FORM 2 THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
“METHOD FOR QUANTIFICATION OF AN INGREDIENT USED IN VACCINE MANUFACTURING & FORMULATIONS”
Name of the Applicant:
SERUM INSTITUTE OF INDIA PRIVATE LIMITED
Address of the Applicant:
SERUM INSTITUTE OF INDIA PRIVATE LIMITED 212/2, Off Soli Poonawalla Road, Hadapsar, Pune - 411028, Maharashtra, India
Nationality: IN
The following specification particularly describes the invention and the manner in which it is to
be performed.

FIELD
The present disclosure relates to analytical methods. Particularly, the present disclosure relates to method for quantification of biological product/vaccine constituent, buffering agent or ingredient content in biological product/vaccine manufacturing processes or vaccine formulations.
BACKGROUND
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Vaccines contain an active component (the antigen) which induces the immune response.
The active component of the vaccine is an ‘antigen’. The active component includes complete or modified or partial/degraded form of the virus, bacteria, other pathogens, as well as the toxins that causes the disease against which the vaccine protects. The vaccine antigen is altered from its original form, no longer causes disease, however the vaccine antigen can induce an immune response when administered to a living being.
The vaccine also contains additional components such as the preservatives, the additives, the adjuvants, the buffering agents and traces of other components.
The adjuvants are used to enhance the immune response to the vaccine antigen. One-way the adjuvants are thought to improve the immune response is by keeping the vaccine antigen(s) near the injection site for ready access to cells of the immune system. The use of aluminium adjuvants in vaccines generally means that less antigen per dose of vaccine is required, and, in some cases, fewer vaccine doses are needed.
The diluent is a liquid provided separately and used to dilute the vaccine to the proper concentration prior to administration. The diluent is usually sterile saline or sterile water.
The additives include the stabilizers that maintain the vaccine’s effectiveness by keeping the antigen and other vaccine components stable during storage. The stabilizers prevent the vaccine components adhering to the side of the vaccine container.
The preservatives are used to prevent fungal and/or bacterial contamination of the vaccines and
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are present in some but not all vaccines. Originally, the preservatives were introduced to prevent bacterial contamination of multi-dose vials. Preservatives have been used in many vaccines for a long time.
The buffering agent/ solution resists changes in pH when small quantities of an acid or an alkali are added to it. The buffering agent maintain the vaccine at a similar pH to that of the body.
Buffering agents are used to maintain the pH of the vaccine preparation generally in a range of about pH 6 to pH 7 throughout the shelf life, which is appropriate for a spectrum of viral and bacterial antigens. The buffering agents usually used include phosphates, carboxylates, and bicarbonates, such as sodium phosphate, potassium phosphate, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, and potassium bicarbonate, etc. The amount of buffering agent used in the vaccine is from 0.00 to 99.9998 percent by weight, preferably from 0.00 to 20.0 percent.
One of the buffering agent used in the vaccine formulation and during its manufacturing process is tris(hydroxymethyl)aminomethane (also known as 2-Amino-2-(hydroxymethyl)- 1,3-propanediol, Tromethamine, TRIS, Tris base, Tris buffer, Trizma, THAM, Trometamol).
TRIS is a commonly used excipient in various approved parenteral medicinal products. TRIS is a biologically inert polar amino alcohol with low toxicity that is extensively used in biochemistry and molecular biology as a component of buffer solutions. TRIS has a pKa of 7.8 at 37°C and is thus an effective buffer in the physiological range that can stabilize and prevent pH fluctuations in solution. TRIS is a solid readily soluble in water. TRIS acts as an osmotic diuretic, increasing urine flow, urinary pH, and excretion of fixed acids, carbon dioxide and electrolytes. TRIS is commonly used as an emulsifying agent in pharmaceutical and cosmetic products or as a counterion for acidic pharmaceutical compounds to generate desired salt forms. TRIS plays a very important role in vaccine formulations. The use of TRIS as a buffering agent enables the stabilization of the vaccine and extends the shelf life of the vaccine by maintaining the pH, hence it is necessary to measure the Tris content.
It has been found that molecular size distribution (MSD) value for Conjugate Meningococcal A polysaccharide – tetanus toxoid conjugate (Men A-TT) was found to decrease at 90 deg C (force degradation study), indicating labile nature of Men A/Men A-TT conjugate.
Also, Meningococcal A polysaccharide – tetanus toxoid conjugate (Men A-TT) was found to
be sensitive to pH below 5 and above 8 (molecular weight preservation study). Applicant found
that Tris (10mM) used for stabilization of Men A-TT both in monovalent Men A-TT conjugate
& Pentavalent Conjugate vaccine having Men A-TT (ACWYX- MenFive) maintained the
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molecular weight of Men A-TT.
Tris analysis in MenFive (ACWYX) (Meningococcal conjugate vaccine comprising polysaccharide from serotypes A and X individually conjugated to Tetanus toxoid (TT) and polysaccharide from serotypes C, W and Y individually conjugated to CRM197) Conjugate vaccine (and in other conjugate vaccines/vaccines) is challenging as it contains five individual conjugates, stabilizers like sucrose and sodium citrate, traces (4-PPY-by product of conjugation reagent CPPT, SDS, Tween -80, glycine, ADH), free polysaccharide, free protein, aggregates (HMW, LMW) all of which may interfere with the TRIS content determination.
Suitable quality control analytical methods are required to quantitate TRIS for regulatory purposes. The European and US pharmacopeia (USP) describe a simple titration against HCl for assay purposes. However, the titration method is not sufficiently sensitive, requires a high sample volume and concentration, as well as suffers from hindrance effects in complex buffered formulations. USP monograph titrimetric assay and a flow injection pseudo titration do not provide specificity. Therefore, a separative method may be a more effective alternative.
TRIS being basic in nature does not contain chromophores or flurophores groups and hence cannot be retained and separated by UV and flurorescence based chromatography methods. To date, only a few chromatography-based methods have been developed for TRIS determination in pharmaceutical products and biological fluids.
An initial study on TRIS derivatization with benzoyl chloride (a chromophore-containing molecule) followed by UV detection revealed that the matrix components caused peak broadening that masked the TRIS derivative, creating difficulties with the quantitation assay. The peak broadening was overcome by using Two reverse-phase (RP) HPLC methods involving chemical derivatization that provide sufficient retention, as well as chromophores or fluorophores for UV/fluorescence detection. (Moran Madmon et.al.; 2023)
Peak broadening emerges from longitudinal diffusion, caused by slow flow rate, void volume within the column and tubing and excessive retention in the column. Peak broadening can cause variation in peak area and also affect peak resolution in case of closely eluting components.
Determination of TRIS as an excipient in Iopamidol injection (a formulated pharmaceutical product) was performed by derivatization and analysed by LC-UV method at a concentration of 1.0 mg/mL. (Naidong W., Ghodbane et.al; 1999)
In Liquid Chromatography Tandem Mass Spectrometry method, initially broad peak of intact
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TRIS was observed under RPLC-MS conditions. To overcome this difficulty and improve the chromatographic retention and peak shape, resolution in RP-LC-ESI-MS/MS, a novel method based on chemical derivatization was developed to improve the RP-LC‒ESI-MS/MS analysis results which included high-dilution step of the sample with an organic solvent (acetone) followed by second high-dilution of the sample with water. The method eliminated ion suppression or enhancement caused by either the matrix or the derivatizing agent. (Moran Madmon et.al.; 2022).
Derivatization alters the melting point, solubility, boiling point, reactivity or the chemical composition of compound to be analyzed. Derivatization mostly leads to increase in the total time of analysis; increases the complexity of post or pre-column reaction and there are chances of error. Derivatization methods necessarily are less straightforward and provide somewhat diminished performance characteristics.
The addition of organic solvents usually reduces the electroosmotic flow and, hence, expands the migration time window. The retention factor is also reduced because the solubility of the analyte into the aqueous phase increases.
For fluorescence, UV and flame ionization detection, derivatization of tromethamine is required, which leads to lengthy sample preparation times.
An alternative approach to realize sufficient retention of the polar TRIS compound is to use hydrophilic interaction chromatography (HILIC) as a normal phase prior to analysis with a refractive index detector. More recently, HILIC has been coupled with a charged aerosol detector. Initial attempts to develop an assay based on HPLC coupled with a UV detector with and without derivatization requires complex sample treatment (two-step derivatization, extractions and evaporation to dryness) and resulted in low performance. (Moran Madmon et.al.; 2022)
In Ion chromatography (IC) followed by conductivity detection, the columns used in the process needs be maintained and the columns may increase the cost of the chromatographic method.
Previously reported methods using HPLC or GC involve lengthy sample and standard preparation, involving derivatization of TRIS. The total analysis time, including sample and standard preparation and run time is more. The other drawbacks include use of large amounts of expensive solvents and chemicals and, in some cases, long times, thereby increasing production costs. Therefore, a comparable method with lower
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consumption of chemicals and costs is needed.
Capillary electrophoresis (CE) separates molecules in an electric field according to size and charge. Capillary zone electrophoresis (CZE) has been reported previously for evaluation or characterization of vaccines, particularly useful for routine quality control of therapeutic proteins, such as monoclonal antibodies. There are some limitations of CZE like injectable sample amount is inherently small along with solubility and purity, among other factors. Moreover, when used with optical detectors, the optical pathway is limited which consequently results in poor detection/concentration sensitivity of sample. The classic capillary zone electrophoresis (CE) method is quite slow and not very useful for high- throughput screening. It also has other limitations: interactions between the compound being studied and the different buffers must be avoided; and it requires strict control of temperature to minimize the Joule effect.
Optimizing the quantitative performance of a method used for quantification of Tris needs consideration of multiple parameters.
Injection precision performance is a well-studied parameter of the commercial capillary electrophoresis (CZE) instrumentation. The injection volumes in CZE are typically 5–50 nL and are achieved by inserting the capillary into a sample solution vial and then pressurizing the vial to force sample solution into the capillary. The volume injected is directly related to the pressure difference and the duration of applying the pressure. Siphoning effects as well as surface tension of the sample solution affect the injection volume and can lead to variable injection volumes and peak areas.
Peak resolution and peak broadening in capillary electrophoresis are dependent on variables, including longitudinal diffusion, injection length, sample adsorption to capillary walls (analyte wall interactions), electrodispersion, Joule heating, and detector cell size. Peak broadening during quantitative measurement can lead to inaccurate results.
All the aforementioned problems can be efficiently eliminated by inclusion of an internal
standard used to prepare the sample and standard solutions. The internal standard should be
chosen to give good peak shape and to migrate reasonably near the solute peak of interest. The
primary solution to improve precision is use of an internal standard which improves linearity
and recovery data. The internal standard is chosen such that it does not react with the substance
to be examined, is stable and does not contain impurities with the same retention time as that of
the substance to be examined. However, unless the internal standard is employed the required
injection precision level is difficult to maintain during long routine injection sequences and/or
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analysis of samples in complex matrices.
Therefore, an unmet need exists to develop a method that overcomes the drawbacks of previously reported methods for quantification of Tris content in vaccine/drugs/monoclonal antibody formulations/manufacturing process or any other biological solution, which provides error free results, sharper peaks, is simple, sensitive, robust, reproducible, easy, accurate, requires short sample/standard run time.
OBJECTS
An object of present disclosure is to provide an improved Capillary Zone Electrophoresis
based method for TRIS quantification in vaccine/drugs/monoclonal antibody
formulations/manufacturing process or any other biological solution, said method exhibits following advantages over previously methods (reported RV-HPLC, LC-UV, Ion Chromatography, HILIC, GC, RPLC- Electrospray Ionization tandem mass spectrometry)-1) Li+ / Lithium hydroxide (instead of Na+) used as cation internal standard provides
good correlation coefficient, reproducibility, resolution of peaks /well resolved
peaks/ provide spike recovery within limits.
2) No interference of sucrose and sodium citrate on Tris migration time (No peak broadening of Tris was observed in presence of other excipients)
3) Sensitive even at lower concentration (i.e. Limit of detection is 3µg/mL); the limit of detection (LOD) 2 µg/ml - 4 µg/ml and limit of quantification (LOQ) 10 µg/ml - 20 µg/ml.
4) Optimized parameters- Coating solution A (comprising polyethyleneimine); Coating solution B (comprising polyacrylic acid); Chromophore containing Cation separation buffer (ultra-pure water, propyl-4-hydroxy-benzoate NaCl, 4-aminopyridine, malic acid, 18-crown- 6) and Conditioner-Na (NaOH solution); separation Voltage of 30KV (1.0 Ramp, normal Polarity; for cation separation to reduce migration time); separation pressure of 20 psi; capillary column ID of 75µm (for symmetrical peak shape of Tris was observed/ no peak broadening), capillary cartridge temperature of 25°C; capillary conditioning time of 0.5 min (to reduce sample run time and to minimize the usage of conditioning reagents), total capillary length of 60 cm and maximum current of 33µA; detection wavelength-200 nm.
5) does not require analytical column, chromatography, analytical grade solvents, carrier
gas, costly derivatizing agents and sample preparation accessories like filters, syringe
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and cartridges
6) devoid of any substantial sample pre-treatment step (except dissolution of lyophilised vial)
7) method does not require acid base hydrolysis thereby ensuring there is no degradation during testing
8) method provides short duration for sample/standard run time – 8 minutes.
9) Total run time for sample/standard analysis is shorter (Process time for one injection program Approx. 13 minutes;
Some of the other objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
Another object of the present disclosure is to provide a method for quantitative measurement of the component/constituent/ ingredient/buffering agent present in the vaccine composition.
Yet another object of the present disclosure is to provide a method for quantitative measurement of the component/constituent/ ingredient/ buffering agent in a monovalent or multivalent polysaccharide-protein conjugate vaccine composition.
Yet another object of the present disclosure is to provide a capillary electrophoresis method for quantitative measurement of the cation component/constituent/ ingredient/ buffering agent in a monovalent or multivalent polysaccharide-protein conjugate vaccine composition.
Yet another object of the present disclosure is to provide a capillary zone electrophoresis
method for quantitative measurement of the cation more preferably TRIS
component/constituent/ingredient/buffering agent in a monovalent or multivalent polysaccharide-protein conjugate vaccine composition using Lithium as cation internal standard.
Still another object of the present disclosure to provide a method for assessing the quality of the polysaccharide-protein conjugate vaccine composition.
Still another object of the present disclosure to provide a simple, safe, accurate, fast,
convenient, cost-effective, highly sensitive, robust and non-destructive method devoid of any
sample processing steps prior to analysis.
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Other objects of the present disclosure are to ameliorate one or more problems of the prior art or to at least provide a useful alternative. Other objects and advantages of the present disclosure will be more apparent from the following description and is not intended to limit the scope of the present disclosure.
SUMMARY:
Surprisingly, it has been found that the above identified objective problems are solved by the method of quantifying content of an ingredient in a vaccine sample, and uses thereof according to the present invention.
The present disclosure provides a method for quantitative measurement of the cation constituent/ingredient/buffering agent in a vaccine composition using a Capillary zone electrophoresis (CZE), also known as free solution capillary electrophoresis, using lithium as cation internal standard.
The present disclosure provides an improved Capillary Zone Electrophoresis based method for TRIS quantification in vaccine/drugs/monoclonal antibody formulations/manufacturing process or any other biological solution.
According to one aspect, the present disclosure is directed to a method of quantifying content of an ingredient in a vaccine sample, the method comprising:
a) providing a vaccine sample;
b) providing an internal standard;
c) subjecting the vaccine sample of step a) and the internal standard of step b) to capillary electrophoresis chromatography comprising a capillary column;
d) identifying the peak of the ingredient and determining the content of ingredient in the vaccine sample;
In some embodiments of the present disclosure, a method of quantifying content of an ingredient in a vaccine sample, the method comprising:
a) providing a vaccine sample;
b) providing an internal standard;
c) subjecting the vaccine sample of step a) and the internal standard of step b) to capillary electrophoresis chromatography comprising a capillary column;
d) identifying the peak of the ingredient and determining the content of ingredient in the vaccine sample;
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wherein the capillary electrophoresis chromatography includes rinsing the capillary column with at least one coating solution, a separation buffer, a conditioner and a rinse solution.
In some embodiments of the present disclosure, a method for quantifying content of an of the ingredient in a vaccine comprises of following non-limiting steps;
a) preparation of working standard spiked with internal standard;
b) preparation of sample to be tested;
c) loading the working standard and sample in a sequence onto a capillary electrophoresis system and applying voltage thereby causing separation of the ions to occur in presence of cation separation buffer and detecting the ions indirectly using a UV/visible photometric detector;
d) plotting the calibration curve using ratio of the area of the working standard to its internal standard against the concentration and calculate slope and intercept of the linear curve;
e) quantifying the ion constituent in the sample.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:
Figure 1 illustrates electropherogram of NmCV5 vaccine displaying peak of Na+ (sodium citrate) at around 3.1 min and Tris at around 4.2 min;
Figure 2 illustrates electropherogram demonstrating Na+ overlap after spiking with NaCl, with increased Na+ intensity as compared to Figure 1;
Figure 3 illustrates distinct peak of Lithium upon spiking Lithium chloride in NmCV5 vaccine;
Figure 4 illustrates the electropherogram displaying the separation of Tris within the NmCV5 vaccine using a 30 KV voltage;
Figure 5 illustrates the electropherogram displaying the separation of Tris within the NmCV5 vaccine using a 25 KV voltage;
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Figure 6 illustrates the electropherogram displaying the separation of Tris within the NmCV5 vaccine using a 20 KV voltage;
Figure 7 illustrates the electropherogram displaying the separation of the NmCV5 vaccine at a capillary cartridge temperature of 30°C;
Figure 8 illustrates the electropherogram displaying the separation of the NmCV5 vaccine at a capillary cartridge temperature of 20°C;
Figure 9 illustrates the electropherogram displaying the separation of the NmCV5 vaccine at a capillary cartridge temperature of 25°C;
Figure 10 illustrates NmCV5 electropherogram at 60 cm total capillary length;
Figure 11 illustrates NmCV5 electropherogram at 80.0 cm total capillary length;
Figure 12 illustrates Current profile at 30KV voltage;
Figure 13 illustrates the electropherogram displaying a peak corresponding to sodium citrate with a migration time of approximately 3.0 minutes;
Figure 14 illustrates the electropherogram displaying sucrose profile;
Figure 15 illustrates the electropherogram displaying a peak corresponding to Tris with a migration time of approximately 4.1 minutes;
Figure 16 illustrates electropherogram of blank injection, wherein height (h) of background noise is 0.2 cm;
Figure 17 illustrates electropherogram of Tris standard 3µg/ml, wherein height of the peak (H) is measuring 0.5 cm;
Figure 18 illustrates electropherogram of a single-dose Hib vaccine, displaying Tris peak elution around 4.1 min (migration time); and
Figure 19 illustrates electropherogram of 5 dose Typhoid bivalent conjugate vaccine displaying Tris peak at 4.1 min (migration time).
DESCRIPTION
Although the present disclosure may be susceptible to different embodiments, certain
embodiments are shown in the drawing and following detailed discussion, with the
understanding that the present disclosure can be considered an exemplification of the principles
of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated
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and disclosed in this description.
Embodiments are provided so as to thoroughly and fully convey the scope of the present
disclosure to the person skilled in the art. Numerous details are set forth, relating to specific
components, and processes, to provide a complete understanding of embodiments of the present
disclosure. It will be apparent to the person skilled in the art that the details provided in the
embodiments should not be construed to limit the scope of the present disclosure. In some
embodiments, well-known composition, well-known processes, and well-known
techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms “a”, “an”, and “the” may be intended to include the plural forms as well, unless the context clearly suggests otherwise.
The terms “comprises”, “comprising”, “including”, and “having” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated, unless stated otherwise. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.
It is understood that each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and/or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or
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combination of features, described herein without having to list every possible combination. Such features or combinations of features apply to any of the aspects of the invention. More particularly, as regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification clearly and unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.
Definitions:
In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification.
As used herein vaccine could be live-attenuated vaccines; inactivated vaccines; subunit vaccines, recombinant vaccines, polysaccharide-protein conjugate vaccines; toxoid vaccines; mRNA vaccines; viral vaccines and viral vector vaccines.
As used herein ‘constituent or ingredient’ refers to a component present in the vaccine formulation which provides significant contribution towards the vaccine’s stability, immunogenicity, potency/efficacy and is used interchangeably throughout the specification.
As used herein ‘working standard’ or ‘working standard solution’ refers to a solution of known quantity of component i.e. ingredient to be quantified (Tris).
As used herein, the expressions ‘internal standard’ or ‘cation internal standard’ or “internal standard solution” are used interchangeably throughout the specification.
As used herein, the expressions ‘NmCv5’ or ‘Menfive’ or ‘Meningococcal conjugate vaccine’ or ‘Meningococcal pentavalent conjugate vaccine’ are used interchangeably throughout the specification
The present disclosure provides a method for quantitative measurement of the constituent, buffering agent or ingredient content in a biological product sample.
The present disclosure provides a method for quantitative measurement of the constituent,
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buffering agent or ingredient content in a biological product sample during in-process manufacturing or at final product stage.
The present disclosure provides a method for quantitative measurement of the constituent, buffering agent or ingredient content in a vaccine sample.
The present disclosure provides a method for quantitative measurement of the vaccine constituent, buffering agent or ingredient content in a vaccine composition.
In some embodiments of the present disclosure, a method for quantitative measurement of the constituent/ingredient/buffering agent in a vaccine composition, is performed using a Capillary electrophoresis.
In some embodiments of the present disclosure, a method for quantitative measurement of the constituent/ingredient/buffering agent in a vaccine composition, is performed using a Capillary electrophoresis, wherein the Capillary electrophoresis technique includes capillary zone electrophoresis, capillary isoelectric focusing electrophoresis, capillary gel electrophoresis, micellar electrokinetic chromatography, Micellar electrokinetic Capillary Chromatography, cyclodextrin electrokinetic chromatography, Capillary isotachophoresis and capillary electrochromatography.
In some embodiments of the present disclosure, a method for quantitative measurement of the cation constituent/ingredient/buffering agent in a vaccine composition, is performed using a Capillary zone electrophoresis (CZE), also known as free solution capillary electrophoresis using lithium as cation internal standard.
In some embodiments of the present disclosure, a method of quantifying content of an ingredient in a vaccine sample, the process comprising:
a) providing a vaccine sample;
b) providing an internal standard;
c) subjecting the vaccine sample of step a) and the internal standard of step b) to capillary electrophoresis chromatography comprising a capillary column;
d) identifying the peak of the ingredient and determining the content of ingredient in the vaccine sample.
In some embodiments of the present disclosure, a method of quantifying content of a buffering agent in a vaccine sample, the process comprising:
a) providing a vaccine sample;
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b) providing an internal standard;
c) subjecting the vaccine sample of step a) and the internal standard of step b) to capillary electrophoresis chromatography comprising a capillary column;
d) identifying the peak of the buffering agent and determining the content of buffering agent in the vaccine sample.
In some embodiments of the present disclosure, a method of quantifying content of an ingredient in a vaccine sample, the process comprising:
a) providing a vaccine sample;
b) providing an internal standard;
c) subjecting the vaccine sample of step a) and the internal standard of step b) to capillary electrophoresis chromatography comprising a capillary column;
d) identifying the peak of the ingredient and determining the content of ingredient in the vaccine sample;
wherein the capillary electrophoresis chromatography includes rinsing the capillary column with at least one coating solution, a separation buffer, a conditioner and a rinse solution.
In some embodiments of the present disclosure, a method for quantitative measurement of the ingredient in a vaccine comprises of following non-limiting steps;
a) preparation of working standard spiked with internal standard;
b) preparation of sample to be tested;
c) loading the working standard and sample in a sequence onto a capillary electrophoresis system and applying voltage thereby causing separation of the ions to occur in presence of cation separation buffer and detecting the ions indirectly using a UV/visible photometric detector;
d) plotting the calibration curve using ratio of the area of the working standard to its internal standard against the concentration and calculate slope and intercept of the linear curve;
e) quantifying the ion constituent in the sample;
In some embodiments of the present disclosure, capillary electrophoresis system comprises of following non-limiting steps;
a) Rinsing of capillary tube with cation coating solution A;
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b) Rinsing of capillary tube with cation coating solution B;
c) Rinsing of capillary tube with cation separation buffer;
d) Dipping of capillary tube in water;
e) Injecting the sample with internal standard in capillary tube;
f) Injecting water in capillary tube;
g) Applying voltage under conditions appropriate for the ions in the mixture to move along the capillary toward one of the electrodes thereby causing separation of the ions to occur in presence of cation separation buffer;
h) Detecting the ions indirectly using a UV/visible photometric detector;
i) Rinsing of capillary tube with conditioning solution
j) Rinsing of capillary tube with rinse solution.
In some embodiments of present disclosure, preparation of standard solution, internal standard solution and sample solution is done using a liquid vehicle selected from but not limited to MilliQ water, ultra-pure water, buffer and combinations thereof.
In some embodiments of present disclosure, preparation of standard solution is done using MilliQ water or ultra-pure water. The standard solutions like stock standard solution and working standard solution are prepared using MilliQ water or ultra-pure water.
In some embodiments of present disclosure, working standard solution comprises of the component similar to the ingredient to be quantified.
In some embodiments of present disclosure, the nature and concentration of the internal standard depends on several factors. The main requirement is that the internal standard should give good peak shape and is resolved from the analytes of interest and any other peaks in the separation. Other requirements for an appropriate internal standard include it is stable in solution, commercially available in a high purity form, readily soluble in the diluent required, possesses acceptably high UV activity at the desired wavelength, is inexpensive and is non-toxic. The use of internal standards improves correlation coefficients obtained for detector linearity during method validation.
In some instances, solvents like organic solvents are used for dissolution of sample to which later internal standard is added. The internal standard in such situation should be sufficiently stable in the sample dissolving solvent to prevent the formation of degradation products, which
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would interfere with the integration results. It should also be chemically stable in the solid state to allow suitable storage.
In some embodiments of the present disclosure, the internal standard solution is prepared by adding the internal standard to MilliQ water or ultra-pure water.
In some embodiments of the present disclosure, the internal standard solution is prepared by using cations such as lithium (Li+), sodium (Na+), magnesium (Mg2+), calcium (Ca2+) and potassium (K+).
In some embodiments of the present disclosure, the internal standard solution is prepared by using cation preferably lithium (Li+).
In some embodiments of the present disclosure, lithium (cation) internal standard is selected from but not limited to lithium hydroxide, lithium chloride and lithium acetate.
In some embodiments of the present disclosure, cation internal standard solution is be prepared by addition of lithium hydroxide to MilliQ water or ultra-pure water.
In some embodiments of present disclosure, internal standard is added with working standard in the ratio of 1:10 to 10:1. The concentrations of working standard used are 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, 100 µg/ml, 120 µg/ml. The internal standard concentration is kept constant as 20 µg/ml against all the concentrations of working standard used.
CZE is mainly performed in the capillary format, typically of 25 to 150 μm inner diameter (ID). Fused silica capillaries with internal diameter (ID) ranging from 10 to 200 μm are commercially available. Typically, capillaries with 25 to 100 μm ID and 350 to 400μm outer diameter (OD) are used. In order to shorten analysis time, capillaries as short as possible should be used. Sensitivity and linear detection range can usually be improved by increasing the inner diameter of the capillary.
