U.S. patent application number 17/351896 was filed with the patent office on 2022-01-13 for heavy peptide approach to accurately measure unprocessed c-terminal lysine in antibodies.
The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Milos Cejkov, Tyler Greer, Ning Li, Reid O'Brien Johnson, Xiaojing Zheng.
Application Number | 20220011318 17/351896 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-13 |
United States Patent
Application |
20220011318 |
Kind Code |
A1 |
Greer; Tyler ; et
al. |
January 13, 2022 |
HEAVY PEPTIDE APPROACH TO ACCURATELY MEASURE UNPROCESSED C-TERMINAL
LYSINE IN ANTIBODIES
Abstract
The present disclosure provides a method for measuring
post-translational modifications in proteins such as antibodies. In
particular, the method may be used to quantify C-terminal
truncation in antibodies that incorporates heavy isotopic standards
for both the unprocessed C-terminal K peptide and the truncated
C-terminal K peptide to build a calibration curve and quantify this
PTM using mass spectrometry. Quantification of post-translational
modifications may occur in a single liquid chromatography tandem
mass spectrometry (LC-MS.sup.2) run.
Inventors: |
Greer; Tyler; (Elmsford,
NY) ; Cejkov; Milos; (Ridgewood, NJ) ; O'Brien
Johnson; Reid; (Hartsdale, NY) ; Zheng; Xiaojing;
(Croton-on-Hudson, NY) ; Li; Ning; (New Canaan,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Appl. No.: |
17/351896 |
Filed: |
June 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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63041015 |
Jun 18, 2020 |
|
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International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 30/72 20060101 G01N030/72 |
Claims
1. A method for quantifying unprocessed C-terminal lysine in a
peptide (K peptide), comprising: mixing a set of heavy C-terminal
peptide standards with a peptide digest; generating a calibration
curve of a peptide signal of the unprocessed C-terminal K response
relative to that of a truncated (des-K) peptide; and analyzing and
quantifying the percentage of K peptide using liquid chromatography
mass spectrometry.
2. The method according to claim 1, wherein the set of heavy
C-terminal peptide standards comprises equimolar concentrations of
.DELTA.4 K peptide:.DELTA.4 des-K peptide, .DELTA.8 K
peptide:.DELTA.4 des-K peptide, .DELTA.12 K peptide:.DELTA.4 des-K
peptide, and .DELTA.16 K peptide:.DELTA.4 des-K peptide.
3. The method of claim 2, wherein the equimolar concentrations are
at molar ratios of 1:1, 1:10, 1:100, and 1:1000 K peptide to des-K
peptide.
4. The method of claim 3, wherein the K peptide standard is SEQ ID
NO:2 and the des-K peptide standard is SEQ ID NO:1.
5. The method of claim 3, wherein the K peptide standard is SEQ ID
NO:4 and the des-K peptide standard is SEQ ID NO:3.
6. The method of claim 1, wherein the percentage of K peptide is
analyzed using liquid chromatography tandem mass spectrometry
(LC-MS.sup.2).
7. The method of claim 4, wherein the unprocessed C-terminal K is
analyzed and quantified in a single LC-MS.sup.2 peptide mapping
run.
8. The method of claim 1, wherein the K peptide is an antibody.
9. The method of claim 8, wherein the antibody is a monoclonal or
bispecific antibody.
10. The method of claim 1, wherein an error of the calibration
curve is less than 10%.
11. A method for quantifying unprocessed C-terminal lysine in a
peptide (K peptide), comprising: digesting a protein with a
protease to produce a peptide digest; mixing the peptide digest
with a set of heavy C-terminal peptide standards, wherein the set
of heavy C-terminal peptide standards comprises equimolar
concentrations of .DELTA.4 K peptide:.DELTA.4 des-K peptide,
.DELTA.8 K peptide:.DELTA.4 des-K peptide, .DELTA.12 K
peptide:.DELTA.4 des-K peptide, and .DELTA.16 K peptide:.DELTA.4
des-K peptide; generating a calibration curve of a peptide signal
of the unprocessed C-terminal K response relative to that of a
truncated (des-K) peptide; and analyzing and quantifying the
percentage of K peptide using liquid chromatography mass
spectrometry.
12. The method of claim 11, wherein the K peptide standard:des-K
peptide standard is SEQ ID NO:2:SEQ ID NO:1 or SEQ ID NO:4:SEQ ID
NO:3.
13. A kit for quantifying unprocessed C-terminal lysine in a
peptide (K peptide), comprising: des-K peptide standards; K peptide
standards; heavy des-K-peptide standards; heavy K-peptide
standards; and instructions for use.
14. The kit of claim 13, further comprising instructions for
calibration, data extraction, analysis, and interpretation.
15. The kit of claim 13, wherein the des-K peptide standards and
heavy des-K peptide standards are SEQ ID NO:1 and the K peptide
standards and heavy K peptide standards are SEQ ID NO:2.
16. The kit of claim 13, wherein the des-K peptide standards and
heavy des-K peptide standards are SEQ ID NO:3 and the K peptide
standards and heavy K peptide standards are SEQ ID NO:4.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 USC .sctn.
119(e) of U.S. Provisional Application No. 63/041,015, filed Jun.
