Journal of Pharmaceutical Sciences 110 (2021) 3811−3818

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Journal of Pharmaceutical Sciences

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

Pharmaceutical Biotechnology

A Highly Sensitive LC-MS/MS Method for Targeted Quantitation of

Lipase Host Cell Proteins in Biotherapeutics
Yunqiu Chena,*, Chong-Feng Xua, Bradley Stanleya, Greg Evangelista,
Alex Brinkmanna, Suli Liua, Sean McCarthyb, Lei Xiongb, Elliott Jonesb, Zoran Sosica,
Bernice Yeunga
a
Biogen, Cambridge, MA, USA
b
SCIEX, Redwood City, California, USA

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

Article history: Identification and accurate quantitation of host cell proteins (HCPs) in biotherapeutic drugs has become
Received 4 May 2021 increasingly important due to the negative impact of certain HCPs on the safety, stability, and other product
Revised 23 August 2021 quality of biotherapeutics. Recently, several lipase HCPs have been identified to potentially cause the enzy-
Accepted 23 August 2021
matic degradation of polysorbate, a widely used excipient in the formulation of biotherapeutics, which can
Available online 28 August 2021
severely impact the stability and product quality of drug products. In this study, we identified three lipase
Edited by Editor Name: Dr K Audus. HCPs that were frequently detected in Chinese hamster ovary (CHO) cell cultures using shotgun proteomics,
including phospholipase B-like 2 (PLBL2), lipoprotein lipase (LPL), and lysosomal acid lipase (LIPA). A targeted
Key Words:
quantitation method for these three lipase HCPs was developed utilizing liquid chromatography coupled
Biopharmaceutical characterization
with tandem mass spectrometry (LC-MS/MS) with high-resolution multiple-reaction-monitoring (MRMhr)
Monoclonal antibody(s)
quantitation. The method demonstrated good sensitivity with low limit of quantitation (LLOQ) around
Liquid Chromatography-Mass Spectrometry
(LC-MS) 1 ng/mL, and linear dynamic range of three orders of magnitude for the three lipase HCPs. It has been applied
Mass spectrometry for the characterization of process intermediates from various in-house monoclonal antibody (mAb) produc-
Proteomic tion. In addition, the method has also been used to evaluate the robustness of clearance for one of the lipase
HCPs, PLBL2, under different column purification process conditions.
© 2021 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/)

Introduction
Host cell proteins (HCPs), the low-level protein impurities derived
from the host cell line that co-purify with protein therapeutics, must
be sufficiently and robustly removed from the final drug product to
an acceptable level. Residual HCPs can have detrimental effects on
biopharmaceutical products, including unwanted immunogenic
effects in patients as well as an impact on stability and efficacy of
drugs.1−2 For example, recently, it has been demonstrated that cer-
tain lipase HCPs can be the major cause for enzymatic degradation of
polysorbate 20 and 80, which are common surfactants present in for-
mulations of biotherapeutics.3−5 Polysorbate 20 and 80 are used in
formulations for both preventing surface adsorption and as stabil-
izers against protein aggregation.6,7 Enzymatic degradation of poly-
sorbate 20 and 80 through hydrolysis of ester bond can result in the
formation of degraded polysorbates and free long-chain fatty acids,
* Corresponding author at: Biogen Inc, 225 Binney Street, Cambridge, MA 02142,
United States.
E-mail address: rachel.chen@biogen.com (Y. Chen).
where the latter in turn can form insoluble particles that affect the
stability and product quality of biotherapeutics.8−10
Several lipase HCPs have been previously identified to cause poly-
sorbate degradation in biotherapeutic formulations, including phos-
pholipase B-like 2 (PLBL2),4 lipoprotein lipase (LPL),3 Group XV
lysosomal phospholipase A2 Isomer X1 (LPLA2),5 phospholipase A2
group VII (PLA2G7),11 and liver carboxylesterase,12 among others. In
addition, there could be other HCPs that potentially possess the lipase
activities that contribute to the degradation of polysorbates, but their
functions are yet to be identified or confirmed.13 These lipase HCPs
are found to often co-purify with many monoclonal antibodies
(mAbs) and other biotherapeutics produced in Chinese hamster
ovary (CHO) cells.14,15 As a result, they can affect drug substance sta-
bility and need to be removed from the drug substance to acceptable
levels. It has been demonstrated that lipases present as low as less
than 1 ppm can cause significant degradation of polysorbates.5
The total amount of HCP is mostly measured by an enzyme-linked
immunosorbent assay (ELISA) method, which is regarded as an
industry “gold standard” method for the total HCP quantitation for
in-process testing and product release.16,17 Though a high-

https://doi.org/10.1016/j.xphs.2021.08.024
0022-3549/© 2021 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/)
3812 Y. Chen et al. / Journal of Pharmaceutical Sciences 110 (2021) 3811−3818