However, choosing the suitable capillary inner diameter (ID) is sometimes difficult. Capillaries with small IDs are preferred for small amounts of sample and has high separation efficiency, due to lower Joule heating. In contrast, capillaries with larger IDs are preferred for on-column UV/Visible detection to generate a better sensitivity due to extended optical path lengths.
Typical capillary tube length is selected based on the separation capacity with applied electric field throughout the tube, which is useful to properly determine the run cycle time and time necessary for fraction collection. Most commonly, the capillary tube length is 25 to 75 cm.
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Joule heating in electrophoresis method refers to the increase in temperature of a liquid or sample as a result of its resistance to an electrical current. This current arises from the electrical voltage that is applied across a capillary tube to generate electroosmosis flow. The increased temperature makes fluid properties non-uniform, and hence alter the applied electric potential field and the flow field.
In some embodiments of present disclosure, the capillary tube used in the method is with an optimum inner diameter of 25µm to 100 µm which leads to improved sensitivity, linear detection range, high separation efficiency. The method does not require any precolumn derivatization step.
In some embodiments of the present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine performed using a Capillary zone electrophoresis (CZE) include optimization of process parameters such as Capillary cartridge temperature; Auto sampler temperature; Total capillary length; Capillary id; Detection wavelength; Separation voltage; Separation pressure; Max current.
In some embodiments of the present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine performed using a Capillary zone electrophoresis (CZE) include optimization of process parameters such as Capillary cartridge temperature in the range of 10°C to 30°C; Auto sampler temperature in the range of 20°C to 30°C; total capillary length in the range of 20 cm to 70 cm; capillary inner diameter (ID) in the range of 25µm to 100 µm ; Detection wavelength in the range of 180 nm to 250 nm; Separation voltage in the range of 5 kV to 40 kV; Separation pressure in the range of 5psi to 30 psi; Max current in the range of 100 µA to 150 µA.
In some embodiments of present disclosure, capillary cartridge temperature is in the range of 10°C to 30°C, 15°C to 30°C and 20°C to 30°C; preferably 25°C. When the capillary cartridge temperature is in the range of 20°C to 30°C, there is no alteration in the actual quantified content of the ingredient however slight variations in migration time is observed in this range.
In some embodiments of present disclosure, auto sampler temperature in the range of 20°C to 30°C, preferably 25°C.
In some embodiments of present disclosure, the internal diameter (ID) of capillary column comprising Fused silica is in the range from 10 to 200 μm, 20 to 200 μm, 20 to 150 μm, 25 to 150 μm, 25 to 100 μm, 50 to 100 μm, preferably 75 μm. The peak broadening is observed when the internal diameter (ID) of capillary column is increased above 75 μm. When the internal
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diameter (ID) of capillary column is reduced below 75 μm, the separation is not observed as the capillary column gets blocked.
In some embodiments of present disclosure, the length of capillary column comprising Fused silica is in the range of 20 cm to 100 cm, 30 cm to 90 cm, 40 cm to 80 cm, 50 cm to 80 cm, 55cm to 65 cm, 20 cm to 70 cm; preferably 60 cm. When the length of capillary column increases above 60 cm, the migration time of ingredient to be quantified gets shifted with higher retention time but does not affect the content of the ingredient.
In some embodiments of present disclosure, detection wavelength in the range of 180 nm to 250 nm, preferably 200 nm.
In some embodiments of present disclosure, separation voltage in the range of 5 kV to 40 kV, 10 kV to 40 kV, 20 kV to 40 kV, 25 kV to 35 kV, preferably 30kV. When separation voltage is reduced below 30kV the retention time of the peak of the cation component to be quantified is shifted and slight broadening is observed. The separation of cation is carried out due to the separation voltage and further the cation is quantified.
In some embodiments of present disclosure, pressure comprises of rinse pressure and inject pressure.
In some embodiments of present disclosure, rinse pressure is in the range of 5psi to 30 psi, 15 psi to 30 psi, 15 psi to 25 psi, preferably 20 psi.
In some embodiments of present disclosure, the inject pressure is in the range of 0.1 psi to 1 psi. Inject pressure for sample injection is 0.5 psi and for water injection is 0.1 psi.
In some embodiments of present disclosure, separation pressure in the range of 5psi to 30 psi, 15 psi to 30 psi, 15 psi to 25 psi, preferably 20 psi.
In some embodiments of present disclosure, the maximum current is in the range of 10 µA to 150 µA, preferably 33 µA.
In some embodiments of the present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine performed using a Capillary zone electrophoresis (CZE) include detectors selected from refractive index detectors, wavelength absorbance detectors (spectroscopy) or UV (Ultra violet) detectors, diode array detectors, chiral detectors, chemiluminescence detectors, circular dichroism detectors, light scattering detectors and fluorescence emission detectors.
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In some embodiments of the present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine performed using a Capillary zone electrophoresis (CZE) include a mode of absorbance signal selected from direct and indirect mode.
Direct mode provides reversed polarity and is used for anion separations. Indirect mode provides normal polarity and is used for cation separations.
In an exemplary embodiment, the mode of absorbance signal for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine is indirect mode.
In some embodiments of present disclosure, the principle of the method is the separation process performed under normal polarity so that the positively charged ions may migrate towards the cathode i.e. negatively charged electrode.
In some embodiments of present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine performed using a Capillary zone electrophoresis (CZE) include preparation of coating solutions, cation separation buffer, conditioning solution.
Various limitations exist for untreated fused silica capillaries, such as narrow pH range, high conductivity, low detection sensitivity, and the ability to analyze only one class of proteins. Altogether, this limits the range of applicability of narrow-bore silica capillaries and does not provide a universal solution for the solute-wall interaction problem. A possible solution to said limitations of untreated silica capillaries is coating the capillary in a suitable way to carry out by modifications, such as dynamic deactivation of the silica surface and permanent modification of the inner capillary wall by covalent, noncovalent layers, and the like.
Ideal coating should exhibit the following features: (1) separation efficiency; (2) analyte recovery (this should approach 100%); (3) reproducibility of migration time from run to run and day to day; and (4) retention of the electroosmotic flow (EOF) so that cationic and anionic species can be separated in the same run; (5) easy to generate; (6) inexpensive; (7) applicable over a wide range of buffer conditions; and (8) should not interfere with detection.
Coating of capillaries overcomes difficulties in carrying out reproducible, homogeneous
chemical derivatization reactions in the capillary lumen. Coating of capillaries do not
eliminate EOF completely but can be easily prepared by rinsing the capillary with a solution of
a polymer, detergent, or multivalent ions; a little amount of the coating material is usually
added to the separation medium to keep the coating on the silica wall surface.
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In some embodiments of the present disclosure, coating solution is used more than once for rinsing the capillary tube.
In some embodiments of the present disclosure, coating solutions used for rinsing of capillary tube is coating solution A and coating solution B.
In some embodiments of the present disclosure, coating solution A used for rinsing of capillary
tube is selected from monoamines, such as triethylamine and propylamine, hydroxylamine
or ethylamine, morpholine, N,N-diethylethanolamine, triethanolamine, as well as the
quaternary base tetramethylamonium chloride; from diamines: cadaverine, putrescine, agmatine
1,3 diaminopropane and ethylenediamine; from polyamines: spermidine, spermine chitosan
and polyethyleneimine (PEI); polyethylene oxide, polyacrylamide, polyvinyl alcohol,
polyvinyl pyrolidone, polyethylene glycol, acrylamidomethylpropylsulfonic acid,
polyacrylic acid and methacrylamidopropyltrimethyl ammonium chloride.
In some embodiments of the present disclosure, coating solution B used for rinsing of capillary tube is selected from monoamines, such as triethylamine and propylamine, hydroxylamine or ethylamine, morpholine, N,N-diethylethanolamine, triethanolamine, as well as the quaternary base tetramethylamonium chloride; from diamines: cadaverine, putrescine, agmatine 1,3 diaminopropane and ethylenediamine; from polyamines: spermidine, spermine chitosan and polyethyleneimine (PEI); polyethylene oxide, polyacrylamide, polyvinyl alcohol, polyvinyl pyrolidone, polyethylene glycol, acrylamidomethylpropylsulfonic acid, polyacrylic acid and methacrylamidopropyltrimethyl ammonium chloride.
In some embodiments of the present disclosure, combination of coating solution A and coating
solution B used for rinsing of capillary tube is selected from monoamines, such as
triethylamine and propylamine, hydroxylamine or ethylamine, morpholine, N,N-
diethylethanolamine, triethanolamine, as well as the quaternary base tetramethylammonium
chloride; from diamines: cadaverine, putrescine, agmatine 1,3 diaminopropane and
ethylenediamine; from polyamines: spermidine, spermine chitosan and polyethyleneimine
(PEI); polyethylene oxide, polyacrylamide, polyvinyl alcohol, polyvinyl pyrolidone,
polyethylene glycol, acrylamidomethylpropyl sulfonic acid polyacrylic acid and
methacrylamidopropyltrimethyl ammonium chloride.
In some embodiments of the present disclosure, coating solution A used for rinsing of capillary tube is prepared by addition of polyethyleneimine stock solution to MilliQ water or ultra-pure water.
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In some embodiments of the present disclosure, coating solution B used for rinsing of capillary tube is prepared by addition of polyacrylic acid stock solution to MilliQ water or ultra-pure water.
In some embodiments of the present disclosure, the conditioning of the column is carried out by rinsing the column with coating solution.
In some embodiments of the present disclosure, the conditioning time required using coating solution A and coating solution B can be optimized considering the baseline separation of sample and cation internal standard.
In some embodiments of the present disclosure, the conditioning time required using coating solution A and coating solution B is 30 seconds. If the time is above 30 seconds the observation of the baseline separation of sample and cation internal standard is same. The short conditioning time provide short sample run time and also minimize the consumption of conditioning reagents.
In some embodiments of the present disclosure, coating solution A and coating solution B is same.
In some embodiments of the present disclosure, coating solution A and coating solution B is different.
The selection of adequate buffer solutions is an important consideration in CE to provide appropriate migration of the analytes and stable conditions for the separation, independent of the sample concentration. The pH, concentration and composition of the buffer have a direct influence on separation performance and selectivity. The pH of the buffer affects both EOF and electrophoretic mobility of the sample, which should remain constant throughout the separation. As the buffer pH is changed, the EOF changes because of changes in ionization of the silanol groups on the capillary inner wall. The more the ionization on capillary wall, the greater is the zeta potential, which is directly related to the EOF. The commonly used buffers for CE are phosphate, acetate, borate and zwitterionic buffers. The buffer and its concentration also play an important role in controlling the EOF, the current produced in the capillary, baseline stability, and peak shape which in turn affects separation efficiency of CE.
In some embodiments of the present disclosure, separation buffer is a cation separation buffer.
In some embodiments of the present disclosure, cation separation buffer comprises of separation solution and at least one coating solution.
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In some embodiments of the present disclosure, separation solution of cation separation buffer used for rinsing of capillary tube is prepared by combining different ingredients like benzoates, pyridines, acid, chromophores.
In some embodiments of the present disclosure, separation solution of cation separation buffer used for rinsing of capillary tube is prepared by combining different ingredients like benzoates, pyridines, acid, chromophores along with at least one coating solution.
In some embodiments of the present disclosure, separation solution of cation separation buffer used for rinsing of capillary tube is prepared by combining different ingredients like benzoates, pyridines, acid, chromophores along with coating solution A.
In some embodiments of the present disclosure, separation solution of cation separation buffer used for rinsing of capillary tube is prepared by combining different ingredients like benzoates, pyridines, acid, chromophores along with coating solution B.
In some embodiments of the present disclosure, separation solution of cation separation buffer used for rinsing of capillary tube is prepared by combining different ingredients like benzoates, pyridines, acid, chromophores along with combination of coating solution A and coating solution B.
In some embodiments of the present disclosure, separation solution of cation separation buffer used for rinsing of capillary tube is prepared by combining propyl-4-hydroxy-benzoate (NaCl) stock solution, 4-aminopyridine, malic acid, 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6).
In some embodiments of the present disclosure, cation separation buffer used for rinsing of capillary tube is prepared by addition of propyl-4-hydroxy-benzoate (NaCl) stock solution, 4-aminopyridine, malic acid, 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6), and said coating solution A to MilliQ water or ultra-pure water.
In some embodiments of the present disclosure, cation separation buffer used for rinsing of capillary tube is prepared by addition of propyl-4-hydroxy-benzoate (NaCl) stock solution, 4-aminopyridine, malic acid, 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6), and said coating solution B to MilliQ water or ultra-pure water.
In some embodiments of present disclosure, the sample is prepared by dissolving the vaccine formulation with MilliQ water or ultra-pure water, under stirring and allowing the vaccine formulation to dissolve for approximately 30 to 90 minutes.
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In some embodiments of present disclosure, the sample preparation step does not involve any pre-treatment and chromatography step.
In some embodiments of present disclosure, the constituent or ingredient of vaccine quantified using a Capillary zone electrophoresis (CZE) may be metal ions or cations.
In some embodiments of present disclosure, the constituent or ingredient or buffering agent of vaccine quantified using a Capillary zone electrophoresis (CZE) is cationic component.
In some embodiments of present disclosure, the constituent or ingredient or buffering agent of vaccine quantified using a Capillary zone electrophoresis (CZE) is cationic component selected from Tris, ammonium sulphate, sodium citrate, sodium chloride and magnesium sulphate.
In some embodiments of present disclosure, the constituent or ingredient o r buffering agent of vaccine quantified using a Capillary zone electrophoresis (CZE) is organic amine compounds such as hydroxylamine and Tris (tromethamine).
In some embodiments of present disclosure, the constituent or ingredient o r buffering agent of vaccine quantified using a Capillary zone electrophoresis (CZE) is Tris (tromethamine).
In some embodiments of present disclosure, the constituent or ingredient or buffering agent of vaccine quantified using a Capillary zone electrophoresis (CZE) is Tris or Tromethamine.
In some embodiments of present disclosure, the constituent or ingredient or buffering agent of vaccine quantified using a Capillary zone electrophoresis (CZE) is Tris cationic derivative component selected from Tris HCl, Tris Buffered Saline (TBS), Tris Acetate EDTA (TAE), Tris Borate EDTA (TBE), Tris Glycine Buffer, Tris-Glycine-Sodium Dodecyl Sulfate (SDS).
In some embodiments of present disclosure, before injecting the sample in the capillary tube, the capillary tube is first rinsed using coating solution A for 40 – 80 seconds duration at 15 - 25 psi pressure followed by rinsing with coating solution B for 40 – 80 seconds duration at 15 - 25 psi pressure.
In some embodiments of present disclosure, before injecting the sample in the capillary tube, the capillary tube is first rinsed using cation separation buffer for 70 – 110 seconds duration at
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15 - 25 psi pressure.
In some embodiments of present disclosure, before injecting the sample in the capillary tube, capillary tube is dipped in water for 10 - 30 seconds duration.
In some embodiments of present disclosure, the sample is injected with internal standard in capillary tube for 3 to 10 seconds duration at 0.1 to 1.0 psi pressure followed by injection of MilliQ water or ultra-pure water for 5 - 15 seconds at 0.1 to 1.0 psi pressure.
Voltage is an important parameter in CZE that greatly affects in both migration times and resolution. The use of high voltages will provide for the greatest efficiency by decreasing the separation time. Both the electroosmotic and electrophoretic velocities are directly proportional to the field strength, so the use of the highest voltages will result in the shortest times for the separation. It is known that short separation times will give the highest efficiencies since diffusion is the most important feature contributing to band/peak broadening. The limiting factor here is Joule heating. Experimentally, the optimal voltage is determined by performing runs at increasing voltages until deterioration in resolution is noted.
In some embodiments of present disclosure, after injection of sample in the capillary tube, an optimum voltage is applied for 6 to 10 minutes duration causing voltage separation with cation separation buffer.
In some embodiments of present disclosure, rinsing of capillary tube is performed using conditioning solution and MilliQ water or ultra-pure for 10 – 50 seconds duration at 15 to 25 psi pressure respectively.
Conditioning of the capillary tube with strong base, acid, or both in sequence, has been recognized as an important parameter or step to obtain reasonable precision in electroosmotic mobility (EOM), and in the migration times of analytes. It provides a capillary tube with reproducible electrophoretic performance, reproducible reduction of wall interactions and may flush out late-migrating components that are of no interest for the analysis.
In some embodiments of present disclosure, rinsing of capillary tube is performed using conditioning solution selected from but not limited to sodium hydroxide, phosphoric acid and others. The conditioning solutions are used to activate the capillary tube by converting silica groups to silanol groups.
In some embodiments of present disclosure, rinsing of capillary tube is performed using 0.1N sodium hydroxide as conditioning solution.
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In some embodiments of present disclosure, rinsing of capillary tube after rinsing with conditioning solution is performed using CE grade water or water for injection (WFI) or filtered ultra-pure water.
In some embodiments of present disclosure, the total run time required for a single standard or sample is in the range from 5 to 20 minutes, preferably from 10 to 15 minutes.
In some embodiments of present disclosure, the run time for single injection of sample/ standard is from 9 to 10 minutes, preferably 8 minutes.
In some embodiments of present disclosure, the total run time considering capillary conditioning, cleaning, and sample injection time, is from 10 to 15 minutes, preferably 13.08 minutes.
In some embodiments of present disclosure, before injecting the sample or running a sample through capillary tube, blank i.e. solution without sample is injected in capillary tube.
In some embodiments of present disclosure, working standards with internal standard and samples with internal standard are run in duplicates using a specific sequence. In some embodiments of present disclosure, lithium (Li+) is added with working standard in the ratio of 1:10 to 10:1.
In some embodiments of present disclosure, after completion of sequence, data for both standard and samples is acquired and integrated using a software.
In some embodiments of present disclosure, after completion of sequence data for both standard and samples is acquired and integrated using a 32 Karat software. Calibration curve of standard concentrations verses ratio of sample and internal standard area may be plotted for calculation of slope and intercept of linear curve.
In some embodiments of present disclosure, the method includes the preparation of set of standards, sample dilution and spike. The series of standards are prepared with the addition of Lithium as internal cation standard. Samples under the test are diluted in order to fit in the standard curve. Samples are spiked with the known quantity of standard i.e. working standard to evaluate the accuracy of assay.
In some embodiments of present disclosure, samples are not spiked with the known quantity of standard i.e. working standard to evaluate the accuracy of assay.
In some embodiments of present disclosure, lithium used as cation internal standard
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provides good correlation coefficient, reproducibility.
In some embodiments of present disclosure, the above method or assay is valid when the correlation coefficient is ≥ 0.99 and spike recovery may be in the range of 80 to 120 %.
In some embodiments of present disclosure, the steps of conditioning the capillary tube using coating solutions and rinsing the capillary with conditioner Na solution and ultrapure water are repeated for each injection of sample with internal standard, standard with internal standard injection and sample with spike.
In some embodiments of present disclosure, the method is validated with respect to different parameters including but not limited to linearity, limit of detection (LOD), limit of quantification (LOQ), precision i.e. assay precision, and accuracy as per the ICH guideline.
In some embodiments of present disclosure, the method is validated with respect to linearity which is in the range of 10 µg/ml to 130 µg/ml; limit of detection (LOD) which is in the range of 2 µg/ml to 4 µg/ml, preferably 3 µg/ml; limit of quantification (LOQ) which is in the range of 10 µg/ml to 20 µg/ml preferably 15 µg/ml; precision i.e. assay precision which is in the range of 5 to 20 %; and accuracy which is in the range of 80 % to 120 %.
In some embodiments of present disclosure, the quantification of ingredient or constituent content is calculated using below formula;
Tris (mg/vial or mg/ml) = (Average of ratio of sample/Li – intercept) x dilution
Slope x 1000 % Spike Recovery = Practical concentration x 100
Theoretical concentration Where, Practical conc. (µg/ml) = (Average of ratio of Tris/Li in Spike – Intercept)
Slope
Theoretical conc. (µg/ml) = (Average of ratio of Tris/Li in Spike – Intercept) + 40
Slope Note: 40 is the known amount of Tris in µg spiked in sample to estimate % spike recovery In some embodiments of the present disclosure, a method for quantitative measurement of the
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constituent or ingredient or buffering agent content of vaccine performed using a Capillary zone electrophoresis (CZE) comprises Lithium as cation internal standard; Coating solution A (comprising polyethyleneimine); Coating solution B (comprising polyacrylic acid); Chromophore containing Cation separation buffer (ultra-pure water, propyl-4-hydroxy-benzoate NaCl, 4- aminopyridine, malic acid, 18-crown-6 and coating solution B); Conditioner-Na (NaOH solution).
In some embodiments of present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine using capillary zone electrophoresis requires minimum volume of sample i.e. 80 µg/ml to 120 µg/ml, preferably 100 µg/ml.
In some embodiments of present disclosure, the use of Lithium as cation internal standard provides good correlation coefficient, reproducibility, resolution of peaks (well resolved peaks with no peak broadening) and provide spike recovery within limits.
In some embodiments of present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine is devoid of derivatization of sample and may avoid the issues related to derivatization.
In some embodiments of present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine using capillary zone electrophoresis provide sharp peaks of Tris i.e. no peak broadening in the presence of other excipients. There is no interference of excipients like sucrose and sodium citrate in the quantification process.
In some embodiments of present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine using capillary zone electrophoresis does not require analytical column, chromatography, analytical grade solvents, carrier gas, costly derivatizing agents and sample preparation accessories like filters, syringe and cartridges.
In some embodiments of present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine using capillary zone electrophoresis does not require acid base hydrolysis thereby ensuring there is no degradation during testing.
In some embodiments of present disclosure, a method for quantitative measurement of the
constituent or ingredient or buffering agent content of vaccine is applicable to single dose or
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multiple dose vaccine vials.
In some embodiments of present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine is applicable to bulk or final fill vaccine formulation.
In some embodiments of present disclosure, a method for quantitative measurement of the constituent or ingredient or buffering agent content of vaccine is applicable to any vaccine containing Tris as a constituent or ingredient of vaccine formulation.
Polysaccharide component of polysaccharide-protein conjugate vaccine component selected from Streptococcus spp. such as Group A Streptococcus, Group B Streptococcus (group Ia, Ib, II, III, IV, V, VI, VII, VII, VIII, and IX); Streptococcus pneumoniae (1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48), Streptococcus pyogenes; Streptococcus agalactiae; Streptococcus viridans; Salmonella spp. such as, Salmonella typhi; Salmonella paratyphi; Salmonella enteritidis; Salmonella typhimurium; Shigella spp. such as Shigella sonnei, Shigella flexneri, Shigella dysenteriae; Shigella boydii; E.coli; Neisseria meningitidis (serotypes such as A, B, B16, B6, C, D, E29, H, I, K, K454 L, M, W135, X, Y, and Z etc); Neisseria gonorrhoeae; Haemophilus influenzae; Haemophilus pneumonia; Helicobacter pylori; Chlamydia pneumoniae; Chlamydia trachomatis; Ureaplasma urealyticum; Mycoplasma pneumoniae; Staphylococcus spp. such as Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8; Enterococcus faecalis; Enterococcus faecium; Bacillus anthracis; Vibrio cholerae; Pasteurella pestis; Pseudomonas aeruginosa; Campylobacter spp. such as jejuni; Clostridium spp. such as Clostridium difficile; Mycobacterium spp. such as Mycobacterium tuberculosis; Moraxella catarrhalis; Klebsiella pneumoniae; Treponema spp.; Borrelia spp.; Borrelia burgdorferi; Leptospira spp.; Hemophilus ducreyi; Corynebacterium diphtheria; Bordetella pertussis; Bordetella parapertussis; Bordetella bronchiseptica; Ehrlichia spp.; and Rickettsia spp.
Preferably estimating TRIS content in Men A, Men A-C, Men CWY, Men AC-Hib, Men ACWY, Men ACWYX, Men ABCWYX & Hib conjugate vaccines
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The carrier protein of polysaccharide-protein conjugate vaccine is selected from group comprising but not limited to CRM197, diphtheria toxoid, tetanus toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, recombinant full-length tetanus toxin with eight individual amino acid mutations (8MTT), pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins (Tbp-B, Tbp-A etc), pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), PhtA, PhtB, PhtE, pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, M. catarrhalis uspA, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), C5a peptidase group A or group B Streptococcus, human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), Cholera toxin subunit B, fHbp, Por A and Por B.
The virus for viral vaccine is selected from the group comprising poxvirus (e.g. orthopoxviruses; avipoxviruses), morbillivirus (e.g. measles), mumps virus, rubella virus, alphavirus (e.g. sendai virus, sindbis virus and semliki forest virus (SFV), ross river virus, encephalitis virus, flavivirus (e.g. yellow fever virus, dengue virus, Japanese encephalitis (JE) virus, a chimeric dengue virus (yellow fever-dengue) virus, a chimeric YF-WN (yellow fever-West Nile virus) virus, a chimeric YF-JE (yellow fever-Japanese encephalitis) virus, Kunjin virus, West Nile (WN) virus, tick-borne encephalitis (TBE) virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Zika virus), rhabdovirus (e.g. vesicular stomatitis virus (VSV)), retrovirus (e.g. RNA tumor viruses), adenovirus (e.g. human adenovirus, bovine adenovirus, a canine adenovirus, a non-human primate adenovirus, a chicken adenovirus, or a porcine or swine adenovirus), adeno-associated viruses, influenza virus type A (H1N1), lentiviral e.g., human immunodeficiency viruses (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV)), herpes simplex virus, cytomegalovirus, picornavirus (e.g. Rhinovirus, Poliovirus etc), baculovirus vectors (autographacalifornica multiple nucleopolyhedrovirus (AcMNPV), hepatitis B virus (HBV), rubula virus (new castle disease virus), parainfluenza virus, influenza virus, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), respiratory Coronavirus (CoV), human SARS Coronavirus, Ebola, Marburg, Nipah, Chikungunya, Chandipura virus, Rotavirus, Human papilloma virus, Herpes simplex virus, herpes simplex virus type 1 (HSV-1), Hepatitis A, Hepatitis C, Hepatitis B, Hepatitis E, Poliovirus, Variola Virus (e.g. smallpox, Monkeypox) and Varicella virus antigens.
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In some embodiments of present disclosure, a method of quantifying content of an ingredient in a biological sample, the method comprising:
a. providing a biological sample;
b. providing an internal standard;
c. subjecting the biological sample of step a) and the internal standard of step b) to
capillary electrophoresis chromatography comprising a capillary column;
d. identifying the peak of the ingredient and determining the content of ingredient in
the biological sample;
wherein the internal standard is a cation selected from lithium (Li+), sodium (Na+), magnesium (Mg2+), calcium (Ca2+) and potassium (K+).
In some embodiments of present disclosure, the vaccine or the vaccine sample is Neisseria meningitis conjugate vaccine, Salmonella conjugate vaccine, Haemophilus influenzae type b (Hib) and Streptococcal conjugate vaccine.
In some embodiments of present disclosure, the method of quantifying content of Tris in Neisseria meningitis conjugate vaccine sample, the method comprising:
a. providing the Neisseria meningitis conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography;
d. identifying the peak of Tris and determining the content of Tris in the Neisseria
meningitis conjugate vaccine sample.
In some embodiments of present disclosure, the method of quantifying content of Tris in Haemophilus influenza type B conjugate vaccine sample, the method comprising:
e. providing the Haemophilus influenza type B conjugate vaccine sample;
f. providing a cation comprising lithium (Li+) as internal standard;
g. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography;
h. identifying the peak of Tris and determining the content of Tris in the Haemophilus influenza type B conjugate vaccine sample.
In some embodiments of present disclosure, the method of quantifying content of Tris in Salmonella conjugate vaccine sample, the method comprising:
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i. providing the Salmonella conjugate vaccine sample;
j. providing a cation comprising lithium (Li+) as internal standard;
k. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography; l. identifying the peak of Tris and determining the content of Tris in the Salmonella
conjugate vaccine sample.
In some embodiments of present disclosure, the method of quantifying content of Tris in Streptococcal conjugate vaccine sample, the method comprising:
a. providing the Streptococcal conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography;
d. identifying the peak of Tris and determining the content of Tris in the Streptococcal
conjugate vaccine sample.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps and can mean "includes”, "including”, and the like; “consisting essentially of” or “consists essentially” likewise is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
The use of the expression “one or more” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Variations or modifications to the composition of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this disclosure.
The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned
32

