DESC:TECHNICAL FIELD
The present disclosure relates to the field of biotechnology, more particularly, it relates to a method for the analysis and quality control of compositions that includes unconjugated or free carrier protein more particularly recombinant CRM197.
BACKGROUND
Isolated bacterial capsular polysaccharides from Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis have been successfully used as vaccines in adults. Vaccines based on capsular polysaccharides stimulate humoral immunity but fail to induce immunologic memory, and thus are ineffective in young infants.
For a vaccine to be effective and induce long lasting immunity, induction of T cell memory is very important. Increased immunogenic response of an antigen can be achieved by converting the T-independent antigen to T-dependent antigen. The conjugation of polysaccharide to carrier protein renders the antigenic molecule to undergo the process of antigen presentation, and enhance the polysaccharide immunogenicity by eliciting the T-cell dependent response. The proteins used for conjugation to polysaccharides include CRM197, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane complex, Haemophilus influenzae protein D, Pneumolysin, etc.
The meningococcal meningitis is caused by Neisseria meningitidis which is an aerobic Gram-negative encapsulated bacterium. To date, more than 10 serotypes of meningococcal have been characterized by differences in the polysaccharide capsule. The polysaccharides from serotype A, C, Y, W, and X have been conjugated to various carrier proteins to prepare a conjugated vaccine (Menactra®, Menveo®), effective against infection by these serotypes.
Similarly for Streptococcus pneumoniae, more than 90 distinct serotypes have been identified throughout the world (WHO); of which a small number of serotypes accounts for most diseases in infants. Pneumococcal conjugate vaccines containing poly-saccharides from 7+ serotypes i.e. Prevnar® & Synflorix® are already into market. The polysaccharides from Pneumo serotype have been conjugated to various carrier proteins i.e. CRM197, Protein D, TT, DT, etc. to prepare a conjugated Ps-Pr vaccine against these serotypes.
Polysaccharide-protein conjugate composition are known to be associated with contaminants such as un-reacted polysaccharide (free polysaccharide), un-reacted carrier protein (free protein), low molecular weight conjugates, and other chemicals used for affecting conjugation such as linkers, coupling agents etc. Such contaminants are highly undesirable in a product which is intended for use as a vaccine.
The amount of free polysaccharide and free carrier protein in the vaccine are key quality control specifications [Refer Peeters et al. 1992]. According to the WHO’s recommendations for the production & control of pneumococcal conjugate vaccines, the total protein content of the conjugate should be determined by means of an appropriately validated assay. [Ref: 54th meeting of the WHO Expert Committee on Biological Standardization, 17-21 November 2003]. Similarly, WHO’s Recommendations to assure the quality, safety and efficacy of group A meningococcal conjugate vaccines require each batch of bulk conjugate to be tested for conjugated and unconjugated protein by the methods agreed with the national regulatory authority which include ion exchange chromatography, capillary electrophoresis and indicate the amounts of group A meningococcal polysaccharide and carrier protein contained in each single human dose labelled on the carton or the leaflet accompanying the container. [Ref: WHO Expert Committee on Biological Standardization Fifty-seventh report; WHO Technical Report Series No. 962, 2011].
Pneumococcal polysaccharides conjugate vaccine (e.g. Prevnar® 7 valent, Prevnar 13 ® valent, and Synflorix®) have been analyzed for free carrier protein content along with free polysaccharide content.[Ref: FDA package insert for Prevnar 7 and Prevnar 13; EMEA Assessment report for Synflorix; 2009]. Thus, particularly it is a statutory requirement to quantify the concentration of free or unconjugated carrier protein available in final vaccine formulation because it is a key marker of process consistency an indirect measure of covalency, and could interfere with the immune response against the glycoconjugate. [Ref: Brian K. Nunnally, Vincent E. Turula, Robert D. Sitrin; Vaccine Analysis: Strategies, Principles, and Control; Springer-Berlin (2014)].
Techniques for estimation of protein concentration in a sample are known in the art. The physico-chemical techniques like Lowry method, Bradford method, Biuret method, BCA etc. are the most widely used assays for the estimation of “total protein” concentration. However, these methods are non-specific and usually estimate total protein content in the final vaccine composition or conjugate bulks irrespective of the type of carrier protein.
Use of SDS-PAGE method for detecting free carrier Protein in the Streptococcus pneumoniae polysaccharide-protein conjugates has been previously reported. However, the above method did not adequately resolve higher molecular weight Proteins. [Ref: Turner AEB, et al Synthetic and Systems Biotechnology (2017)]
Use of micellar electrokinetic chromatography (MEKC) for detection and quantitation of free diphtheria toxoid carrier protein in the presence of Neisseria meningitidis polysaccharide–diphtheria toxoid protein conjugate vaccine has been previously disclosed. [Ref: Lamb et al 2000; Journal of Chromatography, 311–318]
Use of DOC or HCI-induced precipitation for evaluating the protein content of the Hib conjugate bulk and the Hib conjugate product has been previously reported. [Refer Hyun Sung Kim et al 2006]
EP2999709 discloses use of anion exchange chromatography to determine free protein in a polysaccharide-protein conjugate vaccine. However the above method enables the separation of similar types of molecules. Further, the above method involves multiple steps and the separation of molecules is dependent on the salt containing mobile phase or it is pH dependent.
EP2659912 discloses a method of purifying saccharide antigen-carrier protein conjugates from a mixture, comprising contacting said mixture with hydroxyapatite resin and collecting the free saccharide antigen-carrier protein conjugates. The unbound carrier protein may optionally be eluted from the hydroxyapatite resin and re-used in a conjugation reaction. The unconjugated CRM197 bound to the resin while the conjugated protein was protected by the conjugated saccharide and so was found in the flow through.
Previously reported methods have been utilized either to determine the concentration of “total protein” or “free protein” or “removal of free protein”. Method like MEKC is less sensitive and does require sample processing prior to testing. The methods like CZE & SDS-PAGE involve use of harsh condition (such as high pH buffer, high voltage, detergent precipitation, high temperature). The presence of high concentration of excipient may interfere with the determination of free or unconjugated protein content in a conjugate bulk, which necessitates a method where the mere presence of excipients doesn’t affect the final outcome of the results.
Hence, there remains a significant need of a simple, accurate, repeatable, fast, and convenient assay for estimation of the concentration of free carrier protein in a monovalent or multivalent polysaccharide- protein conjugate preparation.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
It is an object of the present disclosure to provide a method for determining unconjugated or free protein content in a carbohydrate-protein conjugate preparation
It is another object of the present disclosure to provide a method for determining unconjugated/free recombinant CRM197 carrier protein content in a monovalent or multivalent meningococcal polysaccharide-protein conjugate preparation, wherein Men-C Men-Y and Men-W conjugated to CRM¬¬197
It is yet another object of the present disclosure to provide a method for assessing the quality of the carbohydrate-protein conjugate
Still another object of the present disclosure to provide a simple, accurate, repeatable, fast, and convenient, highly sensitive and non-destructive method devoid of any sample processing steps prior to analysis and generates consistent reproducible results.
Other objects of the present disclosure is 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, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure provides a method to determine free recombinant CRM197 carrier protein content in a monovalent or multivalent meningococcal polysaccharide-protein conjugate preparation, wherein Men-C Men-Y and Men-W conjugated to CRM¬¬197
The method comprises of following steps:
1) Total carrier protein concentration (C1) was estimated by Micro-bicinchoninic acid protein assay (Micro-BCA) from bacterial capsular polysaccharide-protein conjugate sample to be tested.