18, 2020
SEQUENCE LISTING
[0002] An official copy of the sequence listing is submitted
concurrently with the specification electronically via EFS-Web as
an ASCII formatted sequence listing with a file name of
"10760US01_SEQ_LIST_ST25.txt" created on Jun. 14, 2021 and having a
size of 1 KB. The sequence listing contained in this ASCII
formatted document is part of the specification and is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] Aspects of the invention are generally directed to methods
for characterizing antibody sequence fidelity. More specifically,
the present disclosure provides methods for identifying and
quantifying a post-translational modification (PTM) in the Fc
region after antibody synthesis, such as C-terminal lysine (K)
truncation (clipping), using heavy isotopic standards to establish
a calibration curve and quantify the PTM in a single liquid
chromatography tandem mass spectrometry (LC-MS.sup.2) run.
BACKGROUND OF THE INVENTION
[0004] Therapeutic monoclonal antibodies (mAbs) and bispecific
antibodies (bsAbs) play a key role in treating many disorders. The
advantages of this class of drugs, including high specificity and
affinity to an expansive variety of molecular targets, warrant
their continued development and have led to approvals for treatment
of health conditions like asthma, rheumatoid arthritis, and
elevated low density lipoprotein cholesterol, among many others.
While the commercial and scientific success of therapeutic
antibodies is unprecedented, their inherent benefits are tempered
by their large size, complexity, and chemical heterogeneity,
necessitating that a host of methods be used to evaluate their
safety and efficacy.
[0005] A significant fraction of these methods is devoted to
evaluating PTMs, a product quality attribute (PQA) and major source
of mass and charge heterogeneity. The PTM complement of a single
antibody is diverse, but common modifications are shared among
almost all mAbs and bsAbs, such as C-terminal lysine truncation,
glycosylation, N-terminal pyro-Glu formation, oxidation, amidation,
deamidation, succinimide intermediate formation, glycation,
isomerization, cysteinylation, and trisulfide bonding.
[0006] Careful monitoring of these PTM levels enables their control
through predefined acceptance criteria and has become a common
strategy for two distinct reasons: (1) numerous reports have shown
that PTMs, especially when located in a complementarity determining
region (CDR), can affect the stability and bioactivity of an
antibody, and (2) variability in PTM levels could indicate a lack
of process control.
[0007] Post-translational modifications are assayed at the global
level with chromatographic and electrophoretic techniques,
including methods like size exclusion chromatography multi angle
laser light scanning (SEC-MALLS), capillary electrophoresis sodium
dodecyl sulfate (CE-SDS), imaged capillary isoelectric focusing
(cIEF), and cation exchange chromatography (CEX). Such methods have
enjoyed wide acceptance but typically identify only the most
abundant modifications without determining their specific locations
within the amino acid sequence.
[0008] For example, the acidic species in a CEX chromatogram will
most likely contain PTMs like deamidation, glycation, and
cysteinylation, and the basic species will be comprised of
modifications like unprocessed C-terminal K, oxidation, and
isomerization. However, as the amino acid locations of these PTMs
are indeterminable in global analyses, it is challenging to
determine if they are located in a CDR and at what abundance.
[0009] The integration of highly sensitive mass spectrometer
detectors with an ever-increasing number of liquid chromatography
column chemistries and enzymatic treatment conditions has resulted
in a mature suite of PTM characterization methods. Intact mass
analysis of an antibody via liquid chromatography mass spectrometry
(LC-MS) does not yield site-specific PTM data, but it requires
minimal sample preparation and can provide an analysis of larger
PTMs with the additional benefit of mass identification.
[0010] Disulfide bond reduction and/or limited digestion with
enzymes like IdeS, papain, GingisKHAN.RTM., and FabALACTICA.RTM.
marginally increase sample preparation time but enable subunit
level resolution of PTM localization that can be further increased
by fragmenting each subunit using electron transfer dissociation
(ETD) or another tandem mass spectrometry (MS.sup.2) approach.
However, site-specific localization and quantification of PTMs
across a wide dynamic range are most commonly performed from the
"bottom-up" using a technique called peptide mapping.
[0011] Peptide mapping methods require enzymatic digestion of the
antibody, yielding a peptide mixture that is separated by liquid
chromatography and detected by ultraviolet/visible (UV/Vis)
absorbance before being ionized and infused into a mass
spectrometer. Full MS spectra are acquired, and peptides are
selected and fragmented to produce MS.sup.2 spectra that are used
to validate a peptide's identity or localize a PTM on a peptide
containing more than one potential modification site. While peptide
mapping can potentially induce preparation-related artifacts onto
the antibody sequence and significantly increases the time and
complexity of an experiment, it is the most sensitive PTM
characterization method and is site specific.
[0012] Quantification of each modification can be performed using
UV or extracted ion chromatograms (XICs), but UV quantification is
obfuscated by co-eluting peptides and is inherently less sensitive
than modern mass spectrometers. Because of this, XIC-based
quantification is routinely performed, and an MS-based peptide
mapping assay allows for identification, localization, and
quantification of all relevant PTMs with a detection limit of less
than 0.1% under optimal conditions.
[0013] The advantages of PTM quantification by XIC are accompanied
by some unique disadvantages that affect the method's accuracy and
precision. Many of these issues are related to differences in the
ionization of an unmodified peptide versus the modified form due
to: (1) ion suppression of one or both peptide forms from
co-eluting peptide peaks, (2) the difference in solvent environment
between the two peptide forms eluting at separate retention times,
(3) the disparity in ionization efficiency between the modified
peptide relative to the unmodified, and (4) variability among mass
spectrometers. Peptide mapping quantification of all PTMs is
influenced by these factors, but the C-terminal K truncation
(des-K) value is particularly impacted due to differences in
ionization efficiencies and a reduction in the peptide's
predominant charge state to 1+ compared to the unprocessed form (K,
z=2+).