to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary.
Similarly, the components used in purification, e.g., filters, columns, are not intended to be in any way limiting or exclusionary, and can be substituted for other components to achieve the same purpose at the discretion of the practitioner.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustration of the disclosure and not as a limitation.
TECHNICAL ADVANTAGES
Applicant’s improved Capillary Zone Electrophoresis based method for TRIS quantification in vaccine/drugs/monoclonal antibody formulations/manufacturing process (particularly in polysaccharide-protein conjugate vaccine/MenFive formulations & process) or any other biological solution provides following advantages over previously reported methods (RV-HPLC, LC-UV, Ion Chromatography, HILIC, GC, RPLC- Electrospray Ionization tandem mass spectrometry)-1) Li+ / Lithium hydroxide (instead of Na+) used as cation internal standard provides
good correlation coefficient, reproducibility, resolution of peaks /well resolved
peaks/ provide spike recovery within limits
2) No interference of sucrose and sodium citrate on Tris migration time (No peak broadening of Tris was observed in presence of other excipients)
3) Sensitive even at lower concentration (i.e. Limit of detection is 3µg/mL); the limit of detection (LOD) 2 µg/ml - 4 µg/ml and limit of quantification (LOQ) 10 µg/ml - 20 µg/ml.
4) Optimized parameters- Coating solution A (comprising polyethyleneimine); Coating
solution B (comprising polyacrylic acid); Chromophore containing Cation separation
buffer (ultra-pure water, propyl-4-hydroxy-benzoate NaCl, 4-aminopyridine, malic
acid, 18-crown- 6) and Conditioner-Na (NaOH solution); separation Voltage of
30KV (1.0 Ramp, normal Polarity; for cation separation to reduce migration
time); separation pressure of 20 psi; capillary column ID of 75µm (for
33

