Journal of Pharmaceutical Sciences 111 (2022) 2445−2450

Contents lists available at ScienceDirect

Journal of Pharmaceutical Sciences

jo urn a l h om ep ag e : w ww.jp harms ci.o rg

Pharmaceutical Biotechnology

Observation of Heavy-Chain C-Terminal Amidation in Human

Endogenous IgG
Bhavana Shaha, Ming Lib, Jette Wypycha, Marisa K. Jouberta, Zhongqi Zhanga,*
a
Process Development, Amgen Inc. One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b
Quality, Amgen Inc. One Amgen Center Drive, Thousand Oaks, CA 91320, USA

A R T I C L E I N F O A B S T R A C T

Article history: Therapeutic IgG mAbs expressed from Chinese hamster ovary (CHO) cells are known to contain three C-ter-
Received 15 April 2022 minal variants in their heavy chains, namely, the unprocessed C-terminal lysine, the processed C-terminal
Revised 10 June 2022 lysine, and C-terminal amidation. Although the presence of C-terminal amidation in CHO-expressed IgGs is
Accepted 10 June 2022
well studied, the biological impact of the variant on the safety and efficacy of biotherapeutics has not been
Available online 16 June 2022
well understood. To further our biological understanding of C-terminal amidation, we analyzed a series of
IgG samples, including both endogenous human IgGs as well as recombinant IgGs of different subclasses
Keywords:
expressed from both CHO and murine cell lines, for their heavy-chain C-terminal variants by LC-MS/MS
IgG
C-Terminal Amidation
based peptide mapping. The results demonstrate that heavy-chain C-terminal amidation is a common variant
C-Terminal Lysine occurring in IgG of all four subclasses (IgG1, IgG2, IgG3 and IgG4). The variant is generally present in recombi-
C-Terminal Variant nant IgG mAbs expressed from CHO cell lines but not in IgG mAbs expressed from murine cell lines, whereas
Safety the IgGs expressed from murine cell lines contain a much larger amount of unprocessed C-terminal lysine.
Endogenous IgG Additionally, a significant amount of heavy-chain C-terminal amidation is observed in endogenous human
Chinese hamster ovary IgGs, indicating that small amount of the variant present in therapeutic IgGs does not pose a safety concern.
Murine © 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open
Mass Spectrometry
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction Most IgG-based biomolecules contain an Fc region, which is com-

Monoclonal antibodies (mAb) and mAb-related therapeutics have
become the most important class of biopharmaceuticals for diverse
indications such as various types of cancer, autoimmune disease,
cardiovascular disease, and infectious diseases.1 Most of these mAb-
related therapeutics are IgG-based biomolecules, produced in mamma-
lian cell lines through recombinant technology.2 During production,
these molecules undergo various post-translational modifications
(PTMs), causing structural heterogeneities in the therapeutic
product.3,4 Additionally, chemical modifications may also occur during
manufacturing and storage of the product,5 contributing further to its
heterogeneity. Some of these modifications may impact the safety
and/or efficacy of the product. Modifications having impact on safety
and/or efficacy are considered critical quality attributes (CQAs) and
must be properly understood, monitored, and controlled.6
Abbreviations: IgG, immunoglobulin gamma; CHO, Chinese hamster ovary; LC, liquid
chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; mAb,
monoclonal antibody; PAM, peptidylglycine a-amidating monooxygenase; BPC, base
peak chromatogram.
* Corresponding author at: Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA
91320, USA.
E-mail address: zzhang@amgen.com (Z. Zhang).
posed of two heavy chains ending with -X-glycine-lysine residues.
Due to the actions of carboxypeptidases the C-terminal lysine on
most heavy chains is removed in mature antibody molecules,7-10
exposing the glycine as the C-terminal residue. In addition, it has
been reported that in a small fraction of most IgG1 and IgG4
molecules expressed in Chinese hamster ovary (CHO) cells, the
heavy-chain C-terminal glycine is converted to an amide group
(C-terminal amidation).11-13
The Fc C-terminal amidation is catalyzed by peptidylglycine
a-amidating monooxygenase (PAM).14-16 Utilizing its two active
domains, peptidylglycine alpha-hydroxylating monooxygenase (PHM)
and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), PAM
catalyzes the hydroxylation of the glycine residue at the C-terminus
of a peptide chain, followed by removal of the glyoxylate from the
hydroxyglycine residue, leaving the C-terminus amidated. It has been
shown that the Fc-amidation level of an expressed antibody can be
controlled by modulating the expression level of PAM in CHO cells.17
Fc-amidation in an expressed antibody can also be modulated by cell
culture parameters, such as copper concentration.12
Although the presence of heavy-chain C-terminal amidation in
CHO-expressed IgG1 and IgG4, as well as its biological process of for-
mation, is well studied, its biological impact on the safety and efficacy

