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FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patent Rules 2003
COMPLETE SPECIFICATION
(see sections 10 & rule 13)
1. TITLE OF THE INVENTION:
CHROMATOGRAPHIC DETECTION, ESTIMATION AND
SEPARATION OF AMINO ACIDS IN HYDROLYSATES OF
MONOCLONAL ANTIBODIES FIELD OF INVENTION
2. APPLICANT(S)
NAME NATIONALITY ADDRESS
ZELLE
BIOTECHNOLOGY
PVT. LTD.
INDIAN A-7 M.I.D.C., Mira Industrial Area,
Western Express Highway, Mira
Road, Dist. Thane- 401 104, INDIA
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner
in which it is to be performed
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CHROMATOGRAPHIC DETECTION, ESTIMATION AND SEPARATION
OF AMINO ACIDS IN HYDROLYSATES OF MONOCLONAL ANTIBODIES
FIELD OF INVENTION
[001] The present invention relates to chromatographic detection, estimation and
separation of amino acid in protein hydrolysates, more specifically monoclonal5
antibody hydrolysates.
BACKGROUND
[002] Protein and peptide-based drugs such as monoclonal antibodies are a rapidly
expanding class of therapeutic agents that are used to treat a wide variety of health
conditions, including cancer, metabolic and auto-immune diseases such as rheumatoid10
arthritis, viral infections such as HIV, and more. Biologic drugs, such as monoclonal
antibodies, are derived from living organisms and are usually very expensive.
However, of late many biologics are coming off patents, and the market is now ready
for cost-saving biosimilars. All proteins, including monoclonal antibodies, have
complex structures and amino acids are the most basic moieties that make up and15
determine their function. A difference in amino acid composition changes the intrinsic
structure of proteins and therefore can alter biological activity leading to changes in
safety and efficacy of the drug.
[003] Determining amino acid composition therefore becomes necessary to
characterize and confirm the protein identity and integrity. The amino acid composition20
of a protein hydrolysate can be availed to get a comparison with the available gene
sequence of the desired product. Such amino acid data can also be used to determine
the protein content.
[004] Derivatization of amino acids becomes essential during chromatographic
analysis studies as the underivatized amino acids absorb weakly in the low UV (200-25
220 nm). At these UV ranges and wavelengths, sample mixtures and chromatographic
mobile phases interference is also seen. Thus, it is necessary to derivatize the
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hydrolysed amino acids to allow separation and detection. Highly stable derivatives are
also favorable as they allow a sample to be reanalyzed, if so desired, without assaying
another sample.
[005] At present, a number of derivatization procedures including pre-column and
post-column are available to permit the assay of amino acids by electrochemical5
analysis, gas chromatography, ion exchange chromatography, high performance liquid
chromatography and capillary electrophoresis. Mass spectrometry (GC-MS) analysis
method is to use the anion formed by the carboxyl group in the amino acid molecule in
a strong alkali environment to achieve different separation time on the anion exchange
resin. The method is sensitive with high resolution and good repeatability. However,10
this method is not cost effective as it requires high-end instruments and highly skilled
personnel. Ion exchange chromatography separation and post column derivatization
with ninhydrin results into a purple product through an ultraviolet-visible
spectrophotometer test. However, this method destroys the inherent structure of amino
acids, has a long analysis time, complicated hardware configuration, specific use, and15
poor flexibility.
[006] Pre-column derivatized chemicals and some of the drawbacks are listed here 1)
o-phthalaldehyde (OPA)/mercaptan – adduct is unstable and does not form a derivative
with secondary amino acids; 2) 9-fluorenylmethylchloroformate (FMOC method) -
tryptophan and cystine cannot be quantitated easily, the reagent itself fluoresces and20
hence must be removed, it has also been reported to form multiple derivatives with
histidine, is hazardous to work with as it is corrosive; 3) phenylisothiocyanate method
(PITC) - derivatives are not fluorescent; 4) dansyl chloride method generates multiple
derivatives.