symmetrical peak shape of Tris was observed/ no peak broadening), capillary cartridge temperature of 25°C; capillary conditioning time of 0.5 min (to reduce sample run time and to minimize the usage of conditioning reagents), total capillary length of 60 cm and maximum current of 33µA; detection wavelength-200 nm.
5) Does not require analytical column, chromatography, analytical grade solvents, carrier gas, costly derivatizing agents and sample preparation accessories like filters, syringe and cartridges
6) Devoid of any substantial sample pre-treatment step (except dissolution of lyophilized vial)
7) Method does not require acid base hydrolysis thereby ensuring there is no degradation during testing
8) Method provides short duration for sample/standard run time – 8 minutes.
9) Total run time for sample/standard analysis is shorter (Process time for one injection program Approx. 13 minutes.
The present invention is illustrated in more detail by the following embodiments and combinations of embodiments which result from the corresponding dependency references and links:
I. A method of quantifying content of an ingredient in a biological sample, the method
comprising:
a. providing a biological sample;
b. providing an internal standard;
c. subjecting the biological sample of step a) and the internal standard of step b) to
capillary electrophoresis chromatography comprising a capillary column;
d. identifying the peak of the ingredient and determining the content of ingredient in
the biological sample;
wherein the internal standard is a cation selected from lithium (Li+), sodium (Na+),
magnesium (Mg2+), calcium (Ca2+) and potassium (K+).
II. The method as disclosed in embodiment I, wherein the ingredient is selected from Tris, Tris
HCl, Tris Buffered Saline (TBS), Tris Acetate EDTA (TAE), Tris Borate EDTA (TBE), Tris Glycine Buffer, Tris-Glycine-Sodium Dodecyl Sulfate (SDS), ammonium sulphate, sodium citrate, sodium chloride and magnesium sulphate.
34

