose (an allergen)

Deamidation is not an enzyme-mediated reaction so the specific cell line used

does not have a significant effect on the rates of deamidation.

Product quality and

heterogeneity assessed.
Charge variants via
isoelectric focusing and ion
exchange chromatography

Top 4-6 clones are grown in

bioreactors and further
assessments are performed
before the final clone is
selected.

Upstream Production
16, 17
Culturing
Conditions
Considerations
Cell growth and productivity has the biggest influence on the
culture conditions and are optimally maintained by controlling:
Dissolved oxygen
pH ~7
And temperature 37 C
CHO cells, as well as other optimal mAb production cell lines,
can be grown as suspension cultures in chemically defined,
serum-free medium. For mAb production, fed batch cultures
Figure 5: Chinese Hamster Ovary Cells
or perfusion cultures can be used but their effects on
deamidation rates are minimal. Culturing techniques can have effects on other
aspects of mAbs such as improved glycosylation from using perfusion
cultures.

Recovery Process

18,19

Once the cell culturing is completed, all subsequent steps are typically
performed in a lower pH (~6-5) environment so as to deter unwanted
modifications, such as deamidation.

First, cells and cell debris are removed by large-scale filtration,

then subsequently, depth filtration.

Protein A chromatography specifically binds the Fc regions of

IgGs and purifies the MAbs from other cellular components.

Viral inactivation via low pH assures safety of products produced

by cell culture.

Following the low pH hold, the product is neutralized. This

creates turbidity, which is removed by additional polishing
chromatography.

Chromatography steps reduce host cell protein impurities, high

molecular weight aggregates, low molecular weight clipped
species, DNA, and leached Protein A.

Purification &
Concentration

Affinity Chromatography - A quick and robust operation with a yield of (>)99%.

18, 20

Additional chromatography can be considered as an intermediate polishing stage.

Purification processes are designed based on the properties of antibody such as pI, stability,
hydrophobicity, tendency to form aggregates, etc. Ideally purification processes should be
suitable for a wide variety of MAbs thereby eliminating expensive molecule-specific
purification technology.

Ion Exchange Chromatography MAbs exhibit a higher pI than host cell proteins (HCP). This
characteristic makes IEC a suitable technique for purification. Chromatographic medium is
selected to bind MAbs, while most DNA and HCPs are washed out with the buffer solution.
Ultrafiltration A pressure drive membrane process that is used for protein concentration and
buffer exchange.
Separation is achieved based on protein size; species larger than membrane pores are
retained while smaller species pass through.
Buffer exchange is achieved in diafiltration mode in which a buffer of desired composition is

Downstream Process

Figure 6: Schematic of Downstream Processing of MAbs

16

Formulation

22

Preformulation development begins with a research phase in which general information about the
protein and dosage are gathered to frame the approach for subsequent laboratory studies.
Once candidate formulations have been identified, forced degradation and formulation
development studies are utilized to further characterize the candidates and arrive at the final
selected composition. Forced degradation studies may include additional stress conditions such
as photolysis, acid/base hydrolysis, agitation, freeze/thaw, forced oxidation, and deamidation.
The design of formulation development studies will vary depending on the dosage form and route of
administration, and may include Tangential Flow Filtration (TFF) process development,
lyophilization process development, and materials compatibility studies (e.g., filters, pumps and
tubing, container/closure systems, syringe/needle configurations)
The process of lyophilization or freeze-drying involves removal of bulk water (>95%) from the
product, significantly reducing the rate of degradation due to contact with the aqueous phase.
Lyophilization is comprised of three main steps: freezing, primary drying, and secondary drying.
During the freezing process, bulk water undergoes phase separation via formation of ice crystals.

Formulation
Considerations
Variable

22,23
Conditions to minimize
Asn Deamidation

pH

Optimal pH range for deamidation resistance is 5-8

Storage Temperature

Deamidation rates follow an Arrhenius relationship with

temperature. Activation energy of 21.7 kcal/mol

Excipients

Adding organic solvents such as poly vinyl pyrolidone (PVP),

or glycerol reduces the solutions dielectric constant which
destabilizes the ionic intermediates of deamidation
The increased viscosity from addition of PVP helped reduce
deamidation rates 10 fold

Physical State

The shelf life was significantly improved by lyophilizing the

final product in PVP matrices in the glassy state.

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