throughput, sensitive and robust method, ELISA-based HCP quantita-
tion is highly dependent on the HCP coverage of the capture antibod-
ies and can miss the detection of certain HCP species.16−18 More
recently, an orthogonal liquid chromatography coupled with a tan-
dem mass spectrometry (LC-MS/MS)-based proteomics method has
been increasingly utilized in the profiling of HCP population and
abundance during process development and characterization.19−21
The proteomics-based method has the advantage of both identifying
individual HCP species and providing relative quantitation of their
abundance. Since the identification and quantitation can be per-
formed simultaneously on thousands of HCP species, the proteomics-
based method also has the benefit of providing information on
critical HCPs. However, despite continuous improvement on the rela-
tive quantitation capability of the shotgun proteomics, it remains a
challenge to accurately quantify individual HCPs, especially HCPs at
extremely low levels (<10 ppm), in a fast and high-throughput
manner.22,23
Due to the criticality and high risk of the lipase HCPs, several tar-
geted quantitation methods have been developed to specifically mea-
sure the level of lipase HCPs in process and drug substance samples.
These include lipase activity assays that measure the hydrolysis rate
of lipid-mimic substrates with fluorescent or colorimetric detec-
tion,24 immuno-assays including ELISA and Western blot methods
that target specific lipases,25 activity-based protein profiling (ABPP)
approach for serine hydrolase enrichment and identification,11 as
well as LC-MRM (multi-reaction monitoring)-based targeted quanti-
tation methods.3,5,12,25 The LC-MRM-based method has long been
used as method of choice for selective, sensitive and accurate quanti-
tation to support pharmacokinetic assessment of small molecules
and biotherapeutics. Recently, Gao et al. has demonstrated the sensi-
tivity, robustness and accuracy of an LC-MRM-based quantitation
method for two lipase HCPs, PLBL2 and LPLA2, and that the results
were comparable to the ELISA-based method.25
In this work, a high-resolution LC-MRM (LC-MRMhr) based tar-
geted quantitation method was developed for the quantitation of
three potentially high-risk lipase HCPs, including PLBL2, LPL and
lysosomal acid lipase (LIPA), which have been detected by a shot-
gun proteomics method in multiple CHO-based mAb processes.
With synthesized peptides used as quantitation standards and
heavy-isotope-labeled peptides as internal standards, the method
demonstrated sensitivity around 1 ng/mL and dynamic range
across three orders of magnitude. The reproducibility and accuracy
of the method were also thoroughly evaluated. The method has
been applied in process development and characterization for the
monitoring of lipase clearance in downstream processes, as well
as to demonstrate the robustness of lipase HCP clearance in a
spike-in study.
Material and Methods
Materials
The peptide standards used for lipase quantitation were synthe-
sized from New England Peptide (Gardner, MA, USA). A total of 18
peptides were synthesized, including nine “light” peptides (not
labeled) and their heavy-isotope-labeled (13C6,15N2-Lysine or
13
C6,15N4-Arginine) counterparts, which serve as internal standards
(IS) (Table 1). The recombinant PLBL2 protein was obtained from
MyBiosource, Inc. (San Diego, CA, USA). Urea, Tris, dithiothreitol
(DTT), and iodoacetamide (IAM) were from Sigma-Aldrich (St. Louis,
MO, USA). Trypsin/Lys-C mix (mass spec grade) was from Promega
(Madison, WI, USA). Trifluoroacetic acid (TFA), formic acid (FA), water
and acetonitrile were LC-MS grade and from Thermo Fischer Scien-
tific (Waltham, MA, USA). The protein drug substance and process
intermediate samples, including cell culture harvest sample to be
loaded onto Protein A affinity column (ProA load), Protein A column
purified product (ProA eluate), the purified product from Column 2
and Column 3, were produced at Biogen.
Sample Digestion
Samples (»300 mg therapeutic proteins) were denatured and
reduced by adding 75 mL of 8 M urea, 5 mL of 1 M Tris, pH 8.0, and
1 mL of 500 mM DTT to a final volume of 100 mL. They were incu-
bated at room temperature (RT) for about 30 min. After alkylation by
adding 8 mL of 200 mM IAM and incubation at RT for 30 min in the
dark, the samples were diluted 6-fold by adding 492 mL of 50 mM
Tris (pH 8.0), and digested with 20 mg Trypsin/Lys-C mix overnight
at 37°C. Six mL of TFA was added to 0.1% (v/v) to stop the digestion. A
mAb drug substance (mAb-A) was also prepared in the same way
and used as matrix for standard curve.

Table 1
Three lipase HCPs Identified From Shotgun Proteomics Analysis, and the Optimized MRMhr Transitions Used in the LC-MS/MS Quantitation Method.

Protein Size (kDa) Peptide Retention Peptide Sequence MRM Transition Collision Energy (eV)
Number Time (min)
Precursor(charge) Product Ion (fragment type)