[0014] This PTM readily occurs because of carboxypeptidase activity
during production from mammalian tissue culture cells, and the
resulting predominant form in a mAb or bsAb is des-K. Therefore,
the percent relative abundance of unprocessed C-terminal K is
typically calculated in relation to the sum of K and des-K.
Unprocessed C-terminal K is not thought to be an efficacy or safety
concern in antibodies since it is not in a CDR and has been shown
to be rapidly lost upon injection with a half-life of roughly one
hour, but careful monitoring of this PTM demonstrates process
control, and it has been reported that antibodies with more basic
pl values may also have increased tissue uptake and blood
clearance.
[0015] For these reasons, unprocessed C-terminal K measurement is
still critical, and previous efforts found that the percentage of K
is overestimated during peptide map quantification as the
additional K on the C-terminus of the peptide sequence increases
the ionization efficiency relative to the des-K peptide. Some
attempts to minimize this error include using only the most
abundant charge state to calculate the XIC area under the curve
(AUC) for each peptide or using a correction factor determined by
injecting equal molar amounts of each peptide onto the LC column
and gauging the mass spectrometer response.
[0016] However, while the first method decreases the magnitude of
the unprocessed C-terminal K value, it does so with no empirical
knowledge of how much this value should be decreased by, and the
second assumes that the correction factor remains static across the
possible concentration range of unprocessed C-terminal K, in the
presence of potentially co-eluting peptides, and among different
mass spectrometers.
[0017] Accordingly, it would be desirable to provide a simple
method for accurate and precise quantification of a PTM, such as
quantification of an unprocessed C-terminal lysine (K), in
antibodies and other proteins.
[0018] Therefore, an object of the invention is to provide an
improved method for the precise quantification of a PTM, such as
quantification of an unprocessed C-terminal lysine (K), in
antibodies and other proteins.
SUMMARY OF THE INVENTION
[0019] The present invention provides methods useful for the
accurate and precise characterization of PTMs in proteins, such as
antibodies, using a heavy peptide approach. More specifically, the
present disclosure provides quantification of unprocessed
C-terminal lysine (K) in antibodies. Methods of quantifying
C-terminal K using a heavy peptide approach are provided.
[0020] In one exemplary embodiment of the present invention, a
method of quantifying unprocessed C-terminal K involves mixing a
set of heavy C-terminal peptide standards to a peptide digest,
generating a calibration curve of the unprocessed C-terminal K
peptide's signal response relative to that of the des-K peptide,
and analyzing and quantifying the mass of the unprocessed
C-terminal K. In one embodiment, the mass of the unprocessed
C-terminal lysine (K) is analyzed and quantified in a single
LC-MS.sup.2 peptide mapping experiment.
[0021] In another embodiment, the resulting response curve may span
a concentration ratio range of 1:1000-1:1 K to des-K peptide. In
other embodiments, the resulting response curve has an error of
less than 10%. In one embodiment, the peptide is an antibody. For
example, the method is useful for analyzing and quantifying the
mass of the unprocessed C-terminal lysine (K) in monoclonal
antibodies (mAbs).
[0022] In another embodiment, the disclosed method is useful for
analyzing and quantifying the mass of the unprocessed C-terminal
lysine (K) in bispecific antibodies (bsAbs).
[0023] Advantages of the invention include but are not limited to:
a robust and highly accurate assay using analytical chemistry
designed for the detection of C-terminal lysines (K) at the end of
an antibody using heavy peptides in a single comparative step; a
robust and highly accurate assay using analytical chemistry for
high speed and accurate manufacture of therapeutic antibodies;
therapeutic antibodies produced to a higher level of confidence as
a result of the above as part of manufacturing train; wide
application for perfecting the manufacture of antibodies in
clinical development and in commercial use, and kits containing
heavy peptides, comparative standards, and instructions for use in
carrying out the assays of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0024] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosure and together with the description,
serve to explain the principles of the disclosure.
[0025] FIGS. 1A and 1B. FIG. 1A is a schematic of the assay of the
invention showing a peptide representing the C-terminus of an
antibody fully processed "clipped" (.i.e., lacking a C-terminal
lysine (K)) mixed with four (4) other peptides representing the
C-terminus of an antibody that is unprocessed "unclipped" (i.e.,
having a C-terminal lysine (K)) to form a response curve peptide
mix. This response curve peptide mix is then mixed with a sample
representing a potential antibody manufacturing sample (mAb
Digest), sufficient to accurately quantitate the amount of
C-terminal lysine (K) present. FIG. 1B represents the analytical
peaks observed for each of the species when subjected to Liquid
Chromatography Mass Spectroscopy (M/Z).
[0026] FIGS. 2A-2E show the structures of five heavy peptide
standards. FIGS. 2A-2D show four SLSLSLGK (SEQ ID NO:2) "unclipped"
heavy peptides standards containing .sup.13C and .sup.15N isotopes
(indicated by .cndot.), .DELTA.4, .DELTA.8, .DELTA.12, and
.DELTA.16 K peptides, respectively. FIG. 2E shows the heavy
isotopic SLSLSLG (SEQ ID NO:1) standard, .DELTA.4 des-K. containing
.sup.13C and .sup.15N isotopes (indicated by .cndot.).