III. The method as disclosed in embodiment I, wherein the biological sample is spiked with a working standard.
IV. The method as disclosed in embodiment I, wherein the biological sample is not spiked with a working standard.
V. The method as disclosed in embodiment III-IV, wherein the working standard is a
component similar to the ingredient to be quantified.
VI. The method as disclosed in embodiment I-V, wherein internal standard is mixed with
working standard in the ratio of 1:10 to 10:1.
VII. The method as disclosed in embodiment I, wherein the capillary column comprises of fused
silica with internal diameter ranging from 25 µm to 100 µm, preferably 75 µm.
VIII. The method as disclosed in embodiment I, wherein the capillary electrophoresis
chromatography is performed using separation voltage ranging from 5kV to 40kV,
preferably 30kV.
IX. The method as disclosed in embodiment I, wherein the capillary electrophoresis
chromatography is performed using capillary cartridge temperature ranging from 10°C to
30°C, preferably 25°C.
X. The method as disclosed in embodiment I, wherein the capillary electrophoresis
chromatography is performed using separation pressure ranging from 5psi to 30 psi,
preferably 20 psi.
XI. The method as disclosed in embodiment I, wherein the capillary column is rinsed with at
least one coating solution comprising:
a. monoamines selected from triethylamine and propylamine, hydroxylamine,
ethylamine, morpholine, N,N-diethylethanolamine, triethanolamine, quaternary base
tetramethylamonium chloride;
b. diamines selected from cadaverine, putrescine, agmatine 1,3 diaminopropane and
ethylenediamine;
c. polyamines selected from spermidine, spermine chitosan and polyethyleneimine
(PEI);
d. polyethers selected from polyethylene glycol, polyethylene oxide;
e. polymers selected from polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid;
f. quarternized amino functional monomer such as methacrylamidopropyltrimethyl
ammonium chloride;
g. sulfonic acid acrylic monomer such as acrylamidomethylpropylsulfonic acid.
35