lipoprotein lipase (LPL) 52.8 1 11.1 GLGDVDQLVK 522.29 (2+) 873.46 (y8) 23
GLGDVDQLVK* (IS) 526.30 (2+) 881.48 (y8)
2 17.8 LSPDDADFVDVLHTFTR 649.99 (3+) 661.34 (y5) 35
LSPDDADFVDVLHTFTR* (IS) 653.32 (3+) 671.35 (y5)
3 16.7 SIHLFIDSLLNEENPSK 652.67 (3+) 817.36 (y7) 30
SIHLFIDSLLNEENPSK* (IS) 655.35 (3+) 825.38 (y7)
lysosomal acid lipase/ 45.6 4 18.8 WPEVIIEDLFGHK 528.28 (3+) 698.87 (y122+) 25
cholesteryl ester WPEVIIEDLFGHK* (IS) 530.95 (3+) 702.88 (y122+)
hydrolase-like (LIPA) 5 7.3 VNVYTSHSPAGTSVQNLR 643.99 (3+) 858.94 (y162+) 27
VNVYTSHSPAGTSVQNLR* (IS) 647.33 (3+) 863.94 (y162+)
6 7.2 GNTWSLK 403.22 (2+) 533.31 (y4) 20
GNTWSLK* (IS) 407.22 (2+) 541.32 (y4)
putative phospholipase 65.4 7 12.5 LTLLQLK 414.78 (2+) 614.43 (y5) 20
B-like 2 (PLBL2) LTLLQLK* (IS) 418.79 (2+) 622.44 (y5)
8 11.2 SVLLDAASGQLR 615.34 (2+) 817.41 (y8) 30
SVLLDAASGQLR* (IS) 620.35 (2+) 827.42 (y8)
9 8.4 AFIPNGPSPGSR 600.31 (2+) 868.41 (y9) 25
AFIPNGPSPGSR* (IS) 605.32 (2+) 878.43 (y9)
* heavy-isotope-labeled peptides (13C6,15N2-Lysine or 13C6,15N4-Arginine) used as internal standards
 Y. Chen et al. / Journal of Pharmaceutical Sciences 110 (2021) 3811−3818
Shotgun Proteomics for HCP Identification
Six hundred mg of samples were spiked with 0.1 mg (»167 ppm)
of yeast enolase, which serves as an internal standard for label-free
quantitation. The samples were digested in a similar way as described
in the above section. After digestion, 185 mL of the digest was puri-
fied using Bravo desalting RPS cartridges (Agilent AssayMap). The
desalted samples were speed vacuumed to dryness and re-consti-
tuted in 0.1% (v/v) formic acid. Twenty mL of sample (50 mg) was
loaded for LC-MS analysis using an Orbitrap Fusion mass spectrome-
ter (Thermo Scientific, MA, US) and the LC separations were per-
formed using a Waters Acquity CSH column (1.8 mm, 2.1 mm £ 15
cm) using 0.1% (v/v) formic acid buffers containing water and aceto-
nitrile. The LC gradient started with a 5 min hold at 2% B, a 85 min
gradient elution from 3% to 25% B, ramping to 35% B within 20 min,
followed by washing at 80% B for 5 min and re-equilibration for
10 min. Electrospray ionization (ESI) parameters on the Orbitrap
mass spectrometer included spray voltage at 3.5 kV, ion transfer tube
temperature at 325°C, vaporizer temperature at 275°C, sheath gas
and auxiliary gas at 35 and 10 arbitrary unit, respectively. The mass
spectrometer detection included a full scan in the orbitrap (m/z 300
to 1500) with resolution at 120k and data-dependent MS2 scans on
the top abundant ions within a cycle time of 1 s. Precursor ions were
fragmented using HCD at 25% collision energy and detected in the
ion trap. The LC-MS raw files were searched against a custom data-
base that comprises the CHO proteome database (CHO_refseq down-
loaded from https://www.chogenome.org/) and the sequences of
yeast enolase and protein therapeutics, using the Sequest search
engine with the Proteome Discoverer software (ver. 2.1, Thermo Sci-
entific). The HCPs were quantitated by a “Hi3” label-free method,
where the top-three most abundant peptides from each HCP were
used for quantitation. The peak areas of HCPs were compared with
the spiked-in yeast enolase and an estimated ppm value was calcu-
lated.
Internal Standard and Standard Curve Preparation
Nine heavy-isotope-labeled peptides were dissolved in
50:50 ACN/water and mixed to a final concentration of 5 mM as the
IS stock solution. The nine light peptides were prepared in a similar
way to a 5 mM standard stock solution. The IS stock was further
diluted to 100 nM using matrix digest during sample preparation.
After sample digestion, 6 mL of IS solution (100 nM) was spiked in to
600 mL of sample digest or matrix digest to achieve 1 nM final con-
centration. The standard curve was prepared by spiking light peptide
standard stock into the digested matrix using serial dilution to the
following concentrations: 6.25, 12.5, 25, 50, 100, 250, 500, 1000,
2500 pM. The QC peptides were prepared from a separate stock with
serial dilution to the following concentrations: 16, 80, 400 and
2000 pM.
Targeted Lipase LC-MS/MS Analysis
The digested samples were either directly injected into LC-MS or
purified using an Agilent Bravo desalting RPS cartridge before injec-
tion. For the desalting step, 185 mL of the digest was purified using
RPS cartridges, speed vacuumed to dryness and re-constituted in
56 mL of 0.1% (v/v) formic acid, which equated to 3.3-fold concentra-
tion of the sample. Twenty mL of samples or standards, containing
10 mg (direct injection) or 33 mg (Bravo-desalted) protein, were
loaded for analysis on a TripleTOFÒ 6600 mass spectrometer (SCIEX,
CA, USA) coupled with a Shimadzu HPLC system. Both standards and
samples are analyzed in duplicate. LC separation was performed on a