https://doi.org/10.1016/j.xphs.2022.06.012
0022-3549/© 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
2446 B. Shah et al. / Journal of Pharmaceutical Sciences 111 (2022) 2445−2450

of biotherapeutics has not been well understood. First, it has been
unknown whether the C-terminal amidation happens to all IgG sub-
classes. Second, it has been unclear whether the modification occurs
in antibody molecules expressed in other mammalian cells. Third,
there has been no conclusive data on whether endogenous human
IgGs also undergo the same modification.
To further our biological understanding of these C-terminal var-
iants, we designed this study to address the above questions. Instead
of performing a resource-demanding clinical experiment to assess
the safety profile of the variant, we collected “real-world” evidence
by analyzing a series of IgG samples including both endogenous
human IgGs as well as recombinant IgGs of different subclasses
expressed from both CHO and murine cell lines. Tryptic digestion
was performed on each sample, followed by high-resolution liquid
chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
LC-MS/MS is capable of confidently identifying the attributes of inter-
est and quantifying their relative abundance with high specificity.18
Materials and Methods
Materials
The 13 recombinant IgG1, IgG2 and IgG4 samples expressed from
CHO or murine cell lines were obtained from Amgen and other
respective manufacturers (Table 1). The 5 human myeloma IgGs and
7 human plasma IgGs were obtained from Sigma-Aldrich, Alpha Diag-
nostic International, Athens Research and Technology, Abchem, and
Creative Biomart as shown in Table 1.
Tryptic Digestion
IgG samples were digested with trypsin prior to LC-MS/MS analy-
sis. First, to reduce the disulfide bonds, each sample was treated with
8 mM dithiothreitol (DTT, Sigma-Aldrich) at 37°C for 30 min in a
denaturing solution containing 6.0 M guanidine hydrochloride
(Sigma-Aldrich), 2 mM EDTA, and 0.15 M Tris at pH 7.5. The reduced
IgG sample was then alkylated with 14 mM iodoacetic acid (Sigma-
Aldrich) at 25°C for 25 minutes in the dark. Alkylation was quenched
with 6 mM DTT. The reduced/alkylated sample (»0.25 mg/mL IgG
concentration) was transferred to a Microcon 30-kDa filter (Millipore
Sigma) and spun at 14,000 £ g to exchange from the denaturation
buffer into a 0.1 M tris buffer at pH 7.5. After buffer exchange, an
appropriate amount of recombinant trypsin (Roche) was added onto
the filter to achieve an enzyme:substrate ratio of 1:10, followed by
incubation at 37°C for 2 hrs. After digestion peptides were collected
in the flowthrough by spinning the filters at 14,000 £ g. To each filter,
an equal volume of 0.25 M acetate buffer (pH 4.8) in 6 M guanidine
hydrochloride was added as a quench buffer, and the quench buffer
flowthrough was pooled in each respective sample. Final peptide
concentration in the digest was »0.5 mg/mL, assuming 100% peptide
recovery.
LC-MS/MS Data Collection
Each proteolytic digest was analyzed by reversed-phase liquid
chromatography on an Agilent 1290-II HPLC directly connected to a
Thermo Fisher Scientific Q-Exactive HF high-resolution mass spec-
trometer. Peptides were eluted using a Waters Acquity Peptide CSH