[0027] FIG. 3 shows a calibration curve (CC) exhibiting a
proportional relationship between "clipped" and "unclipped"
peptides.
[0028] FIG. 4 shows an exemplary response curve using heavy chain
(HC) C-terminal peptides in accordance with an embodiment of the
present invention.
[0029] FIGS. 5A and 5B. FIG. 5A shows a UV chromatogram of an
equimolar mixture of "unclipped" SLSLPGK (SEQ ID NO:4) and
"clipped" SLSLSPG (SEQ ID NO:3). FIG. 5B shows an extracted ion
chromatogram (XIC) of equimolar amounts of the SLSLPGK (SEQ ID
NO:4) and SLSLSPG (SEQ ID NO:3) reagent set in accordance with an
embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0030] The term "C terminal lysine (K)" or "K peptide" refers to an
amino acid lysine residue or "K" residue that can be present or
absent on the end of the heavy chain of an antibody.
[0031] The term "truncated peptide" or "(des-K)" refers to a
representative portion of a protein having the C-terminal amino
acid sequence of an antibody missing a C-terminal lysine (K).
[0032] The term "analyzing and quantifying the percentage of K
peptide" refers to comparing the difference between a first and
second assay signal sufficient to ascertain the difference between
an antibody, or representative peptide thereof, which shows the
presence of absence of a C-terminal lysine (K).
[0033] The term "analytical chemistry or chemistries" refers to
quantitative analysis of molecules for the purpose of carrying out
the invention, and in particular liquid chromatography mass
spectrometry.
[0034] The term "heavy peptides" refers to any peptide of the
invention, or equivalents thereof, wherein at least one or more
carbon or nitrogen atoms of the peptide is a heavy isotope thereof,
for example, .sup.13C and .sup.15N isotopes.
[0035] The term "peptide digest" refers to peptide mix resultant
from exposing an antibody, as described herein, when incubated with
one or more enzymes capable of digesting an antibody protein
sequence such that a polypeptide sequence representative of the
C-terminus of the antibody is released.
[0036] The term "unclipped" refers to an antibody C-terminal
sequence or a representative polypeptide sequence thereof, wherein
the C-terminal sequence is has a terminal lysine (K) amino acid
residue.
[0037] The term "clipped" refers to an antibody C-terminal sequence
or a representative polypeptide sequence thereof, wherein the
C-terminal sequence is missing a terminal lysine (K) amino acid
residue.
[0038] The term "antibody" refers to a therapeutic immunobinder,
e.g., a monoclonal antibody, bi- or multi-specific antibody, that
is suitable for introducing into a subject for modulating a disease
or disorder, for example, an immune or oncological disorder. A
"drug antibody" can be, for example, a bispecific antibody that can
bind to two (2) targets.
[0039] The term "antibody" is to be construed broadly as describing
monoclonal antibodies, bispecific antibodies, antibody compositions
with multi-specificity, as well as antibody fragments (e.g., Fab,
F(ab')2, scFv and Fv), antibody derivatives, variants, and
analogs.
[0040] Unless defined otherwise, all terms and phrases used herein
include the meanings that the terms and phrases have attained in
the art, unless the contrary is clearly indicated or clearly
apparent from the context in which the term or phrase is used.
2. Improved Assays for Antibody C-Terminal Lysine (K) Analysis
[0041] The invention provides a peptide-based for assay for
accurately quantitating the undesirable amount of antibody
C-terminal lysine (K). The assays of the invention are essential
quality control tools for evaluating an antibody candidate, for
example, in clinical trials or in commercial use.
[0042] Typically, the assay of the invention is structured as shown
in FIGS. 1A and 1B where five (5) heavy peptides are co-incubated
in the presence of an antibody digest to produce a detectable
signal. The detectable signal can indicate an accurate measure of
the "clipped" and "unclipped" C-terminal lysine (K).
[0043] The assay of the invention, using a novel set of heavy
peptides and analytical chemistries (e.g., liquid chromatography
and mass spectrophotometry) can be calibrated to provide highly
accurate measurements. This assay fidelity is key for the
manufacture of complex protein molecules, in particular,
therapeutic antibodies designed to be introduced into human
patients.
3. Assay Kits
[0044] The invention also provides kits for carrying out the assay
of the invention. A key step in the assay for determining accurate
and true measures of the presence of C-terminal lysines (K) is the
use of one or more heavy peptides of sufficient plurality, that
when admixed with appropriate standards and a sample, provide a
readable signal. The signal is typically measured using analytical
chemistries, for example, Liquid Chromatography Mass Spectroscopy
(LCMS).
[0045] Accordingly, exemplary components of the kit consist of:
1. standard peptides "clipped" 2. standard peptides "unclipped" 3.
standard heavy peptides ("clipped" and "unclipped") including one
or more of the following exemplary peptides disclosed herein; and
4. instructions for use, including instructions for calibration,
data extraction, analysis, and interpretation.
[0046] Accordingly, the invention provides for a convenient test
kit and instructions for perfecting an important antibody
manufacturing chemistry, manufacturing, and controls (CMC)
endpoint.
5. Wide Application of the Invention
[0047] It should be appreciated that current invention provides for
the accurate determination of the fine structure and exact amino
acid sequence of a therapeutic antibody. Accordingly, the invention
compliments and improves the CMC (Chemistry, Manufacturing, and
Controls) of any commercially produced therapeutic antibody.
[0048] For example, the invention allows for perfecting the
manufacture and safeguarding of a number of antibody therapies.