Waters Acquity CSH C18 column (2.1 £ 100 mm, 1.7 mm, 130 A)
using 0.2 mL/min flow rate and column temperature at 55°C. Mobile
3813
phases consisted of 0.1% (v/v) formic acid in water (A) and in acetoni-
trile (B). The LC gradient starts with 2% B for 2 min, then increases to
10% B within 1 min, ramping up to 35% B within 19 min, followed by
1 min wash step at 80% B and re-equilibration at 2% B for 10 min. The
LC eluate before 4 min and after 20 min was diverted to waste. The
mass spectrometer is operated in positive mode using a DuoSprayTM
Turbo V Ion Source with the following parameters: curtain gas at 30;
ion source gas 1 and 2 at 50; temperature at 600°C; ion spray voltage
at 5500 eV. The mass spectrometry acquisition starts with a full scan
in the TripleTOFÒ 6600 System with 0.1 s acquisition time, followed
by “Product Ion” scans with 200 ms acquisition time using high sensi-
tivity mode, with “enhance mass” around the m/z of the targeted
product ion (detailed in the Results and Discussion section). The MS/
MS acquisitions were scheduled to occur within three LC time peri-
ods: 4 to 10 min; 10 to 15 min; 15 to 20 min. MRMhr transitions
were multiplexed according to the peptide elution time, and three
pairs of peptides (3 light and 3 heavy-labeled) are scheduled within
each LC time period. The data was processed by extracting the tar-
geted product ions using 0.1 Da mass window using MultiQuantTM
Software 3.0 (Sciex, CA). Calibration curves were calculated based on
the linear regression of peak area ratio (peak area of analyte divided
by IS) against the analyte concentration, applying a weighting factor
of 1/x. Assuming a complete digest, the obtained peptide concentra-
tion can be used to calculate the lipase HCP concentration using the
following equation:
HCP concentration ðg=LÞ ¼ peptide concentrationðmol=LÞ  size of
HCP ðg=molÞ
The relative content of HCP compared with mAb protein
(expressed as ng/mg or ppm) can be further calculated by dividing
the HCP concentration (ng/mL) by the mAb concentration (mg/mL) in
the digest.
Results
Identification of Lipase HCPs Using Shotgun Proteomics
To identify the lipase HCPs present in the in-house mAb processes
using the CHO host, a shotgun proteomics characterization was per-
formed on cell culture harvest and Protein A column eluate samples
from multiple in-house produced mAbs. The data was searched
against the CHO proteome database. Several HCPs with potential
enzymatic activities were identified and three lipase-related HCPs
were identified from multiple mAb samples, including lipoprotein
lipase (LPL), lysosomal acid lipase/cholesteryl ester hydrolase-like
(LIPA) and putative phospholipase B-like 2 (PLBL2), as shown in
Table 1. These three lipase HCPs are commonly present in multiple
mAb cell culture samples. PLBL2 and LPL have previously been
reported to be associated with polysorbate degradation,3−4 and LIPA
was recently identified from an activity-based protein profiling
(ABPP) study as an active hydrolytic enzyme that may cause polysor-
bate degradation.11 Thus we decided to develop a platform, MRM-
based quantitation method for the three lipase HCPs in order to
achieve better sensitivity of detection and higher throughput to sup-
port process development.
LC-MRMhr Method Development for Lipase HCP Quantitation
From the list of peptides identified in the shotgun proteomics
experiment, three tryptic peptides from each lipase HCP were
selected for targeted quantitation (Table 1). These peptides were
selected as they had high MS signal intensity, low propensity to post-
translational modifications and no mis-cleavages. These peptides
were also confirmed to be unique peptides for each HCP protein in
the CHO proteome through database searching by Blastp. To achieve
the purpose of absolute quantitation, the selected peptides were
3814 Y. Chen et al. / Journal of Pharmaceutical Sciences 110 (2021) 3811−3818
Fig. 1. A scheme of scheduled MRM. The LC-MS time is divided into three periods and
MRM scans of the 9 peptides are scheduled according to their elution time. (A) Total
ion chromatogram (TIC) of the matrix digest spiked with 10 nM synthetic peptides. (B)
Extracted ion chromatograms (XICs) of the nine peptides with peaks labeled by pep-
tide number.
chemically synthesized along with their heavy-isotope-labeled coun-
terparts (13C6,15N2-Lysine or 13C6,15N4-Arginine labeled), which are
used as quantitation standards and internal standards, respectively.
We chose to use synthetic tryptic peptides as standards to develop
the method, as many HCPs are not commercially available as purified
recombinant proteins and attempting to purify individual HCPs in-
house can be resource-intensive, especially when trying to produce
and characterize the heavy-isotope labeled HCP proteins. Synthetic
peptides, on the other hand, can be easily designed and synthesized
to achieve a quick development timeline.
An LC-MS/MS-based MRMhr quantitation workflow was devel-
oped for the nine tryptic peptides on a TripleTOFÒ mass spectrometer
coupled with reversed phase LC separation. The MRM transitions
were optimized for the nine pairs of peptides, including 9 target pep-
tides and their heavy-isotope labeled counterparts, as shown in
Table 1. We optimized the choice of precursor and product ions, as