XII. The method as disclosed in embodiment I, wherein the capillary column is rinsed with a
separation buffer comprising a mixture of separation solution and atleast one coating solution.
XIII. The method as disclosed in embodiment XII, wherein the separation solution comprises benzoates, pyridines, acids, chromophores, or combination thereof.
XIV. The method as disclosed in embodiment I, wherein the biological sample is a vaccine sample.
XV. The method as disclosed in embodiment XIV, wherein the vaccine sample can be selected
from but not limited to live-attenuated vaccines; inactivated vaccines; subunit vaccines;
recombinant vaccines; polysaccharide-carrier protein conjugate vaccines; polysaccharide
vaccines; toxoid vaccines; mRNA vaccines; viral vaccines and viral vector vaccines.
XVI. The method as disclosed in embodiment XV, wherein the vaccine sample is
polysaccharide-carrier protein conjugate vaccines.
XVII. The method as disclosed in embodiment XV, wherein the polysaccharide-carrier protein
conjugate vaccine sample includes polysaccharide selected from Streptococcus spp. as Group A Streptococcus, Group B Streptococcus selected from group Ia, Ib, II, III, IV, V, VI, VII, VII, VIII, and IX; Streptococcus pneumoniae serotype selected from 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48, Streptococcus pyogenes; Streptococcus agalactiae; Streptococcus viridans; Salmonella spp.;, Salmonella typhi; Salmonella paratyphi; Salmonella enteritidis; Salmonella typhimurium; Shigella spp. as Shigella sonnei, Shigella flexneri, Shigella dysenteriae; Shigella boydii; E.coli; Neisseria meningitidis serotypes selected from A, B, B16, B6, C, D, E29, H, I, K, K454 L, M, W135, X, Y, and Z; Neisseria gonorrhoeae; Haemophilus influenzae; Haemophilus pneumonia; Helicobacter pylori; Chlamydia pneumoniae; Chlamydia trachomatis; Ureaplasma urealyticum; Mycoplasma pneumoniae; Staphylococcus spp.; Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8; Enterococcus faecalis; Enterococcus faecium; Bacillus anthracis; Vibrio cholerae; Pasteurella pestis; Pseudomonas aeruginosa; Campylobacter spp.; jejuni; Clostridium spp.; Clostridium difficile; Mycobacterium spp.; Mycobacterium tuberculosis; Moraxella catarrhalis; Klebsiella pneumoniae; Treponema spp.; Borrelia
36

spp.; Borrelia burgdorferi; Leptospira spp.; Hemophilus ducreyi; Corynebacterium
diphtheria; Bordetella pertussis; Bordetella parapertussis; Bordetella bronchiseptica;
Ehrlichia spp.; and Rickettsia spp.
XVIII. The method as disclosed in embodiment XVI, wherein the carrier protein is selected from
the group comprising CRM197, diphtheria toxoid, tetanus toxoid, Neisseria meningitidis
outer membrane complex, fragment C of tetanus toxoid, recombinant full-length tetanus
toxin with eight individual amino acid mutations (8MTT), pertussis toxoid, protein D of H.
influenzae, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, outer
membrane complex c (OMPC), Outer Membrane Protein T2554 from Salmonella spp, Pilus
proteins from Streptococcus spp, porins, transferrin binding proteins (Tbp-B, Tbp-A),
pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A
(PsaA), PhtA, PhtB, PhtE, pneumococcal PhtD, pneumococcal surface proteins BVH-3 and
BVH-11, M. catarrhalis uspA, protective antigen (PA) of Bacillus anthracis and detoxified
edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet
hemocyanin (KLH), C5a peptidase group A or group B Streptococcus, human serum
albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD),
Cholera toxin subunit B, fHbp, Por A and Por B.
XIX. The method as disclosed in embodiment XVI - XVIII, wherein the vaccine sample is
Neisseria meningitis conjugate vaccine, Salmonella conjugate vaccine, Streptococcal
conjugate vaccine, Haemophilus influenzae type b (Hib) conjugate vaccine.
XX. The method of quantifying content of Tris in Neisseria meningitis conjugate vaccine sample
as disclosed in embodiment XIX, the method comprising:
a. providing the Neisseria meningitis conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography comprising a capillary column;
d. identifying the peak of Tris and determining Tris content in the Neisseria meningitis
conjugate vaccine sample.
XXI. The method of quantifying content of Tris in Haemophilus influenza type B conjugate
vaccine sample as disclosed in embodiment XIX, the method comprising:
a. providing the Haemophilus influenza type B conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography comprising a capillary column;
37

d. identifying the peak of Tris and determining the content of Tris in the Haemophilus
influenza type B conjugate vaccine sample.
XXII. The method of quantifying content of Tris in Salmonella conjugate vaccine sample as
disclosed in embodiment XIX, the method comprising:
a. providing the Salmonella conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography comprising a capillary column;
d. identifying the peak of Tris and determining the content of Tris in the Salmonella
conjugate vaccine sample.
XXIII. The method of quantifying content of Tris in Streptococcal conjugate vaccine sample as
disclosed in embodiment XIX, the method comprising:
a. providing the Streptococcal conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography comprising a capillary column;
d. identifying the peak of Tris and determining the content of Tris in the Streptococcal
conjugate vaccine sample.
XXIV. The method as disclosed in embodiment I-XXIII, wherein the vaccine sample is spiked with
Tris as working standard.
XXV. The method as disclosed in embodiment I-XXIII, wherein the vaccine sample is not spiked
with Tris as working standard.
XXVI. The method as disclosed in embodiment I-XXIII, wherein lithium (Li+) is mixed with Tris
as working standard in the ratio of 1:10 to 10:1.
XXVII. The method as disclosed in embodiment I-XXIII, wherein the capillary column is rinsed
with at least one coating solution selected from polyethyleneimine, polyacrylic acid or
combinations thereof.
XXVIII. The method as disclosed in embodiment I-XXIII, wherein the capillary column is rinsed
with a separation buffer comprising propyl-4-hydroxy-benzoate (sodium chloride) stock solution, 4-aminopyridine, malic acid, 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6) in combination with polyacrylic acid.
38

EXAMPLES:
The foregoing description of the embodiments has been provided for purposes of illustration and not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment, but, are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.
The present invention is further described in light of the following examples which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure.
EXAMPLE 1: A) Strains and source: Table 1 provides the details about the biological material used in the vaccine composition and their source.
Table 1: Biological material and their source

Serogroup
Men A

Strain
Neisseria
meningitidis Strain M1027

Source of strain
Center for Biologics Evaluation and Research, FDA, USA

Men C

Neisseria
meningitidis Strain C11

Center for Biologics Evaluation and Research, FDA, USA

Men Y

Neisseria
meningitidis Strain M10659

CDC, USA

Men W
Men X

Neisseria
meningitidis Strain S877
Neisseria
meningitidis Strain M8210

Center for Biologics Evaluation and Research, FDA, USA
Center for Biologics Evaluation and Research, FDA, USA

Salmonella enterica typhi ATCC 19430; C6524
S. typhi

Identified by Geneombio Technologies Private
serovarLimited, Pune and isolated from stool sample of
typhoid confirmed patient at Villoo Poonawalla
Memorial Hospital, Pune

S. paratyphi A

Salmonella ATCC 9150

paratyphi

A:Chromachemie Bangalore

Laboratory

Private

Limited,

type b
CRM
DT
Isolated at Academic Medical Centre (AMC),
H. influenzaeHaemophilus influenzae
University of Amsterdam. This strain was transferred type
b, strain 760705
to SIIPL as a part of collaboration between SIIPL and
Netherlands Vaccines Institute (NVI, The
Netherlands)
Pseudomonas fluorescens
Pfenex Inc. USA expressing CRM197
Park-Williams strain
The strain was obtained from Wellcome Research
Cornynebacterium diphtheriaeLaboratory by the National Control Authority C.R.I.
Number 8Kasauli, in lyophilized form in the year 1973. The
strain was revived and further lyophilized under “Master Seed Lot - C. diphtheriae CN2000 A1” at
39

C.R.I. Kasauli. 25 copies of the Master Seed Lot were obtained from National Control Authority C.R.I. Kasauli by Serum Institute of India.
TT Originally obtained from The Rijks Institute Voor de Clostridium tetani Harvard
Volksgezondheid (Netherlands) by the National 49205
Control Authority C.R.I. Kasauli,
B) Composition details:
Composition details of Pentavalent Meningococcal conjugate vaccine, Bi-valent Typhoid Conjugate vaccine and Haemophilus influenzae type b (Hib) conjugate vaccine are provided below (Refer Tables 2, 3 and 4).
a) Meningococcal (Pentavalent) conjugate vaccine:
The quantities of inactive ingredients post reconstitution, for both 5 & single dose is 5 µg each for ACYWX per dose of 0.5 ml.
Table 2: Composition of Meningococcal (Pentavalent) conjugate vaccine

Excipient Single Dose 5-Doses
Sucrose 15 mg/vial 15 mg/vial
Sodium Citrate (Dihydrate) 2.5 mg/vial 2.5 mg/vial
Tris Buffer 0.61 mg/vial 0.61 mg/vial
b) Typhoid (Bi-valent) Conjugate vaccine:
Table 3: Composition of Typhoid (Bi-valent) Conjugate vaccine

Excipient 5-Doses
Tris Buffer 0.3 mg/dose
Mannitol 25 mg/dose
c) Haemophilus influenzae type b (Hib) conjugate vaccine:
Table 4: Composition of Haemophilus influenzae type b (Hib) conjugate vaccine

Excipient Single dose
Tris Buffer 0.73 mg/dose
Sucrose 25.9 mg/dose
EXAMPLE 2:
40

A) Impact of tris on stability:
The pH stability of each polysaccharide in Menfive vaccine in a solution at different pH was checked by keeping the polysaccharide solutions at room temperature for 24Hr. From Table 5 it was observed that the average molecular weight of meningococcal polysaccharides C, Y, W and X did not show any significant change at the end of 24hrs. While meningococcal A polysaccharide was found sensitive to pH below 5 and above 8. Thus, it was concluded to make stabilizer mixture for lyophilization in a buffer which has suitable buffering range by tris component minimum buffer 10mM tris. The same approach was considered for final formulation Menfive vaccine.
Table 5: Avg Molecular Weight of polysaccharides (Men A, Men C, Men Y, Men W, Men
X) at different pH

Polysaccharide Serogroup Initial (kDa) Avg Molecular Weight After 24 hrs treatment at RT (kDa)

pH 4.0 pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0
Men A 378 258 285 348 353 340 326
Men C 418 405 410 423 430 428 432
Men Y 342 330 347 346 358 368 357
Men W 342 335 342 348 357 355 358
Men X 137 136 145 149 151 148 153
Table 6: Molecular weight of conjugates by SEC-HPLC (Mw) Kda

Bulk Conjugate Molecular weight by SEC-HPLC (Mw) Kda

10mM Tris Without TRIS
Men A -TT 618 562
Men C-CRM 899 889
Men Y-CRM 817 800
Men W-CRM 942 932
Men X-TT 952 944
From above data (refer Table 6), it was observed that MenA -TT conjugate with tris buffer has higher molecular weight than that of conjugate devoid of tris as stabilizer.
B) Force degradation study
Force degradation study was performed at 90°C and molecular size distribution of Neisseria Meningitis bulk conjugates was evaluated. It was observed that there is decrease in MSD value of Men A-TT conjugate at 90°C as the degradation study proceeds from 0 hr to 20 hrs. (Refer Table 7)
41

Table 7: Force degradation at 900C study

Bulk conjugates Molecular size distribution (MSD) of Meningococcal bulk conjugate

Force degradation at 900C % MSD ELUTION BEFORE 0.5kd
Men A-TT 0HR 96
2HR 74
20HR 56

MEN C-CRM 0HR 96
3HR 95
6HR 93
24HR 80
EXAMPLE 3: A) Solutions/ Buffers/ Reagents:
1. Stock standard solution (Tris 1mg/ml): 50 mg of Tris (Trizma base) was dissolved in 50 ml milliQ water or ultra-pure water
2. Working standard solution (Tris 200 µg/ml): 10 ml of stock standard solution was taken in volumetric flask and final volume was make up to 50 ml with MQW or ultra-pure water
3. Cation internal standard solution (1388 ppm Lithium (Li) ion): 20-fold diluted internal standard was used for both standard and samples.
Set of Tris standard and internal standard were prepared as follows (refer Table 8):
Table 8: Preparation of Tris standard and internal standard

Sr No
1 2 3 4 5 6 TRIS Working Standard (µl) MQW Li (internal std) 20F Diluted (µl) Concentration (µg/ml) Total Volume