[0049] Such antibody therapies include:
[0050] abciximab, adalimumab, adalimumab-atto, ado-trastuzumab
emtansine, alemtuzumab, alirocumab, atezolizumab, avelumab,
basiliximab, belimumab, bevacizumab, bezlotoxumab, blinatumomab,
brentuximab vedotin, brodalumab, canakinumab, capromab pendetide,
certolizumab pegol, cetuximab, daclizumab (Zenapax), daclizumab
(Zinbryta), daratumumab, denosumab, dinutuximab, dupilumab,
durvalumab, eculizumab, elotuzumab, evolocumab, golimumab,
golimumab, ibritumomab tiuxetan, idarucizumab, infliximab,
infliximab-abda, infliximab-dyyb, ipilimumab ixekizumab,
mepolizumab, natalizumab, necitumumab, nivolumab, obiltoxaximab,
obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab,
palivizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab,
ranibizumab, raxibacumab, reslizumab, rituximab, secukinumab,
siltuximab, tocilizumab, tocilizumab, trastuzumab, ustekinumab,
vedolizumab, sarilumab, rituximab and hyaluronidaseguselkumab,
inotuzumab ozogamicin, adalimumab-adbm, gemtuzumab ozogamicin,
bevacizumab-awwb, benralizumab, and emicizumab-kxwh.
trastuzumab-dkst, infliximab-qbtx, ibalizumab-uiyk,
tildrakizumab-asmn, burosumab-twza, and erenumab-aooe.
[0051] Other therapeutic antibodies of interest for various
indications subject to the invention include: aflibercept, for
treating eye disorders; rilonacept for treating blindness and
metastatic colorectal cancer; alirocumab for treating familial
hypercholesterolemia or clinical atherosclerotic cardiovascular
disease (ASCVD); dupilumab for treating atopic dermatitis;
sarilumab for treating rheumatoid arthritis and COVID-19;
cemiplimab for treating PD-1 related disease; and antibodies for
treating Ebola.
EXAMPLES
[0052] The examples below are provided for illustrative purposes
and should not be construed as limiting the invention which is
defined by the appended claims. All references and patents recited
within the present application are included herein by
reference.
Materials and Methods
[0053] The present invention, when practiced by the person skilled
in the art, may make use of conventional techniques in the field of
pharmaceutical chemistry, immunology, molecular biology, cell
biology, recombinant DNA technology, and assay techniques, as
described in, for example, Sambrook et al. "Molecular Cloning: A
Laboratory Manual", 3.sup.rd ed. 2001; Ausubel et al. "Short
Protocols in Molecular Biology", 5.sup.th ed. 1995; "Methods in
Enzymology", Academic Press, Inc.; MacPherson, Hames and Taylor
(eds.). "PCR 2: A practical approach", 1995; "Harlow and Lane
(eds.) "Antibodies, a Laboratory Manual" 1988; Freshney (ed.)
"Culture of Animal Cells", 4.sup.th ed. 2000; "Methods in Molecular
Biology" vol. 149 ("The ELISA Guidebook" by John Crowther) Humana
Press 2001, and later editions of these treatises (e.g., "Molecular
Cloning" by Michael Green (4.sup.th Ed. 2012) and "Culture of
Animal Cells" by Freshney (7.sup.th Ed., 2015), as well as current
electronic versions.
[0054] Methods useful for quantifying and analyzing PTMs in
proteins are provided within the disclosure. More specifically, the
present disclosure provides methods for quantifying and analyzing
C-terminal lysine (K) in proteins, for example, antibodies. The
methods include applying a set of heavy C-terminal peptide
standards to a digested protein. The protein may be digested by
proteases such as trypsin and other suitable enzymes.
[0055] The method may involve spiking calibration curves into
antibody digests and injecting approximately equimolar amounts of
heavy des-K peptide to digested des-K peptide onto a column in each
LC-MS.sup.2 run. Unprocessed C-terminal K may be quantified in a
single LC-MS.sup.2 peptide mapping experiment.
[0056] The method may involve generating a calibration curve
spanning a ratio range of 1:1000-1:1 K to des-K peptide. The
calibration curve may have an error of less than 10%, less than 9%,
or less than 8%. Mass spectra may be quantified using various
spectrometers, such Thermo Q-Exactive Plus 3, Q-Exactive Plus 4 or
Orbitrap Fusion Lumos mass spectrometers.
[0057] The following working examples demonstrate exemplary methods
for identifying and quantifying PTMs after antibody synthesis.
Example 1
Assay Design and Methods for Calibration
[0058] This example shows the experimental design of the assay of
the invention for calibrating the understanding of antibody
C-terminus lysine (K) structure.
[0059] All light and heavy isotopic peptide standards were
purchased from New England Peptide (Gardner, Mass.).
Trifluoroacetic acid (TFA), formic acid (FA), tris
[2-carboxylethyl] phosphine hydrochloride (TCEP-HCl), and Optima
LC/MS grade acetonitrile (ACN) were obtained from Thermo Fisher
Scientific (Rockford, Ill.) while glacial acetic acid and
iodoacetamide (IAM) were procured from Sigma-Aldrich (St. Louis,
Mo.). Sequencing grade modified trypsin, ultrapure urea, and
ultrapure 1 M Tris(hydroxymethyl)aminomethane hydrochloride
(Tris-HCl) were purchased from Promega (Madison, Wis.), Alfa Aesar
(Haverhill, Mass.), and Invitrogen (Carlsbad, Calif.),
respectively. Milli-Q water was purified by a Millipore Milli-Q
Advantage .DELTA.10 Water Purification System.