well as the collision energy to achieve the optimal signal intensity
and selectivity. A scheduled MRM strategy was applied, where the
20-min LC gradient was divided into three 5-min windows and pepti-
des are monitored only during their expected elution windows
(Fig. 1). The scheduled MRM was used to accommodate multiplexing
of peptides and maintain adequate scan time to ensure reproducibil-
ity of peak integration and quantitation.
This LC-MRMhr workflow utilizes a hybrid triple quadrupole
time-of-flight (TOF) mass spectrometer, where the peptide precur-
sors were selected in the triple quadrupole and product ions are
detected in the TOF analyzer with high mass accuracy. While the MS/
MS scans are acquired in full scan mode in the TOF system, one prod-
uct ion from each scan was selected for monitoring using the
“enhancement” function of the tripleTOF instrument. With enhance-
ment mode, ions are briefly trapped in the collision cell and released
as a packet. The release of trapped ions is timed so that the m/z of
interest arrives in the accelerator precisely in time to be pushed into
the TOF. In this way it is possible to increase the duty cycle of a partic-
ular m/z to close to 100%, which in turn increases the signal intensi-
ties of product ion and boosts method sensitivity. We found the
enhancement function increased the signal intensities of the selected
peptides by 2- to 4-fold across the concentration range from 50 pM
to 10 nM (Supplementary figure 1). At the same time, the high
resolving power of the TOF analyzer can minimize the background
interference from matrix and allow quantitation based on a single
isotopic ion.
Matrix selection is very important for the LC-MRM method devel-
opment, as the selected matrix used for standard curve should
represent the samples to be analyzed and contain minimal interfer-
ence with quantitation. We screened several in-house and commer-
cially available purified mAbs and found that an in-house mAb-A
drug substance provided a relatively clean matrix with lowest back-
ground lipase detected. The mAb-A digestion matrix showed no
interference with peptide 5 and peptide 7 in the extracted ion chro-
matograms (XICs). It exhibited a background signal at around 10% of
LLOQ level for peptide 1, thus the impact on the quantitation is
deemed minimal (Supplementary Figure 2).
The MRMhr-based quantitation method demonstrated good sen-
sitivity for the selected lipase peptides. The low limit-of-quantitation
(LLOQ) of the nine selected peptides range from 6.25 pM to 100 pM,
as shown in Table 2. The peptide with the best sensitivity and optimal
linear range was chosen as the quantifier peptide for each lipase, i.e.,
peptide 1 (GLGDVDQLVK) for LPL, peptide 5 (VNVYTSH-
SPAGTSVQNLR) for LIPA, and peptide 7 (LTLLQLK) for PLBL2. The
other peptides were also monitored simultaneously to provide addi-
tional confirmation of the quantitation results. The LLOQ for the
quantifying peptides was determined to be 12.5 pM for peptide 1
(LPL), 25 pM for peptide 5 (LIPA), and 6.25 pM for peptide 7 (PLBL2).
The signal-to-noise (S/N) ratio for the quantifying peptides is at least
10 at the LLOQ level, as shown in Supplemental Figure 2. The assay
showed a linear dynamic range around three orders of magnitude for
all three lipases, with regression coefficient (r) above 0.99 (Fig. 2). A
detailed examination of the standard curve demonstrated that the
measured concentrations of peptide standards are within 80-115% of
the theoretical values for peptide concentration above LLOQ, and
within 130% accuracy at the LLOQ level, with variation less than 20%
among triplicate analysis as shown in Supplementary Table 1.
The LC-MRM method was evaluated for its intra-run and inter-run
reproducibility and quantitation accuracy using QC peptides as well
as a mAb process intermediate sample. QC peptides were prepared at
four different levels, including 16, 80, 400 and 2000 pM to cover a
wide concentration range. They were prepared for two separate runs,
with three replicates for Run 1 and five replicates for Run 2. As shown
in Supplementary Table 2, for peptide 1 (LPL), the accuracy of the QC
peptides was within 75%-110% for the intra-run, and 80%-105% for
inter-run. The precision was within 7% coefficient of variation (CV)
both intra-run and inter-run for peptide concentration 80-2000 pM,
with higher variation within 25% CV for QC peptide at 16 pM. For
peptide 7 (PLBL2), the accuracy of the QC peptides was within 75-
115% for the intra-run, and 80%-105% for inter-run. The precision
was within 15% CV intra-run and inter-run for peptide concentration
80-2000 pM, while the precision for 16 pM level was within 25% CV.
For peptide 5 (LIPA), the accuracy of the QC peptides was within 75-
115% for the intra-run, and 75%-105% for inter-run. The precision
was within 15% CV intra-run and inter-run for peptide concentration
80-2000 pM. In addition to QC peptides, to assess the reproducibility
of sample preparation including enzyme digestion, a mAb process
intermediate sample was digested and analyzed in two different
runs, each with six separate preparations. The quantitation results of
peptide 1 (LPL), peptide 7 (PLBL2), and peptide 5 (LIPA) are 915 pM,
174 pM, and 108 pM, respectively. The intra-run variation is within