C18 column (2.1 £ 150 mm, 1.7 mm particle, 130 A pore size) at a
flow rate of 0.25 mL/min with the column temperature maintained at
50°C. Mobile phase A was 0.1% (v/v) formic acid in water, and mobile
phase B was 0.1% (v/v) formic acid in acetonitrile. After an initial hold
at 0.5% B for 5 minutes, mobile phase B was increased linearly to 36%
in 72 minutes. Column wash was achieved by increasing mobile
phase B to 90% in 5 minutes with hold for 3 minutes. Column was
equilibrated with 0.5% B for 14 min.
The mass spectrometer was set up to collect a high-resolution MS
scan at resolution setting of 120,000 and automatic gain control (AGC)
target of 1E6, followed by five data-dependent higher-energy collision
dissociation (HCD) MS/MS (normalized collision energy = 27%), with
dynamic exclusion, of the most abundant ions.
Data Processing
All MS data were processed on MassAnalyzer,18 a custom program
developed in-house (available in Biopharma FinderTM from Thermo
Fisher Scientific). MassAnalyzer performs component detection,
retention time alignment,19 peptide identification, peak integration
as well as attribute quantitation in an automated fashion. For reliable

Table 1
Determined Abundance of the Two Minor Variants for the 25 IgG Samples Analyzed in this Study.

Sample IgG Subclass Source/Cell line Manufacturer/Vendor (Catalog #) DesK Sequence Amidation SLSLSX-NH2 Lysine SLSLSXGK

1 IgG1k CHO Amgen SLSLSPG 0.35% 2.03%

2 IgG2k CHO Amgen SLSLSPG 0.34% 4.55%
3 IgG2λ CHO Amgen SLSLSPG 0.10% 2.05%
4 IgG4k CHO Merck SLSLSLG 2.86% 4.49%
5 IgG4k CHO Bristol-Myers Squibb SLSLSLG 0.24% 3.41%
6 IgG1k Murine NIST (RM 8671) SLSLSPG <0.001% 15.1%
7 IgG1k Murine Lilly SLSLSPG <0.001% 41.2%
8 IgG1k Murine Janssen SLSLSPG <0.001% 60.5%
9 IgG4k Murine Biogen SLSLSLG <0.001% 7.34%
10 IgG2/4k Murine Alexion (US lot 1) SLSLSLG 0.004% 21.2%
11 IgG2/4k Murine Alexion (US lot 2) SLSLSLG 0.001% 16.4%
12 IgG2/4k Murine Alexion (EU) SLSLSLG 0.004% 20.4%
13 IgG2/4k Murine Alexion (Germany) SLSLSLG 0.002% 19.9%
14 IgG1k Human myeloma Sigma-Aldrich (I5154) SLSLSPG 0.12% 0.001%
15 IgG2k Human myeloma Sigma-Aldrich (I5404) SLSLSPG 0.020% 0.016%
16 IgG2λ Human myeloma Sigma-Aldrich (I5279) SLSLSPG 0.33% 0.001%
17 IgG4k Human myeloma Alpha Diagnostic (20007-G4-500) SLSLSLG 1.13% 0.011%
18 IgG4k Human myeloma Athens (16-16-090707-4M) SLSLSLG 1.20% 0.012%
19 IgG1 Human plasma Athens (16-16-090707-1) SLSLSPG 0.15% 0.006%
20 IgG2 Human plasma Athens (16-16-090707-2) SLSLSPG 0.14% 0.012%
21 IgG3 Human plasma Athens (16-16-090707-3) SLSLSPG 0.11% <0.001%
22 IgG3 Human plasma Alpha Diagnostic (20007-G3-1P) SLSLSPG 0.10% <0.001%
23 IgG4 Human plasma Athens (16-16-090707-4) SLSLSLG 0.10% 0.007%
24 IgG4 Human plasma Abchem (ab183266) SLSLSLG 0.24% 0.031%
25 IgG4 Human plasma Creative Biomart (IgG4-232H) SLSLSLG 0.22% 0.032%
 B. Shah et al. / Journal of Pharmaceutical Sciences 111 (2022) 2445−2450 2447