[0060] Isotopic HC C-terminal peptide standards were used to
normalize the mass spectrometer response between corresponding
light unprocessed and truncated peptides for quantification of
percent lysine. Peptide standards included SLSLSLG (SEQ ID NO:1),
SLSLSLGK (SEQ ID NO:2), SLSLSPG (SEQ ID NO:3) and SLSLSPGK (SEQ ID
NO:4).
[0061] FIG. 1A is a schematic of the assay of the invention showing
a peptide representing the C-terminus of an antibody fully
processed "clipped" (.i.e., lacking a C-terminal lysine (K)) mixed
with four (4) other peptides representing the C-terminus of an
antibody that is unprocessed "unclipped" (i.e., having a C-terminal
lysine (K)) to form a response curve peptide mix. This response
curve peptide mix is then mixed with a sample representing a
potential antibody manufacturing sample (mAb Digest), sufficient to
accurately quantitate the amount of C-terminal lysine (K) present.
FIG. 1B represents the analytical peaks observed for each of the
species when subjected to Liquid Chromatography Mass Spectroscopy
(M/Z).
[0062] FIGS. 2A-2E show heavy isotopic standards. .sup.13C and
.sup.15N are indicated by .cndot.. FIGS. 2A-2D show heavy isotopic
SLSLSLGK (SEQ ID NO:2) .DELTA.4, .DELTA.8, .DELTA.12, and .DELTA.16
K peptides, respectively. FIG. 2E shows the heavy isotopic SLSLSLG
(SEQ ID NO:1) standard, .DELTA.4 des-K. The peptide standards were
dissolved in 10% ACN, 0.1% TFA and combined into two calibration
curve sets according to the C-terminal sequence (LGK or PGK). Each
set contained equimolar concentrations of .DELTA.4 des-K and K
peptide as well as .DELTA.8, .DELTA.12, and .DELTA.16 K peptides at
molar ratios of 1:10, 1:100, and 1:1000 K to des-K, respectively.
The mixture was analyzed by XIC as shown in FIG. 3.
[0063] An equimolar mixture of SLSLSPGK (SEQ ID NO:4) and SLSLSPG
(SEQ ID NO:3) was quantified by UV chromatography, as shown in FIG.
4A. Corresponding K AUC/des-K AUC values for PGK peptides were
1.08. Similarly, K AUC/des-K AUC values for LGK peptides were 1.07.
Equimolar amounts of the SLSLSPGK (SEQ ID NO:4) and SLSLSPG (SEQ ID
NO:3) reagent set were quantified by XIC, as shown in FIG. 4B.
Heavy AUC/light AUC values for PGK and LGK peptides are shown in
Table 1.
TABLE-US-00001 TABLE 1 Heavy/light Heavy AUC/ Peptide isotope light
AUC SLSLSPGK .DELTA.4/.DELTA.0 0.98 .DELTA.8/.DELTA.0 1.00
.DELTA.12/.DELTA.0 0.99 .DELTA.16/.DELTA.0 1.00 SLSLSPG
.DELTA.4/.DELTA.0 0.99 SLSLSLGK .DELTA.4/.DELTA.0 1.02
.DELTA.8/.DELTA.0 1.04 .DELTA.12/.DELTA.0 1.06 .DELTA.16/.DELTA.0
1.03 SLSLSLG .DELTA.4/.DELTA.0 1.00
[0064] As can be seen, the values of the heavy peptides were
approximately equal to the corresponding light peptides.
[0065] To determine the accuracy of the method, known quantities of
light des-K and K were spiked into the reagent sets across the
1:10-1:1000 K to des-K peptide ratio range and measured using the
calibration curve corrected method.
[0066] As shown in Table 2, the calibration curve corrected values
were closely aligned with the expected % lysine.
TABLE-US-00002 TABLE 2 SLSLSLGK (SEQ ID NO: 2) SLSLSPGK (SEQ ID NO:
4) CC CC Expected Corrected % Corrected % % K % K Difference % K
Difference 50.0 49.6 0.8 50.8 1.5 9.1 8.9 2.0 9.3 2.5 1.0 0.9 8.9
1.0 7.1 0.1 0.1 2.0 0.1 3.5
Example 2
[0067] Unprocessed C-Terminal Lysine Quantification of mAbs
[0068] This example shows the experimental design of the assay of
the invention for understanding the antibody C-terminus lysine (K)
structure.
[0069] For antibody analysis, the calibration curves were spiked
into antibody digests so that an approximately equimolar amount of
heavy des-K peptide to digested des-K peptide was injected onto the
column in each LC-MS.sup.2 run.
Antibody Digestion
[0070] Equal weights of five IgG4 mAb samples were buffer exchanged
into 5 mM acetic acid and 5 mM TCEP-HCl before denaturation and
reduction at 80.degree. C. for ten minutes. The samples were
further denatured in 4 M urea/0.1 M Tris-HCl, pH 7.5 and alkylated
with 5 mM IAM at room temperature in the dark for 30 minutes. Urea
concentration was lowered to 1 M by adding 0.1 M Tris-HCl, pH 7.4,
and the antibodies were digested at a 1:20 antibody to trypsin
ratio at 37.degree. C. for 4 hours. Enzymatic activity was quenched
by acidifying the samples in 0.2% TFA.