20% CV (n = 6), and the inter-run variability is with 15% for peptide 1
and 7, and 24% for peptide 5 (n = 12) (Supplementary Table 3).
The lipase HCP concentration in the sample can be calculated
based on the peptide concentration and the size of HCP, assuming
a complete digest of HCP. Based on the size of three lipase HCPs
(Table 1), the LLOQ of the lipase HCPs that can be quantified by
the LC-MRM method is estimated to be 0.7 ng/mL for LPL,
0.4 ng/mL for PLBL2, and 1.1 ng/mL for LIPA, as shown in Table 2.
In HCP analysis, the relative content of HCP compared with the
therapeutic protein, expressed as ng HCP per mg of mAb (ng/mg,
or ppm), is very critical for monitoring HCP clearance during pro-
cess development and for risk assessment of human exposure to
 Y. Chen et al. / Journal of Pharmaceutical Sciences 110 (2021) 3811−3818
Table 2
Low Limit of Quantitation (LLOQ) and Linear Range of the Lipase HCP Quantitation Method.
Protein Peptide Number Peptide LLOQ (pM)
a
Lipoprotein lipase (LPL) 1 12.5
2 25
3 50
Lysosomal acid lipase (LIPA) 4 100
5a 25
6 25
putative phospholipase B-like 2 (PLBL2) 7a 6.25
8 12.5
9 25
a
Peptides denoted are quantifiers for each HCP.
b
LLOQ of HCP is calculated based on peptide LLOQ and the size of HCP, assuming a complete digestion.
Fig. 2. Standard curves for (A) peptide 1 (GLGDVDQLVK), (B) peptide 5 (VNVYTSHSPAGTSVQNLR) and (C) peptide 7 (LTLLQLK) quantitation. The linear regression (using 1/x weight-
ing) and the R values are shown.
HCP. We further calculated the relative concentration of lipase
HCPs compared with mAb in the digested sample. In the LC-MRM
method, the digested sample contains »0.5 mg/mL of mAb; it can
be further concentrated using a desalting cartridge by 3.3-fold
resulting in »1.67 mg/mL of mAb in the digested sample. For the
direct injection runs, the calculated LLOQ of the three lipase HCPs
are between 0.8 to 2.2 ppm, and for the runs with sample pre-con-
centration, the LLOQ can reach <1 ppm level (supplementary
Table 4). We have examined that the standard curve for the direct
injection runs and concentrated sample runs have no significant
difference in terms of LLOQ and dynamic range. The peptide at
LLOQ level can be reliably detected with sufficient S/N ratio with-
out being affected by the concentration of matrix.
The accuracy of the method was also assessed by spiking known
amounts of recombinant PLBL2 into a mAb drug substance at four
levels, including 1, 5, 10, and 100 ppm, before digestion and the LC-
MS/MS analysis. The quantitation results of PLBL2 based on peptide 7
is shown in Table 3. The accuracy falls between 100% and 121% for
the spike-in range between 1 ppm and 100 ppm, and the %CV calcu-
lated based on triplicate analysis are generally within 15%. It is worth
mentioning that the quantitation results obtained from the other two
peptides from PLBL2, peptide 8 and 9, are highly consistent with
quantitation by peptide 7 without a significant peptide-to-peptide
bias (Supplementary Table 5). This spike-in experiment demon-
strated that the method could provide accurate quantitation of PLBL2
lipase HCP for mAb drug substance samples across a wide concentra-
tion range. Due to limited commercial availability, the quantitation
accuracy for the other two lipase HCPs were not assessed in this
study.
3815
Linear Range (pM) Calculated LLOQ of HCP (ng/mL)b
12.5-2500
25-2500 0.7
50-2500
100-2500
25-2500 1.1
25-2500
6.25-2500
12.5-2500 0.4
25-1000
Analysis of Process Intermediate Samples to Understand Lipase HCP
Clearance
The targeted HCP quantitation method was applied to monitor the
abundance of lipase HCPs during each step of the downstream purifi-
cation process for several in-house produced mAbs. The mAb purifi-
cation process typically consists of three column steps, including the
Protein A affinity column and two additional polishing columns. In-
process samples, including Protein A column load and eluate, the
products from second and third columns, were analyzed by the LC-
MRMhr method for lipase HCP monitoring. The quantitation results
for process intermediates from four monoclonal antibodies, including
two IgG1 mAbs (A and B), one IgG1/IgG4 hybrid mAb (C) and one
IgG4 mAb (D) are shown in Fig. 3.
The quantitation results showed that the 3-column process in
general demonstrated the capability of effectively removing the three
lipase HCPs to a level lower or close to LLOQ for the four mAbs used in
the analysis. However, a close examination of the lipase clearance
revealed that, for different mAbs and processes, the starting abun-
dance of lipase HCPs and the clearance at each column step can be
highly divergent. For example, PLBL2 was detected with an abun-
dance ranging from 200 to 800 ppm in the Protein A column load,
and its abundance in the Protein A column eluate was detected any-
where between <1 ppm to more than 100 ppm for the four mAbs.
For mAb A and mAb B (both IgG1 mAbs), the level of PLBL2 was
detected at lower than 5 ppm in the Protein A column eluate, which
can be translated to approximately 100-fold reduction by the Protein
A column purification. On the other hand, PLBL2 was present at »100
and »20 ppm for mAb C and mAb D after Protein A column. After the
3816 Y. Chen et al. / Journal of Pharmaceutical Sciences 110 (2021) 3811−3818