20 160 20 20 200

40 140 20 40 200

60 120 20 60 200

80 100 20 80 200

100 80 20 100 200

120 60 20 120 200
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4. Sample preparation: Vaccine vial was dissolved in 1ml of MQW or ultra-pure water and vortexed. It was allowed to dissolve for approximately for 1hr and the sample were diluted 2-fold with MQW.
5. Sample with spike of Tris: Vaccine vial was dissolved in 1ml of MQW or ultra-pure water and vortexed. It was allowed to dissolve for approximately for 1hr and the sample were diluted 2-fold with MQW. Tris working standard (40µl) was added to the 2-fold diluted sample solution
Sample and its spike are prepared as follows (refer Table 9):
Table 9: Preparation of Sample and its spike solutions

Sample MQW
(µl) Sample (2-Fold Diluted) (µl) TRIS
Working STD (µl) Li (internal std) (20F Diluted) (µl) Total Volume (µl)
Sample 155 25 0 20 200
Sample +Spike 115 25 40 20 200
100 µl of each standard, sample and spike sample were transferred into respective PCR vial and cover with cap and label each vial.
6. Cation coating solution A:
a) Polyethyleneimine (1% wt) stock solution preparation: Dilute 200 mg of the Polyethyleneimine (50% wt) to a final volume of 10 ml with filtered deionized water. Use a stir bar to mix well.
b) In a graduated cylinder, add 105 µl of the prepared Polyethyleneimine (1% wt) stock solution. Add water until the total volume reached 100 ml.
7. Cation coating solution B:
a) Polyacrylic acid (~MW. 450,000) stock solution preparation (0.5% wt.): Weigh 50 mg of Polyacrylic acid (~MW. 450,000) into a glass vial and add 10 ml of filtered deionized water. Cap the glass vial and seal it well. Put the glass vial onto a rotator and allow the polymer to slowly dissolve minimum overnight.
b) Mix 1 ml of the prepared polyacrylic acid, stock solution (0.5% wt.) with 49 ml of filtered deionized water.
8. Cation separation buffer:
43

a) Propyl-4-hydroxybenzoate (NaCl) stock solution preparation: Weigh out 25 mg of Propyl-4-hydroxybenzoate (NaCl) and dissolve the solid in 10 ml of filtered deionized water in a 15 ml conical tube. Cover the conical tube and mix well by inverting it multiple times.
b) In a graduated cylinder with 70 ml of filtered deionized water, while stirring, add 1 ml of Propyl-4-hydroxybenzoate (NaCl) stock solution, 188 mg of 4-aminopyridine, 254 mg of malic acid, 450 mg of 18-crown-6 and 260µl of prepared cation coating solution B. Cover with parafilm and invert to mix until all solids are fully dissolved. After inverting, add water until the total volume reaches 100 ml.

9. Conditioner-Na: 01.N NaOH solution
10. Rinse solution: CE grade water/ WFI (filtered ultra-pure water)
B) Instrumentation:
a. Capillary cartridge temperature: 25°C
b. Auto sampler temperature: 25°C
c. Total capillary length: 60cm (50 cm from injection site to detector)
d. Capillary ID: 75µm
e. Detector: UV (i.e. Ultra violet)
f. Detection wavelength: 200nm
g. Separation voltage: 30kV (1.0 Ramp, normal Polarity)
h. Separation pressure: 20psi
i. Max current: 33µA
j. Auxiliary data channels: Current
k. Absorbance signal: Indirect mode
C) Run Program: Separation time programme
Table 10 provides the sequential steps of the sample run alonwith Separation time programme
Table 10: Separation time programme

Sr No Event Value (psi) Duration (time) Method Comments
1 Rinse pressure 20 1 min Rinse with Cation coating solution A
2 Rinse pressure 20 1 min Rinse with Cation coating solution B
3 Rinse pressure 20 1.5 min Rinse with Cation separation
44

buffer
4 Wait 20 sec Water dip
5 Inject pressure 0.5 5 sec Sample injection
6 Inject pressure 0.1 10 sec Water injection
7 Separation voltage 30KV 8 min Separation voltage with Cation separation buffer
8 Rinse pressure 20 0.5 min Rinse with conditioner Na
9 Rinse pressure 20 0.5 min Rinse with rinse solution
Total run time single standard/ sample is 13.08 minutes. Capillary is conditioned using the above separation programme.
D) Injection sequence:
Standards and samples were run in duplicate using above mentioned separation programme. Sequence of injecting standards and samples are created as follows (Refer Table 11).
Table 11: Injection sequence

Sr No Standard/ Sample (with Lithium internal standard) Method
1 Blank CATION SEPARATION
2 STD 1 INJECTION - 1 CATION SEPARATION
3 STD 2 INJECTION - 1 CATION SEPARATION
4 STD 3 INJECTION - 1 CATION SEPARATION
5 STD 4 INJECTION - 1 CATION SEPARATION
6 STD 5 INJECTION - 1 CATION SEPARATION
7 STD 6 INJECTION - 1 CATION SEPARATION
8 SAMPLE INJECTION 1 CATION SEPARATION
9 SAMPLE INJECTION 2 CATION SEPARATION
10 SAMPLE WITH SPIKE INJECTION 1 CATION SEPARATION
11 SAMPLE WITH SPIKE INJECTION 2 CATION SEPARATION
12 STD 1 INJECTION – 2 CATION SEPARATION
13 STD 2 INJECTION – 2 CATION SEPARATION
14 STD 3 INJECTION – 2 CATION SEPARATION
15 STD 4 INJECTION – 2 CATION SEPARATION
16 STD 5 INJECTION – 2 CATION SEPARATION
17 STD 6 INJECTION - 2 CATION SEPARATION
45

E) Chromatographic integration:
- After completion of sequence, integrate the acquired data both standard and samples using proper processing method in 32Karat software.
- Plot the calibration curve of Tris standard concentration Vs ratio of Tris area and lithium area (Concentration Vs Tris/ Lithium).
- Calculate slope and intercept of the linear curve. Assay is valid when the correlation coefficient is ≥0.99 and spike recovery is between 80 - 120%.
F) Calculation:
Tris (mg/vial or mg/ml) = (Average of ratio of sample/Li - intercept) x dilution
Slope x 1000 % Spike Recovery = Practical concentration x 100
Theoretical concentration Where, Practical conc. (μg/ml) = (Average of ratio of Tris/Li in Spike - Intercept)
Slope Theoretical conc. (μg/ml) = (Average of ratio of Tris/Li in Spike - Intercept) + 40
Slope Note: 40 is the known amount of Tris in μg spiked in sample to estimate % spike recovery
Calculation of Tris estimation of NmCv5 1 dose vial: Tables 12, 13 and 14 provides the readings/ values for calculation Tris content, Practical concentration, Theoretical concentration
and % Spike recovery.
Table 12: Calculation Part I

Std (µg/mL) Injection Corrected Tris Area Corrected Lithium Area Tris/Lithium Ratio Avg.
Tris/Lithium
Ratio
20 Inj-1 910 4547 0.2001 0.1967

Inj-2 749 3876 0.1932

40 Inj-1 1721 4453 0.3865 0.3691

Inj-2 1462 4156 0.3518

60 Inj-1 2829 4543 0.6227 0.6031

Inj-2 2441 4183 0.5836

46

80 Inj-1 3403 4406 0.7724 0.7635

Inj-2 3376 4474 0.7546

100 Inj-1 4123 4088 1.0086 0.9908

Inj-2 4376 4497 0.9731

120 Inj-1 4377 3751 1.1669 1.1787

Inj-2 5113 4295 1.1905

Slope 0.01
Intercept -0.01
Correlation Coefficient (r) 0.999
Table 13: Calculation Part II

Sample Name Injection Sample Tris Area Sample Lithium Area Tris/Lithium Ratio Avg.
Tris/Lithium
Ratio
NmCV5 1 Dose Inj-1 1565 3756 0.4167 0.4161

Inj-2 1757 4229 0.4155

Table 14: Calculation Part III

Spike Recovery Sample Name Injection Spike Tris Area Spike Lithium Area Tris/Lithium Ratio Avg.
Tris/Lithium
Ratio
NmCV5 1 Dose Inj-1 3034 3740 0.8112 0.8151

Inj-2 2889 3528 0.8189

1) Tris (mg/vial or mg/ml) = (Average of ratio of sample/Li - intercept) x dilution
Slope x 1000 Tris (mg/vial or mg/ml) = (0.4161 - (-0.01) x 16
0.01 x 1000 Tris (mg/vial or mg/ml) = 0.682
2) Practical conc. (μg/ml) = (Average of ratio of Tris/Li in Spike - Intercept)
Slope Practical conc. (μg/ml) = (0.8151 - (-0.01)
0.01 Practical conc. (μg/ml) = 82.51
3) Theoretical conc. (μg/ml) = (Average of ratio of Tris/Li in Spike - Intercept) + 40
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Slope Note: 40 is the known amount of Tris in μg spiked in sample to estimate % spike recovery Theoretical conc. (μg/ml) = (0.4161 – (-0.01) + 40
0.01 Theoretical conc. (μg/ml) = 82.61
4) % Spike Recovery = Practical concentration x 100
Theoretical concentration % Spike Recovery = 82.51 x 100
82.61 % Spike Recovery = 99.88
EXAMPLE 4: A) LOD, LOQ, Linearity, Assay precision, Accuracy:
The method validation considering below parameters was performed as per ICH guideline Q2 (R1) “text and methodology on validation of analytical procedures”. (Refer Tables 15 and 16)
Table 15: Method validation parameters

Sr No Parameters Values
1 LOD 3.0µg/ml
2 LOQ 15.0 µg/ml
3 Linearity 20 µg/ml to 120 µg/ml
4 Assay precision ≤ 10%
Accuracy:
Table 16: % Accuracy

Sr No Spike Concentration level % Accuracy
1 30 µg 100.99
2 60 µg 103.97
3 80 µg 96.93
Observation: % Accuracy carried out during validation at three concentration level in final fill vaccine was found between 80 to 120% (refer Table 16). Moreover, method does not require acid base hydrolysis or derivatizing agents for separation which in turn proves that there is no degradation takes place during analysis.
B) Comparison of internal standard Li+ Versus Na+ for TRIS quantification in Neisseria meningitis conjugate vaccine
Using conventional Na+ as an internal standard is not feasible due to potential co-elution or
48

overlap with Sodium citrate in the final fill vaccine (refer Figures 1, 2 and 3). This interference could affect the accurate quantitation of Tris. Instead, we opted for Lithium hydroxide/lithium chloride as an internal standard, as its retention time differs from that of Tris, preventing peak overlap.
Generally, sodium ion is a routine ion found in most of the vaccine formulations or compositions in the form of a component such as sodium chloride, sodium citrate and other sodium salts. Sodium is not used as internal standard as there can be overlapping of the peaks of sodium from vaccine component and peak of sodium from internal standard. This overlapping will not be able to provide the accurate quantification of the component to be analyzed. When conventional sodium cation is used as an internal standard, the potential co-elution and overlap with the sodium ion of the excipient in the vaccine could affect the accurate quantification results. Same can be observed with magnesium, calcium and potassium. Thus, lithium may be considered as suitable cation internal standard which may avoid the overlapping of peaks. Lithium cation of lithium salts as internal standard provide a peak having a retention time different from that of the cation to be quantified and prevents the overlapping of peak.
C) Comparison of Conditioner (Li+ versus Na+) in Neisseria meningitis conjugate vaccine
Since Tris quantification relies on Lithium as an internal standard, using LiOH (0.1N) as a conditioner is not feasible due to potential Li+ carryover. This carryover could interfere with accurate quantification. Therefore, NaOH (0.1N) has been chosen as the conditioner for method development.
D) Comparison of Separation Voltage (30KV, 25KV, and 20KV) considering retention
time:
In this study separation voltages at 30KV, 25KV, and 20KV were examined. From Figure 4, 5 and 6 it was observed that as voltage decreased, retention time shifted, and there was slight peak broadening at 20KV observed.
E) Comparison of Separation Voltage (15KV, 20KV and 30KV) considering spike
recovery:
Table 17: Comparison of Separation Voltage (15KV, 20KV and 30KV) considering
spike recovery

Sr No Sample Name Voltage % Spike Recovery (80 -120%) Correlation (≥ 0.99)
49

1 5D Menfive vaccine 15KV 71 0.95
2
20 KV 77 0.97
3
30KV 95 0.99
Observation: From Table 17 it was observed that separation optimized at 30 KV complies minimum assay validity criteria of spike recovery (95%) and correlation coefficient (0.99).
F) Comparison of Capillary column ID (50µm, 75µm, 100µm):
Although spike recovery fits within the expected range for 50µm, 75µm and 100µm, there was noticeable peak broadening was observed for 100µm ID capillary column (refer Table 18).
Table 18: Comparison of Capillary column ID (50µm, 75µm, 100µm)