LC-MS and LC-MS.sup.2 Parameters
[0071] Aliquots of 5 .mu.g of antibody digest was injected onto a
2.1 mm.times.150 mm Waters Acquity Ultra Performance Liquid
Chromatography (UPLC) Charged Surface Hybrid (CSH) C18 column with
1.7 .mu.m particles. Peptides were separated on this column with a
Waters Acquity I-Class UPLC set to a flow rate of 250 .mu.L/min and
column temperature of 40.degree. C. The gradient consisted of a
0.1-35% increase of organic mobile phase (ACN and 0.1% FA) relative
to water and 0.1% FA over 95 minutes.
[0072] Mass data was acquired using a Thermo Q-Exactive Plus using
QE Plus 3 and 4 systems and/or Orbitrap Fusion Lumos mass
spectrometer. Full mass scans were performed on the Q-Exactive Plus
acquired an m/z range of 300-2000 at 140,000 resolution (m/z 200)
for an ion population limited by an automatic gain control (AGC)
target set to 1.times.106 or a maximum ion injection time (max IT)
of 50 ms.
[0073] Experiments requiring MS.sup.2 identification by data
dependent acquisition (DDA), a single dd-MS.sup.2 loop began by
isolating and fragmenting each of the five most intense peptide
ions with a 1.5 Th window using higher energy collisional
dissociation (HCD) at a normalized collision energy of 30 was
used.
[0074] Fragment ion population data was collected using an AGC
target of 1.times.105 or a max IT of 100 ms and then scanned at
17,500 resolution, at which point the sampled precursor was placed
on an exclusion list for 10 seconds to ensure the analysis of less
intense ions.
[0075] Orbitrap Fusion Lumos parameters for MS.sup.1 acquisition
were the same as for the QE-Plus, with the exceptions being
resolution set to 120,000 (m/z 200) and ACG target to 5.times.105.
Differences in MS.sup.2 settings were: (1) limiting DDA by a cycle
time of one second instead of by number of precursors, (2) setting
AGC target to 2.times.104, (3) controlling max IT with 50 ms but
allowing for continued injection if parallelizable time was
available, and (4) scanning at 15,000 resolution (m/z 200).
[0076] Relevant LC-MS.sup.2 raw files were analyzed with Byonic 3.0
using custom fasta files for each antibody according to the
following parameters: (1) Cleavage Sites--R, K, (2) Cleavage
Side--C-terminal, (3) Digestion Specificity--Fully Specific, (4)
Precursor Mass Tolerance--10 ppm, (5) Fragmentation Type--QTOF/HCD,
Fragment Mass Tolerance--20 ppm, (6) Fixed and Variable
Modifications--Fixed C Carbamidomethyl, Variable M Oxidation,
Variable E/Q to pE, and Variable C-term K Loss, and (7) Glycan
Modifications--50 common biantennary N-glycans. Ion chromatograms
for the 1+ and 2+ charge states of light and heavy C-terminal
peptides were extracted in Thermo Xcalibur 3.1 by the Genesis
algorithm set to a 10 ppm m/z tolerance. Quantitative AUC
measurements were exported to Microsoft Excel, where calibration
curves ranging from 1:1000-1:1 K to des-K were constructed to
calculate the percentage of unprocessed C-terminal K in each
sample.
[0077] Table 3 shows the results obtained using the calibration
curve correction method compared to normal, uncorrected peptide
mapping. As shown in Table 3, the percentage of C-terminal lysine
is overestimated during peptide quantification using uncorrected
peptide mapping in comparison to the CC corrected method of the
present disclosure.
TABLE-US-00003 TABLE 3 CC Corrected C-term Lys % Uncorrected C-term
Lys % Standard Standard Antibody Mean Deviation Mean Deviation mAb
1 5.7 0.2 10.4 0.4 mAb 2 6.9 0.7 11.5 0.7 mAb 3 6.3 0.2 12.0 0.3
mAb 4 9.8 0.4 15.8 0.2 mAb 5 11.8 0.4 19.1 1.1
Example 3
Unprocessed C-Terminal Lysine Quantification of Bispecific
Antibodies (BsAbs)
[0078] This example shows the experimental design of the assay of
the invention for assaying bispecific antibodies (BsAbs).
[0079] Multiple IgG4-based bsAbs (7 seven) (containing both
SLSLSLGK (SEQ ID NO:2) and SLSLSPGK (SEQ ID NO:4) C-terminal
sequences) were digested as described above. Calibration curves
were spiked into the antibody digests and approximately equimolar
amount of heavy des-K peptide to digested des-K peptide was
injected onto the column in each LC-MS.sup.2 run. Corresponding
bsAb digests were subjected to traditional, uncorrected peptide
mapping.
[0080] Table 4 shows the results obtained using the calibration
curve correction method compared to normal, uncorrected peptide
mapping of the PGK C-terminal sequences. As shown in Table 4, the
percentage of C-terminal lysine is overestimated during peptide
quantification using uncorrected peptide mapping in comparison to
the CC corrected method of the present disclosure.