Table 3
Accuracy of quantitation demonstrated by recombinant PLBL2 spike-in study.
Shown are the quantitation results based on peptide 7. Digested samples were pre-
concentrated using a desalting cartridge before injection into LC-MS analysis. Each
spiked sample was analyzed in triplicate with %CV shown.

PLBL2 Measured Calculated %CV (N = 3) %Accuracy

spike-in peptide PLBL2
amount (ppm) 7 conc. (pM) amount (ppm)

1 26.3 1.0 6% 103%

5 155.5 6.1 12% 121%
10 289.9 11.3 4% 113%
100 3004.9 117.0 6% 117%

Column-2, PLBL2 was cleared to <LLOQ level for mAb C, while for
mAb D (IgG4), PLBL2 was still present at »7 ppm. It was further
removed by Column-3 down to a level that is below LLOQ. It is recog-
nized that the process conditions for the four mAbs, such as wash
buffer, pH, loading, etc., are different, and these parameters will affect
individual HCP clearance. Nevertheless, this data suggested that
PLBL2 may possess a slightly different affinity in terms of protein-
protein interactions to mAb C and mAb D as compared to mAb A and
B, which could possibly explain its different clearance pattern during
the purification process. Indeed, the similar observations were
reported by Tran et al. that PLBL2 binds more strongly to IgG4 mAbs
due to protein-protein interactions.26
LPL and LIPA also showed a molecule- and process-dependent
clearance during the purification. LPL was detected in the Protein A
column load between 200 to 1200 ppm from the four mAbs. After
Protein A column, its abundance was reduced to anywhere between
3 to 100 ppm in the Protein A column eluate, and was further cleared
down to close to 1 ppm level after Column-2 and Column-3. On the
other hand, LIPA was detected all below 20 ppm in the Protein A col-
umn eluates and was further removed to below LLOQ after the Col-
umn-2 for the four mAbs.

Robustness of PLBL2 Clearance

Fig. 3. Concentrations of (A) PLBL2, (B) LPL and (C) LIPA in process intermediate sam-
To further understand the robustness of lipase HCP clearance by ples from four mAbs: mAb A and mAb B (IgG1), mAb C (IgG1/IgG4 hybrid) and mAb D
the 3-column process, we carried out a lipase clearance “challenging (IgG4). Samples with HCP level below LLOQ are labeled as “*”.