Figure 1. Base-peak chromatograms (BPC) of tryptic digestion of (A) a CHO-expressed IgG2 mAb (Amgen) and (B) a human plasma IgG1 (Athens). Mass spectra of the three heavy-
chain C-terminal peptide variants are also shown on top of each chromatogram. Each major peak in the BPC is labeled with its determined mass. The retention time range of each of
the three peptide variants is indicated in green. Relative difference between determined mass and theoretical mass for each peptide variant is shown in ppm.

peptide identification, MassAnalyzer compares the experimental MS/
MS to accurately predicted theoretical MS/MS of unmodified and
modified peptides.20-22 When the program detects a modification
that is not provided by the user, it gives the mass change caused by
the modification and the proposed location of the modification
(unrestricted modification search).18 Therefore, C-terminal amidation
and lysine addition are identified automatically as G-58.005 and G
+128.095, respectively, even if the two modifications are not specified
by the user. When no MS/MS is collected in samples with extremely
low variant abundance, the variant is identified based on its retention
time and accurately determined m/z value. The abundance of each
modification was calculated from the peak area of the modified
peptide relative to the total peak area of modified and unmodified
peptides.
Results
A total of 25 samples were analyzed. They include recombinant
IgG1, IgG2, and IgG4 mAbs expressed in CHO cells (5 in total), recom-
binant IgG1, IgG2/4 (hybrid of an IgG2 CH1/hinge and an IgG4 Fc23)
and IgG4 mAbs expressed from murine cells (8 in total), IgG1, IgG2
and IgG4 mAbs from human myeloma (5 in total), and endogenous
polyclonal IgG1, IgG2, IgG3 and IgG4 purified from normal human
plasma (7 in total). Each of the 25 samples were digested with
trypsin, followed by reversed-phase LC-MS/MS analysis. LC-MS/MS
data were processed on MassAnalyzer for identification and relative
quantitation of the heavy-chain C-terminal peptides containing dif-
ferent variants.
Fig. 1 shows the base-peak chromatograms (BPC) of the tryptic
digests of a CHO-expressed IgG2 mAb and a human endogenous
IgG1. Tryptic digestion of IgG1, IgG2 and IgG3 generates a heavy-
chain C-terminal peptide with a sequence of SLSLSPGK. Due to the
actions of carboxypeptidases and PAM, three variants were observed,
including the original SLSLSPGK, the variant with C-terminal lysine
removed (desK, SLSLSPG), and the C-terminal amidated form
(SLSLSP-NH2). DesK (SLSLSPG) is usually the most abundant variant.
When the three peptide variants generated from CHO-expressed
IgG2 and human endogenous IgG1 are compared, it can be seen that
they elute at the same times and have the same m/z that match the
theoretical m/z of each variant. For the original SLSLSPGK variant,
due to its extremely low level in the human endogenous IgG1, its
doubly charged ion at m/z 394.729 is only observable in the mass
spectrum when zoomed to a narrow m/z range.
Similarly, Fig. 2 shows the base-peak chromatograms of the tryp-
tic digests of a CHO-expressed IgG4 mAb and a human endogenous
IgG4. Different from IgG1, IgG2 and IgG3, tryptic digestion of IgG4
generates a heavy-chain C-terminal peptide with a sequence of
SLSLSLGK. Similar to IgG1, IgG2 and IgG3, however, due to the actions
2448 B. Shah et al. / Journal of Pharmaceutical Sciences 111 (2022) 2445−2450

Figure 2. Base-peak chromatograms (BPC) of tryptic digestion of (A) a CHO-expressed IgG4 mAb (Merck) and (B) a human plasma IgG4 (Abchem). Mass spectra of the three heavy-
chain C-terminal peptide variants are also shown on top of each chromatogram. Each major peak in the BPC is labeled with its determined mass. The retention time range of each of
the three peptide variants is indicated in green. Relative difference between determined mass and theoretical mass for each peptide variant is shown in ppm.