Sr No
1 2
3 Sample Name Capillary column ID (µm) % Spike Recovery (80 -120%) Observation

5D
Menfive
vaccine 50 -- Separation did not occur as the capillary column blocked

75 95 Spike recovery was found within range. symmetrical peak shape of Tris was observed

100 92 Spike recovery was found within range but Peak broadening of Tris was observed.
G) Comparison of Capillary cartridge temperature (20°C, 25°C, 30°C):
The study of capillary cartridge temperature was conducted at 20°C, 25°C and 30°C. Although there was no substantial alteration in the actual Tris content, slight variations in migration time were observed at these temperatures. As a result of validation studies, the capillary cartridge temperature was ultimately set at 25°C. Also refer Table 19 and Figures
7, 8 and 9.
Table 19: Tris content at different capillary cartridge temperature

Sr No Sample Name Capillary cartridge temperature Tris content (mg/ vial)
1 5D- Men five vaccine 20 °C 0.665
2
25 °C 0.655
3
30 °C 0.649
H) Comparison of Conditioning time (0.5 min, 1 min and 2 min):
50

The capillary was conditioned using coating buffer A and coating buffer B at 20 psi for 0.5 minutes to shorten sample run time and minimize the consumption of conditioning reagents. Refer Table 20 below.
Table 20: Comparison of Conditioning time (0.5 min, 1 min and 2 min)

Sr No
1 2 3 Sample Name Rinse pressure Coating Buffer Time Results

5D Menfive vaccine 20 psi Coating A and coating B buffer 0.5 min Baseline separation of Tris and Lithium was observed

20 psi
1 min Baseline separation of Tris and Lithium was observed

20 psi
2 min Baseline separation of Tris and Lithium was observed
I) Comparison of Total capillary length (60cm and 80cm):
The study compared total capillary total lengths of 60 cm and 80 cm. Although the migration time of Tris shifted from 4.1 to 6.2, there was no significant alteration in the actual Tris content (Refer Figure 10 and 11).
J) Comparative Data [Generation of Maximum current:
The method separates cations based on voltage, resulting in the generation of approximately 33µA current in response to a 30KV voltage (refer Figure 12).
K) TRIS estimation without interference of excipients:
In the specificity experiment, Figures 13, 14 and 15 indicate that sodium citrate was eluted around 3.0 minutes, while Tris was eluted at approximately 4.1 minutes. Sucrose did not exhibit any peak during the run, and there was no broadening of the Tris peak in the presence of other excipients.
L) Sensitivity comparison:
Method sensitivity was calculated using signal to noise ration formula. A signal to noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit (refer Figure 16 and 17). Calculation: S/N = 2H
h where S/N = Signal to noise H = Height of peak h – peak to peak background noise
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The S/N ratio for 3µg/ml tris standard is 5.
Tris analysis LOD is 3µg/ml.
This calculation showed that 3.0 µg/ml is lowest concentration compared with other
concentrations.
M) TRIS estimation in Hib conjugate vaccine:
In Hib conjugate vaccine formulations, Tris estimation can be performed without interference from excipients as shown in Figure 18. In this context, either lithium hydroxide or sodium chloride can serve as an internal standard.
N) TRIS estimation in Typhi/ Paratyphi bivalent conjugate vaccine:
In the formulation of a 5-dose typhoid bivalent vaccine, Tris estimation can be performed without interference from excipients as shown in Figure 19. In this context, either lithium chloride or sodium chloride can serve as an internal standard.
INFERENCE:
The experimental data mentioned in above in examples shows that the developed TRIS quantification method is robust and reproducible and validated as per ICH guidelines. Also,
• Method does not require acid base hydrolysis or derivatizing agents for separation which in turn proves that no degradation takes place during analysis.
• Lithium hydroxide/lithium chloride as an internal standard prevents peak overlap as its retention time differs from that of Tris.
• Use of NaOH (0.1N) as the conditioner provides accurate quantification without interference.
• Separation optimized at 30 KV voltage, complies minimum assay validity criteria of spike recovery and correlation coefficient with efficient TRIS separation.
• The method provides TRIS estimation without interference of excipients.
52

CLAIMS:
We claim;
1. A method of quantifying content of an ingredient in a biological sample, the method
comprising:
a. providing a biological sample;
b. providing an internal standard;
c. subjecting the biological sample of step a) and the internal standard of step b) to
capillary electrophoresis chromatography comprising a capillary column;
d. identifying the peak of the ingredient and determining the content of ingredient in
the biological sample;
wherein the internal standard is a cation selected from lithium (Li+), sodium (Na+), magnesium (Mg2+), calcium (Ca2+) and potassium (K+).
2. The method as claimed in claim 1, wherein the ingredient is selected from Tris, Tris HCl, Tris Buffered Saline (TBS), Tris Acetate EDTA (TAE), Tris Borate EDTA (TBE), Tris Glycine Buffer, Tris-Glycine-Sodium Dodecyl Sulfate (SDS), ammonium sulphate, sodium citrate, sodium chloride and magnesium sulphate.
3. The method as claimed in claim 1, wherein the biological sample is spiked with a working standard.
4. The method as claimed in claim 1, wherein the biological sample is not spiked with a working standard.
5. The method as claimed in claims 3 - 4, wherein the working standard is a component similar to the ingredient to be quantified.
6. The method as claimed in claims 1 - 5, wherein internal standard is mixed with working standard in the ratio of 1:10 to 10:1.
7. The method as claimed in claim 1, wherein the capillary column comprises of fused silica with internal diameter ranging from 25 µm to 100 µm, preferably 75 µm.
8. The method as claimed in claim 1, wherein the capillary electrophoresis chromatography is performed using separation voltage ranging from 5kV to 40kV, preferably 30kV.
9. The method as claimed in claim 1, wherein the capillary electrophoresis chromatography is performed using capillary cartridge temperature ranging from 10°C to 30°C, preferably 25°C.
10. The method as claimed in claim 1, wherein the capillary electrophoresis chromatography is performed using separation pressure ranging from 5psi to 30 psi, preferably 20 psi.

11. The method as claimed in claim 1, wherein the capillary column is rinsed with at least one
coating solution comprising:
a. monoamines selected from triethylamine and propylamine, hydroxylamine,
ethylamine, morpholine, N,N-diethylethanolamine, triethanolamine, quaternary base
tetramethylamonium chloride;
b. diamines selected from cadaverine, putrescine, agmatine 1,3 diaminopropane and
ethylenediamine;
c. polyamines selected from spermidine, spermine chitosan and polyethyleneimine
(PEI);
d. polyethers selected from polyethylene glycol, polyethylene oxide;
e. polymers selected from polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid;
f. quarternized amino functional monomer such as methacrylamidopropyltrimethyl
ammonium chloride;
g. sulfonic acid acrylic monomer such as acrylamidomethylpropylsulfonic acid.
12. The method as claimed in claim 1, wherein the capillary column is rinsed with a separation buffer comprising a mixture of separation solution and atleast one coating solution.
13. The method as claimed in claim 12, wherein the separation solution comprises benzoates, pyridines, acids, chromophores, or combination thereof.
14. The method as claimed in claim 1, wherein the biological sample is a vaccine sample.
15. The method as claimed in claim 14, wherein the vaccine sample can be selected from but not limited to live-attenuated vaccines; inactivated vaccines; subunit vaccines; recombinant vaccines; polysaccharide-carrier protein conjugate vaccines; polysaccharide vaccines; toxoid vaccines; mRNA vaccines; viral vaccines and viral vector vaccines.
16. The method as claimed in claim 15, wherein the vaccine sample is polysaccharide-carrier protein conjugate vaccines.
17. The method as claimed in claim 16, wherein the polysaccharide-carrier protein conjugate vaccine sample includes polysaccharide selected from Streptococcus spp. as Group A Streptococcus, Group B Streptococcus selected from group Ia, Ib, II, III, IV, V, VI, VII, VII, VIII, and IX; Streptococcus pneumoniae serotype selected from 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48,

Streptococcus pyogenes; Streptococcus agalactiae; Streptococcus viridans; Salmonella
spp.;, Salmonella typhi; Salmonella paratyphi; Salmonella enteritidis; Salmonella
typhimurium; Shigella spp. as Shigella sonnei, Shigella flexneri, Shigella dysenteriae;
Shigella boydii; E.coli; Neisseria meningitidis serotypes selected from A, B, B16, B6, C, D,
E29, H, I, K, K454 L, M, W135, X, Y, and Z; Neisseria gonorrhoeae; Haemophilus
influenzae; Haemophilus pneumonia; Helicobacter pylori; Chlamydia pneumoniae;
Chlamydia trachomatis; Ureaplasma urealyticum; Mycoplasma pneumoniae;
Staphylococcus spp.; Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8; Enterococcus faecalis; Enterococcus faecium; Bacillus anthracis; Vibrio cholerae; Pasteurella pestis; Pseudomonas aeruginosa; Campylobacter spp.; jejuni; Clostridium spp.; Clostridium difficile; Mycobacterium spp.; Mycobacterium tuberculosis; Moraxella catarrhalis; Klebsiella pneumoniae; Treponema spp.; Borrelia spp.; Borrelia burgdorferi; Leptospira spp.; Hemophilus ducreyi; Corynebacterium diphtheria; Bordetella pertussis; Bordetella parapertussis; Bordetella bronchiseptica; Ehrlichia spp.; and Rickettsia spp.
18. The method as claimed in claim 16, wherein the carrier protein is selected from the group comprising CRM197, diphtheria toxoid, tetanus toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, recombinant full-length tetanus toxin with eight individual amino acid mutations (8MTT), pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), Outer Membrane Protein T2554 from Salmonella spp, Pilus proteins from Streptococcus spp, porins, transferrin binding proteins (Tbp-B, Tbp-A), pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), PhtA, PhtB, PhtE, pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, M. catarrhalis uspA, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), C5a peptidase group A or group B Streptococcus, human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), Cholera toxin subunit B, fHbp, Por A and Por B.
19. The method as claimed in claims 16 - 18, wherein the vaccine sample is Neisseria meningitis conjugate vaccine, Salmonella conjugate vaccine, Streptococcal conjugate vaccine, Haemophilus influenzae type b (Hib) conjugate vaccine.
20. The method of quantifying content of Tris in Neisseria meningitis conjugate vaccine sample as claimed in claim 19, the method comprising:

a. providing the Neisseria meningitis conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography comprising a capillary column;
d. identifying the peak of Tris and determining Tris content in the Neisseria meningitis
conjugate vaccine sample.
21. The method of quantifying content of Tris in Haemophilus influenza type B conjugate
vaccine sample as claimed in claim 19, the method comprising:
a. providing the Haemophilus influenza type B conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography comprising a capillary column;
d. identifying the peak of Tris and determining the content of Tris in the Haemophilus
influenza type B conjugate vaccine sample.
22. The method of quantifying content of Tris in Salmonella conjugate vaccine sample as
claimed in claim 19, the method comprising:
a. providing the Salmonella conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography comprising a capillary column;
d. identifying the peak of Tris and determining the content of Tris in the Salmonella
conjugate vaccine sample.
23. The method of quantifying content of Tris in Streptococcal conjugate vaccine sample as
claimed in claim 19, the method comprising:
a. providing the Streptococcal conjugate vaccine sample;
b. providing a cation comprising lithium (Li+) as internal standard;
c. subjecting the vaccine sample of step a) and lithium (Li+) of step b) to capillary
electrophoresis chromatography comprising a capillary column;
d. identifying the peak of Tris and determining the content of Tris in the Streptococcal
conjugate vaccine sample.
24. The method as claimed in claims 1-23, wherein the vaccine sample is spiked with Tris as working standard.
25. The method as claimed in claims 1-23, wherein the vaccine sample is not spiked with Tris as working standard.

26. The method as claimed in claims 1-23, wherein lithium (Li+) is mixed with Tris as working standard in the ratio of 1:10 to 10:1.
27. The method as claimed in claims 1-23, wherein the capillary column is rinsed with at least one coating solution selected from polyethyleneimine, polyacrylic acid or combinations thereof.
28. The method as claimed in claims 1-23, wherein the capillary column is rinsed with a separation buffer comprising propyl-4-hydroxy-benzoate (sodium chloride) stock solution, 4-aminopyridine, malic acid, 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6) in combination with polyacrylic acid.