[007] In addition to the above methods, in 1993, Waters developed the AQC (6-25
aminoquinoiyi-nhydroxysuccinimidyi carbamate) tag method for reversed-phase high
performance liquid chromatography. This method based on pre column derivatization,
attaches a fluorophore to the amino group of an amino acid, separated on high
performance liquid chromatography and then detects it by fluorescence and an
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ultraviolet detector. This method is more sensitive, the separation conditions are easier
to improve, and is highly versatile.
[008] However, the commercially available kit is expensive, and is a bottleneck in
development of cost-saving biosimilars.
[009] An improved method for detecting and accurately measuring the presence and5
amount of amino acid in monoclonal hydrolysate sample that is cost-effective and less
time consuming is required. The present invention provides an effective and
indigenous method for faster separation of amino acid in protein hydrolysates, more
specifically monoclonal antibody hydrolysates, with simpler and cheaper mobile
phases.10
OBJECTIVES OF THE INVENTION
[010] In light of the foregoing, it is therefore an objective of the present invention to
provide an alternative chromatographic (RP-HPLC) method for the separation,
detection and quantification of amino acid in protein hydrolysates, more specifically
monoclonal antibody hydrolysates.15
[011] A particular object of the invention is to provide a new, accurate and sensitive
RP-HPLC method; wherein the method allows a multiple-point calibration curve and
absolute quantification as well as recovery of the amino acid in protein hydrolysates,
more specifically monoclonal antibody hydrolysates.
[012] Yet another objective of the present invention is to provide a novel gradient20
elution procedure with different mobile phases.
[013] Another particular objective of the present invention is to provide a cost
effective in-house mobile phase for gradient elution RP-HPLC separation, detection
and quantification of the amino acid in protein hydrolysates, more specifically
monoclonal antibody hydrolysatesDescription:One aspect of the present invention is to provide a method for separation, detection and quantification of amino acid in protein hydrolysates, more specifically monoclonal antibody hydrolysates, the method comprising hydrolysis of the monoclonal antibodies to obtain an hydrolysate; derivatizing the hydrolysates of monoclonal antibodies and their neutralization; RP-HPLC separation of the hydrolysates using a gradient elution procedure and calculating the monoclonal antibodies and the amino acid content in the hydrolysate by using a standard curve.
Another aspect of the present invention recites a method for hydrolysis of the monoclonal antibodies; the method comprising treating the monoclonal antibodies with an internal standard, an oxygen scavenger, and an acid; purging the hydrolyzed monoclonal antibodies with nitrogen for 30 seconds and incubating the purged hydrolysate at a temperature in the range of 100 to 1200 C for a period of 10 to 17 hours; centrifuging at a speed of 5000 to 15000 rpm and neutralizing the hydrolysate with alkaline solution of 5 to 10 N.
Yet another aspect of the present invention provides a method for derivatizing the hydrolysate by treating the said hydrolysate with a derivatizing agent at a temperature in the range of 40 to 600C for a time period of 5 to 10 minutes.
Yet another particular aspect of the disclosure provides a RP-HPLC method for detection and separation of monoclonal antibodies and their hydrolysares the method involving:
• a high performance liquid chromatography system coupled with ultraviolet and fluorescence detector; fluorescence detection: excitation wavelength 266 nm, emission wavelength 473 nm; UV detection wavelength: 260 nm;
• Column: RP 1.7µm, 2.1* 100mm column; column temperature: 45-55 °C; flow rate in the range of 0.5 to 1 ml/min;
• Mobile phase: mobile phase A comprising 10-75 mM ammonium formate in 3-7% ACN and 0.25-1.5% Formic acid; mobile phase B comprising 10% of Mobile Phase D; mobile phase C comprising of water; and Mobile Phase D comprising 90-99% ACN and 0.25-1.5% Formic acid;
• Gradient elution involving mobile phases A, B, C and D in the gradient ranging as follows: Mobile phase A: 2.0%-9.0%, Mobile phase B: 0.0%-80%, Mobile phase C: 98.0%-11.0%, Mobile phase D: 0.0%-59.7% over 9 minute with combination of quaternary gradients; injection volume 1 µL
Yet another aspect of the present invention is to provide a standard curve for each amino acid by subjecting the derivatized amino acid standard solution to ultraviolet and fluorescence detection.