of carboxypeptidases and PAM, three variants were observed, includ-
ing the original SLSLSLGK, the variant with C-terminal lysine
removed (desK, SLSLSLG), and the C-terminal amidated form
(SLSLSL-NH2). DesK (SLSLSLG) is usually the most abundant variant.
When the three peptide variants generated from CHO-expressed
IgG4 and human endogenous IgG4 are compared, it can be seen that
they elute at the same times and have the same m/z that match the
theoretical m/z of each variant. For the original SLSLSLGK variant,
due to its extremely low level in the human endogenous IgG4, its
doubly charged ion at m/z 402.745 is only observable in the mass
spectrum when zoomed to a narrow m/z range.
Confident identification of the peptide variants is achieved by the
exact match of the determined masses to their theoretical masses
(within 2 ppm, as shown in Figs. 1 and 2). Additionally, the sequence
of each variant is also confirmed from its fragment ions by MS/MS
(Fig. 3). The MS/MS of amidated variants SLSLSX-NH2 (X represents
P for IgG1, IgG2, IgG3, and L for IgG4) from the human endogenous
IgGs match those from CHO-expressed IgGs exactly, while MS/MS of
the SLSLSXGK variants in human endogenous IgGs were not collected
due to their extremely low abundances.
To achieve relative quantitation of each variant, an extracted-ion
chromatogram of each peptide variant was constructed, and the area
under each peak was determined. When constructing an extracted-
ion chromatogram, the singly charged ion (1+) was used for the two
variants without lysine (SLSLSXG and SLSLSX-NH2), and the doubly
charged ion (2+) was used for the variant containing lysine
(SLSLSXGK). As seen in the mass spectra shown in Figs. 1 and 2, these
charge states are the predominant species in each spectrum − ignor-
ing the minor charge state would not significantly alter the quantita-
tion result.
To ensure the observed MS signals for the C-terminal peptides
were not from carryovers of the previous injection, a few blank runs
(with an injection of pure water) were inserted into the sample
sequence periodically to evaluate the level of carryovers of these pep-
tides. No detectable carryovers were observed for the C-terminal
peptides except for the IgG4 desK peptide (SLSLSLG), which has a car-
ryover level of about 0.01% (relative to the same peptide in the previ-
ous injection) or lower.
The relative abundance of each variant is calculated as its peak
area divided by the total peak area of all three variants and is shown
as a percentage. Table 1 shows the determined abundance of the two
minor variants (with C-terminal amidation and unprocessed C-termi-
nal lysine) for the 25 samples. Except for one IgG1 mAb derived from
murine cell lines (sample 8), desK (SLSLSXG) is the most abundant
variant in all IgGs. Limit of detection (LOD) of the two minor variants
were estimated to be »0.001%, as MS signals of corresponding ions
 B. Shah et al. / Journal of Pharmaceutical Sciences 111 (2022) 2445−2450 2449

Figure 3. MS/MS of peptide variants with fragment assignments confirming the sequence of each peptide. Panels A‒D are from the C-terminal peptide variants having sequence of
SLSLSPGK that appears in IgG1, IgG2 and IgG3 (exemplified by the three variants from CHO-expressed IgG2 and the SLSLSP-NH2 variant from human plasma IgG1 as shown in
Fig. 1). Panels E‒H are from the C-terminal peptide variants having sequence of SLSLSLGK that appears in IgG4 (exemplified by the three variants from CHO-expressed IgG4 and the
SLSLSL-NH2 variant from human plasma IgG4 as shown in Fig. 2).