study,” where recombinant PLBL2 was spiked into process intermedi-

ates, including the Column-2 load and Column-3 load. This spike-in
study was intended to create some worst-case scenarios where the
lipase HCPs are present at abnormally high concentration, so that the
downstream process can be evaluated for its robustness to success-
fully clear the HCPs. PLBL2 is one of the best-studied lipase HCPs with
commercial availability and, therefore, was chosen for the robustness
study.
As shown in Fig. 3, PLBL2 was present at very low level (»1 ppm)
after Protein A column purification for mAb A and was further
reduced to <0.2 ppm after the Column-2 purification. To understand
the robustness of the Column-2 process for PLBL2 clearance, recombi-
nant PLBL2 was spiked into the Column-2 load of mAb A to a final
concentration of 74 ppm. After Column-2 purification, PLBL2 concen-
tration was measured to be around 17 ppm in the product (Fig. 4a).
The results indicated that in an extreme condition where PLBL2 is
present at higher-than-normal level in Protein A column eluate, a
second column alone does not have sufficient capacity to clear PLBL2
to a level comparable to the 3-column process for mAb A.
Column-3 is crucial for PLBL2 clearance in the downstream pro-
cess of a few mAbs such as mAb D. The spike-in study was carried out
on the Column-3 process, where recombinant PLBL2 was spiked into
the Column-3 load of mAb A (IgG1) and mAb D (IgG4) at two different
levels. In the lower spike-in level, PLBL2 concentration was measured
to be 80 ppm and 66 ppm, respectively, for mAb A and mAb D load;
after the Column-3 purification, PLBL2 was not detected above LLOQ
(0.8 ppm) for both antibodies. The Column-3 process was further
challenged with higher PLBL2 spike-in at around 500 and 300 ppm
for mAb A and mAb D, respectively. The measured PLBL2 concentra-
tion in the Column-3 product was still below the LLOQ in both cases
(Fig. 4b). This translated to more than 2 logs of reduction of PLBL2 by
the Column-3 process for the two antibodies studied, which demon-
strated that Column-3 is critical and has a strong capacity for PLBL2
clearance in mAb A and mAb D processes.
Discussion
In this study, we demonstrated a workflow starting with shotgun
proteomics to identify potentially high-risk lipase HCPs followed by
developing an LC-MRMhr-based method for the quantitation. We
evaluated the sensitivity, linearity, reproducibility and accuracy of
the lipase HCP quantitation method. The method can achieve sensi-
tivity of 6.25 to 25 pM for the three lipase HCPs, corresponding to
around 1 ng/mL of HCP in the digested sample. The method is sensi-
tive enough to detect lipase HCP present at 1-2 ppm level in a mAb
sample.
ELISA assay has been the gold standard assay for HCP analysis in
the industry owing to its superior specificity, sensitivity, and high
throughput. However, the ELISA assay can be limited by the availabil-
ity of reagents that recognizes specific HCP, hence the development
timeline can be much longer than for LC-MS/MS method. In addition,
 Y. Chen et al. / Journal of Pharmaceutical Sciences 110 (2021) 3811−3818
Fig. 4. PLBL2 clearance robustness study. PLBL2 concentration in column load (L) and product (P) was determined, where recombinant PLBL2 was spiked into (A) Column-2 load of
mAb A and (B) Column-3 load for mAb A and mAb D at different levels.
even when there are commercial HCP specific ELISA kits available,
not all of them are specific for CHO host and often times the cross-
species reactivity is unknown. The targeted LC-MS/MS method, on
the other hand, offers similar selectivity and sensitivity approaching
low-ppm to sub-ppm levels. It was recently demonstrated that quan-
titation results of process intermediate samples from LC-MRM
method and a PLBL2-specific ELISA method showed strong correla-
tion across a wide concentration range for different mAb molecules,25
indicating the two assays can provide comparable results for high-
risk HCP monitoring. There are several advantages of developing an
LC-MS/MS method for targeted HCP quantitation. First, an LC-MS/MS
method has the benefit of versatility that any HCPs that are detect-
able by mass spectrometry can be put through a similar development
process for a targeted quantitation assay. In addition,25 the use of
synthetic peptides as quantitation standards makes absolute quanti-
tation possible, and provides a tool for faster assay development in
response to newly identified HCPs when recombinant protein stand-
ards may not be available. Finally, the targeted LC-MS/MS can be mul-
tiplexed by incorporating multiple HCP targets into one assay. With
proper scheduling of the MS/MS events according to the retention
time of the target peptides, additional MRM transitions can be
included in a 20-min gradient, so that even more HCPs can be moni-
tored by a single method.
Compared with LC-MRM methods using low resolution mass
spectrometers, this study has demonstrated that a LC-MRMhr
method could achieve similar sensitivity, accuracy, and reproducibil-
ity. We found the enhancement function utilized in the LC-MRMhr
method played a critical role to improve the method sensitivity. Com-
paring the standard curves obtained by using enhancement vs. no-
enhancement in the method, the LLOQ was 2- to 10-fold higher if
enhancement was not used, and there was no significant change of
linear range comparing the two setups (Supplementary Table 6). In
addition, the high-resolution mass detection provided additional
level of specificity and selectivity due to the high mass accuracy. As
shown in Supplementary Figure 2, there are very minimal interfer-
ence with the target peptide in the detection window even at LLOQ
level. The use of heavy-isotope-labeled peptides as internal standards
further improves method specificity by facilitating accurate assign-
ment of target peaks, which sometimes can be difficult due to sample
low concentration and presence of interfering peaks. The use of IS
also minimizes potential run-to-run variations due to variance in
sample matrices and instrument performance, which helps improv-
ing method robustness. Finally, using a high-resolution mass spec-
trometry allows to perform both the HCP discovery by shot-gun
proteomics and targeted MRM method development on the same
instrument, which minimizes variability of peptide behavior between
different instrumentation.
3817
We also realize that the targeted HCP method may possess some
limitations. First, the quantitation accuracy can be potentially
affected by the matrix, especially early in-process samples with
higher HCP complexity, such as cell culture harvest samples. None-
theless, it is acknowledged that the accuracy of HCP quantitation is
more important in the drug substance and later process intermedi-
ates, as the residual HCPs will more likely affect product quality and
stability. In addition, the method could still provide relative trend
information of HCP abundance for earlier intermediates. One mitiga-
tion strategy we implemented is to ensure the digestion enzymes are
placed in a sufficiently high enzyme-to-sample ratio to ensure a
near-complete digestion, and to select peptides that are less prone to
mis-cleavage. An alternative to using peptide standards is to use
recombinantly purified HCP proteins as standards, which will com-
pensate for the digestion efficiency and recovery issues to a large
extent.25 However, this approach still cannot eliminate the matrix
effect and also faces issues as many HCPs are not commercially avail-
able, or the purity can be too low to serve as a standard. A third
approach that can potentially minimize matrix effect is to spike
heavy-isotope labeled peptides or proteins into samples, and the
quantitation can be achieved through comparing the signals of target
peptides with the heavy-isotope labeled peptides. This method, how-
ever, will require spiking-in heavy-isotope labeled peptides at a few
different levels to accommodate for the wide dynamic range of the
HCPs in different samples.
In this study, we have shown that the LC-MRMhr method can be
used as a convenient tool to quantify the three lipase HCPs in process
intermediate samples to support process development, where the
clearance of lipase HCPs can be monitored throughout the upstream
and downstream processes. In addition, we also performed a prelimi-
nary spike-in study to demonstrate the robustness of PLBL2 clearance
during various column steps and identified certain column steps and
parameters that are critical for the clearance of this lipase. We antici-
pate that this kind of clearance robustness study will be useful in
understanding HCP removal under different process conditions and
could facilitate decision-making in process development to allow the
most robust HCP clearance.
Conclusions
There has been an ever-increasing interest and urgent need to iden-
tify and quantify HCP abundance, especially high-risk HCP species that
can have a negative impact on product quality, safety, and stability dur-
ing the development and manufacturing of biological therapeutics.
Some problematic HCPs require very sensitive quantitation methods
and differentiation with other HCPs, such as lipases, as even trace
amounts of such HCPs can cause significant product quality and
3818 Y. Chen et al. / Journal of Pharmaceutical Sciences 110 (2021) 3811−3818
stability issues. In this study, we demonstrate that an LC-MRMhr-based
lipase HCP quantitation method can be developed with a streamlined
protocol within a short timeline, with relative ease in securing suitable
standards. The LC-MRMhr method can be used in both early-stage pro-
cess development to guide HCP removal as well as in late-stage process
characterization. We anticipate that the LC-MRMhr quantitation
method could complement the proteomics-based HCP profiling
method by providing more sensitive and accurate quantitation for cer-
tain high-risk, problematic HCP species. The current method can be
further augmented to include other HCP species and used either as a
platform or molecule-specific method for HCP monitoring.
Acknowledgements
The authors are grateful of Christopher Barton for great discussion
and suggestions.
Supplementary Materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.xphs.2021.08.024.
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