Yet another aspect of the present invention provides calculation and determination of the amino acid content in the amino acid hydrolysate by using the standard curve. This invention is not limited in its application to the details of construction and the arrangement of components or the permutation and specific combination of reagents and chemicals as set forth in the following description and examples or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.
An embodiment of the present invention provides a method for analyzing and detecting amino acids in monoclonal antibodies and hydrolysates thereof, the method comprising:
• hydrolyzing the monoclonal antibodies to obtain hydrolysate, wherein the hydrolyzing process comprises treating the monoclonal antibodies with an internal standard, an oxygen scavenger, and an acid; purging the hydrolyzed monoclonal antibodies with nitrogen for 30 seconds and incubating the purged hydrolysate at a temperature in the range of 100 to 1200 C for a period of 10 to 17 hours; centrifuging at a speed of 5000 to 15000 rpm and neutralizing the hydrolysate with alkaline solution of 5 to 10 N;
• derivatizing the amino acid hydrolysate by treating the amino acid hydrolysate with a derivatizing agent at a temperature in the range of 40 to 600C for a time period of 5 to 10 minutes;
• utilizing a RP-HPLC method for detection, estimation and separation of amino acids in the derivatised hydrolysate, the High-performance method comprising:
o A High-performance liquid chromatography system coupled with ultraviolet and fluorescence detector; fluorescence detection: excitation wavelength 266 nm, emission wavelength 473 nm; UV detection wavelength: 260 nm;
o Column: a Reverse Phase 1.7µm, 2.1* 100mm column; column temperature: 45-55 °C; flow rate of 0.7mL / min
o Mobile phases: mobile Phase A comprising 10-75 mM ammonium formate in 3-7% ACN and 0.25-1.5% Formic acid; mobile phase B comprising 10% of Mobile Phase D; mobile phase C comprising of water; and Mobile Phase D comprising 90-99% ACN and 0.25-1.5% Formic acid;
o Gradient elution procedure: Mobile phase A: 2.0%-9.0%, Mobile phase B: 0.0%-80%, Mobile phase C: 98.0%-11.0%, Mobile phase D: 0.0%-59.7% over 9 minute with combination of quaternary gradients; injection volume 1 µL
• Obtaining a standard curve for each amino acid by subjecting the derivatized amino acid standard solution to ultraviolet and fluorescence detection
• Calculating the amino acid content in the amino acid hydrolysate by using the standard curve using line equation y = mx + c. Where, c = intercept m = slope, x and y are variables.
According to a specific embodiment of the invention the mobile A comprises of 50mM Ammonium formate in 7% ACN and 1.5% formic acid.
In yet another embodiment of the invention the mobile phase B is 10% of Mobile Phase D and mobile phase C comprises of water.
In yet another embodiment of the invention the Mobile Phase D comprises of 99% ACN and 1% Formic acid.
In yet another embodiment of the present invention, the monoclonal antibodies are selected from trastuzumab, denosumab. palivizumab, rituximab, aflibercept, etanercept, teriparatide, bovine serum albumin, erythropoitien.
In yet another embodiment of the present invention, the amino acids/ amino acid standards are selected from a group consisting of histidine, serine, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, IS (Norvaline) isoleucine, leucine, phenylalanine, tryptophan.
In yet another embodiment of the present invention, the derivatizing agent is 6-aminoquinoline-N-hydroxysuccinimidyl carboxylate (AQC)
According to an embodiment of the present invention, the derivatizing agent is dissolved in dry acetonitrile.
In yet another specific embodiment of the present invention, the internal standard is Norvaline or any other unnatural amino acid.