(with correct retention time and determined m/z values within 2
ppm) at 0.001% level were marginally distinguishable from noises.
These MS signals were not carryovers from previous injections
because carryovers of the minor variants were determined to be
undetectable.
Discussion
All IgG mAbs expressed from CHO cells contain C-terminal amidation
As shown in Table 1, all recombinant IgG1, IgG2 and IgG4 mAbs
expressed in CHO cells exhibit some level of C-terminal amidation
(0.1% - 2.9%), indicating the action of PAM on IgG is not subclass spe-
cific, even if IgG4 Fc has a slightly different C-terminal sequence. This
conclusion is consistent with the literature reporting the observation
of C-terminal amidation in IgG1s and IgG4s expressed from CHO
cells.11-13
Endogenous human IgGs contain significant level of C-terminal
amidation and trace level of unprocessed C-terminal lysine
Although a previous study has not detected C-terminal amidation
in an endogenous human IgG,13 it is interesting that, in this study,
C-terminal amidation is observed in all endogenous human IgGs of
all four subclasses, ranging from 0.02% to 1.2% in human myeloma
IgGs, and 0.10% to 0.24% in normal human plasma IgGs (Table 1). In
fact, the abundances of C-terminal amidation in endogenous human
IgGs generally agree with those in recombinant IgGs expressed from
CHO cells (Table 1).
Based on the result presented in Table 1, endogenous human IgGs
contain 0.10% to 0.24% of C-terminal amidation in healthy individuals
(human plasma IgG samples). Considering that a typical adult human
body has 12 − 16 g/L of IgG24 in 5 L of blood, also considering that a
healthy individual can have »0.2% C-terminal amidation variant, an
individual can have 120 − 160 mg of C-terminal amidation variant
circulating in their body constantly. If a human body is constantly
exposed to »140 mg of the variant for their entire life without a
safety problem, a relatively small amount of administrated IgG mAb
(usually ≤ 1000 mg/dose) containing a small amount (≤ 140 mg) of
the variant logically should not be a safety concern. The endogenous
and continuous exposure of this variant in humans suggests that the
human body is likely to have high tolerance to this variant and not
see it as foreign to the human immune system. We therefore con-
clude that small amount of C-terminal amidation variant in therapeu-
tic IgG does not pose a safety concern.
As also shown in Table 1, endogenous human IgGs purified from
normal plasma contain trace level of unprocessed C-terminal lysine
(≤ 0.03%), consistent with a previous finding that IgGs rapidly lose
their C-terminal lysine residues in serum.9
IgGs expressed from murine cells contain no C-terminal amidation but
large amount of unprocessed C-terminal lysine
Another interesting observation is that none of the eight IgG mAbs
expressed from murine cell lines exhibit significant amount of C-
2450 B. Shah et al. / Journal of Pharmaceutical Sciences 111 (2022) 2445−2450
terminal amidation. Although the PAM gene is present in the mouse
genome, it is likely not expressed in murine cell lines used for the
mAb production. The lack of C-terminal amidation in IgG products
expressed from murine cell lines poses a challenge to the biosimilar
development of these products using CHO cell lines. This work dem-
onstrates that the small amount of C-terminal amidation present in
CHO-expressed IgG mAbs does not pose a safety concern. In addition,
the variant generally has very low abundance in CHO-expressed
product, and has very low likelihood to affect efficacy and clear-
ance.25 IgGs expressed from murine cells also exhibit much higher
amount of unprocessed C-terminal lysine (7% - 60%, Table 1) than
those expressed in CHO cells. Evidence shown in the literature sug-
gests that the unprocessed C-terminal lysine has low potential
impact on safety and efficacy, due to its rapid processing in vivo,9 and
similarity in potency, pharmacokinetic profile, and effector func-
tions.25-28 It is important to point out that the levels of unprocessed
C-terminal lysine reported in Table 1 are likely overestimated due to
the higher ionization efficiency of the lysine-containing peptide.29 C-
terminal amidation, on the other hand, does not have this bias due to
its similar basicity as the most abundant desK peptide.
Conclusion
Data presented in this work demonstrated that Fc C-terminal ami-
dation is a common variant occurring in IgG of all four subclasses. The
variant is generally present in IgG mAbs expressed from CHO cell
lines but not in IgG mAbs expressed from murine cell lines. Neverthe-
less, the presence of significant amount of C-terminal amidation in
endogenous human IgGs indicates that small amount of the variant
in therapeutic IgGs does not pose a safety concern. This conclusion is
based on low levels of C-terminal amidation typically detected in bio-
therapeutic mAbs. Further studies are needed to assess the potential
impact of much higher levels of C-terminal amidation on safety and
efficacy.
Declaration of Interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
The authors thank Kate Hutterer, Jennifer Liu and Anna Ip for ini-
tial discussions and help in procuring samples for various therapeutic
IgG mAb products. This work is supported by Amgen Inc.
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