TABLE-US-00004 TABLE 4 CC Corrected C-term Lys % Uncorrected C-term
Lys % Antibody Standard Standard (PGK) Mean Deviation Mean
Deviation bsAb 1 14.3 0.1 23.9 0.3 bsAb 2 15.3 0.0 23.5 0.6 bsAb 3
15.8 0.0 27.3 1.0 bsAb 4 16.4 0.2 27.0 0.5 bsAb 5 16.9 0.2 25.7 1.2
bsAb 6 20.0 0.1 30.1 0.6 bsAb 7 26.4 0.3 37.3 0.3
[0081] Table 5 shows the results obtained using the calibration
curve correction method compared to normal, uncorrected peptide
mapping of the LGK C-terminal sequences. As shown in Table 5, the
percentage of C-terminal lysine is overestimated during peptide
quantification using uncorrected peptide mapping in comparison to
the CC corrected method of the present disclosure.
TABLE-US-00005 TABLE 5 CC Corrected C-term Lys % Uncorrected C-term
Lys % Antibody Standard Standard (LGK) Mean Deviation Mean
Deviation bsAb 1 2.0 0.1 3.5 0.1 bsAb 2 2.5 0.1 4.1 0.1 bsAb 3 2.2
0.1 3.7 0.2 bsAb 4 2.5 0.2 4.2 0.1 bsAb 5 2.7 0.0 4.7 0.1 bsAb 6
3.4 0.1 5.7 0.1 bsAb 7 5.1 0.1 8.4 0.2
[0082] Five IgG4 mAbs and one IgG1 mAb were digested as described
above. Calibration curves were spiked into the antibody digests and
approximately equimolar amount of heavy des-K peptide to digested
des-K peptide was injected onto the column in each LC-MS2 run.
Corresponding mAb digests were subjected to traditional,
uncorrected peptide mapping. Mass data were acquired using a Thermo
Q-Exactive Plus and an Orbitrap Fusion Lumos mass spectrometer.
[0083] As shown in Table 6, when using the CC corrected method,
there was zero to little difference in percent lysine when
quantified using either the QE-Plus or Fusion mass spectrometer.
However, greater variability of percent lysine was seen across
instruments when uncorrected peptide mapping was used.
TABLE-US-00006 TABLE 6 Antibody CO Corrected C-term Lys %
Uncorrected C-term Lys % (C-term) QE-Plus Fusion % RSD QE-Plus
Fusion % RSD IgG4 5.5 5.5 0.4 10.0 9.1 6.5 mAb 1 IgG4 6.5 6.5 0.2
10.9 9.8 7.5 mAb 2 IgG4 6.6 6.5 0.7 12.2 10.0 14.0 mAb 3 IgG4 9.7
9.6 0.7 16.0 14.8 5.5 mAb 4 IgG4 12.0 11.6 2.6 20.0 17.7 8.8 mAb 5
IgG1 0.7 0.6 8.6 1.1 0.8 19.2 mAb 1 (PGK)
[0084] Multiple (7) IgG4-based bsAbs (containing both SLSLSLGK (SEQ
ID NO:2) and SLSLSPGK (SEQ ID NOA4) C-terminal sequences) were
digested as described above. Calibration curves were spiked into
the antibody digests and approximately equimolar amount of heavy
des-K peptide to digested des-K peptide was injected onto the
column in each LC-MS2 run. Corresponding bsAb digests were
subjected to traditional, uncorrected peptide mapping.
[0085] Mass data were acquired using a Thermo Q-Exactive Plus and
an Orbitrap Fusion Lumos mass spectrometer. As shown in Table 7,
when using the CC corrected method, there was zero to little
difference in percent lysine when quantified using either the
QE-Plus or Fusion mass spectrometer. However, greater variability
of percent lysine was seen across instruments when uncorrected
peptide mapping was used.
TABLE-US-00007 TABLE 7 CC Corrected Uncorrected C-term Lys % C-term
Lys % C- QE- % QE- % Antibody terminal Plus Fusion RSD Plus Fusion
RSD bsAb 1 PGK 14.2 14.8 2.7 24.1 18.9 17.0 LGK 2.1 2.1 2.6 3.6 3.2
9.2 bsAb 2 PGK 15.3 15.5 1.2 24.2 19.2 16.3 LGK 2.6 2.5 2.5 4.0 4.2
3.3 bsAb 3 PGK 15.8 16.3 2.2 28.1 20.8 20.9 LGK 2.3 2.2 3.8 3.7 3.3
7.7 bsAb 4 PGK 16.3 16.9 2.2 28.1 20.8 20.9 LGK 2.6 2.5 2.1 4.3 3.9
6.2 bsAb 5 PGK 16.7 17.3 2.2 24.8 21.1 11.4 LGK 2.7 2.7 1.2 4.6 4.3
4.2 bsAb 6 PGK 20.1 20.4 1.3 30.6 25.1 14.0 LGK 3.5 3.4 2.6 5.8 5.3
6.0 bsAb 7 PGK 26.1 26.9 2.0 37.6 31.9 11.8 LGK 5.2 5.1 0.9 8.6 8.0
5.0
[0086] While in the foregoing specification this invention has been
described in relation to certain embodiments thereof, and many
details have been put forth for the purpose of illustration, it
will be apparent to those skilled in the art that the invention is
susceptible to additional embodiments and that certain details
described herein can be varied without departing from the basic
principles of the invention.
Sequence CWU 1
1
417PRTArtificial SequenceSynthetic 1Ser Leu Ser Leu Ser Leu Gly1
528PRTArtificial SequenceSynthetic 2Ser Leu Ser Leu Ser Leu Gly
Lys1 537PRTArtificial SequenceSynthetic 3Ser Leu Ser Leu Ser Pro
Gly1 548PRTArtificial SequenceSynthetic 4Ser Leu Ser Leu Ser Pro
Gly Lys1 5
* * * * *