In yet another specific embodiment of the invention the unnatural amino acid are selected from the group consisting of hydroxyproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine and pyroglutamic acid.
According to an embodiment of the present invention, the oxygen scavenger is capable of reducing oxygen
According to an embodiment of the present invention, the oxygen scavenger is phenol.
, C , Claims:We claim:
1. A method for detection, estimation and separation of amino acids in protein and/or monoclonal antibody and their hydrolysates, the method comprising:
i. hydrolyzing the protein and/or monoclonal antibodies to obtain a hydrolysate, wherein the hydrolyzing process comprises treating the protein and /or monoclonal antibodies with an internal standard, an oxygen scavenger, and an acid; purging the hydrolyzed protein and/or monoclonal antibodies with nitrogen for 30 seconds and incubating the purged hydrolysate at a temperature in the range of 100 to 1200 C for a period of 10 to 17 hours; centrifuging at a speed of 5000 to 15000 rpm and neutralizing the hydrolysate with alkaline solution of 5 to 10 N;
ii. derivatizing the hydrolysate by treating the said hydrolysate with a derivatizing agent at a temperature in the range of 40 to 600C for a time period of 5 to 10 minutes to obtain derivatised amino acid hydrolysate;
iii. utilizing a RP-HPLC analysis method for, detection, estimation and separation of amino acids in the derivatised hydrolysate, the RP-HPLC method comprising:
a. a High performance liquid chromatography system coupled with ultraviolet and fluorescence detector; fluorescence detection at an excitation wavelength of 266 nm, emission wavelength of 473 nm; and UV detection at wavelength of 260 nm;
b. a reverse phase 1.7µm, 2.1* 100mm column; with column temperature in the range of 45-55 °C; and a flow rate in the range of 0.5 to 1.0 mL / min
o Mobile phases A, B, C and D wherein mobile phase A comprises 10-75 mM ammonium formate in 3-7% ACN and 0.25-1.5% Formic acid; mobile phase B comprising 10% of Mobile Phase D; mobile phase C comprising of water; and Mobile Phase D comprising 90-99% ACN and 0.25-1.5% Formic acid;
c. A gradient elution procedure of over 9 minutes wherein each of the mobile phases is used in the following ranges: Mobile phase A: 2.0%-9.0%, Mobile phase B: 0.0%-80%, Mobile phase C: 98.0%- 11.0%, and Mobile phase D: 0.0%-59.7% with combination of quaternary gradients and injection volume 1 µL;
iv. Obtaining a standard curve for each amino acid by subjecting the derivatized amino acid standard solution to ultraviolet and fluorescence detection;
v. Calculating the amino acid content in the hydrolysate by using the standard curve.
2. The method as claimed in claim 1 wherein the mobile phase A comprises specifically of 50mM Ammonium formate in 7% ACN and 1.5% Formic acid; mobile phase B is 10% of Mobile Phase D; a mobile phase C of water; and Mobile Phase D is 99% ACN and 1% Formic acid.
3. The method as claimed in claim 1, where the protein and /or monoclonal antibodies are selected from trastuzumab, denosumab. palivizumab, rituximab, aflibercept, etanercept, teriparatide, bovine serum albumin, erythropoitien,
4. The method as claimed in claim 1, wherein the amino acids/ amino acid standards are selected from histidine, serine, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cysteine, lysine, tyrosine, methionine, valine, IS (Norvaline) isoleucine, leucine, phenylalanine, tryptophan.
5. The method as claimed in claim 1, wherein the derivatizing agent is 6-aminoquinoline-N-hydroxysuccinimidyl carboxylate (AQC) and dissolved in dry acetonitrile.
6. The method as claimed in claim 1, wherein the internal standard is Norvaline or any other unnatural amino acid and wherein the unnatural amino acid is selected from hydroxyproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine and pyroglutamic acid.
7. The method as claimed in claim 1, wherein the oxygen scavenger capable of reducing oxygen is specifically phenol.