2) Free carrier protein concentration (C2) was estimated by High performance size exclusion chromatography (HPLC-SEC), by running a standard carrier protein of known concentration under the same conditions as for the conjugate sample comprising the carrier protein to be tested.

3) The percentage of unconjugated or free carrier protein was calculated by dividing the amount of free protein detected by HPLC-SEC by the total amount of protein quanti?ed in the sample by BCA assay.

The assay is applicable for estimating free protein content in “monovalent conjugate preparation” or “multivalent conjugate preparation” wherein one or more polysaccharides are conjugated to same type of carrier protein.
DETAILED DESCRIPTION
The present disclosure relates to a novel and efficient method for estimation of free carrier protein content in a monovalent or multivalent conjugate preparation. As envisaged in the present disclosure, the first step of the method involves estimating the concentration of total carrier protein in the monovalent or multivalent conjugate preparation by Micro BCA, further followed by estimating the concentration of free carrier protein in the monovalent or multivalent conjugate preparation by HPLC-SEC. Wherein the percentage of unconjugated free carrier protein was calculated by dividing the amount of free protein detected by HPLC-SEC by the total amount of protein quanti?ed in the sample by BCA assay. The method of the present disclosure overcomes the limitations of physico-chemical techniques such as Lowry method, Bradford method, Biuret method, DOC HCl precipitation, and other electrophoretic techniques such as SDS-PAGE, MEKC, CZE, for protein quantification and helps to determine the concentration of the “free carrier protein” in a monovalent or multivalent conjugate bulk.
According to a first embodiment of the present disclosure, the method can determine the percentage of free or unconjugated carrier protein in a monovalent or multivalent polysaccharide protein conjugate preparation, wherein one or more polysaccharides are conjugated to same type of carrier protein.
According to a first aspect of the first embodiment, the monovalent or multivalent polysaccharide protein conjugate preparation comprises of one or more polysaccharide selected from groups consisting of gram positive or gram-negative bacteria.
Polysaccharide can be selected from the group consisting of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Vibrio cholerae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Hemophilus influenzae, Escherichia coli, Shigella spp., Ehrlichia spp., and Rickettsia spp. Polysaccharides of Streptococcus pneumoniae type 1, 2, 3, 4,5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F. Polysaccharides of Meningococcal serogroup A, B, C, D, W135, X, Y, Z, 29E, H. influenzae type b, etc.
The polysaccharides of the present disclosure can be conjugated to any one type of carrier protein selected from group consisting of but not limited to CRM197, diphtheria toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11 , protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD).
The biological material used during experimentation were as follows:

Polysaccharides were isolated from:
Neisseria meningitidis A M1027-SynCoBiopartners (Netherlands)
Neisseria meningitidis C C11(60E)- CBER/FDA, USA
Neisseria meningitidis W S877 - CBER/FDA, USA
Neisseria meningitidis Y M10659 - CDC, USA
Neisseria meningitidis X M8210-CBER/FDA, USA

CRM197 was procured from Recombinant Strain CS463-003 (MB101) of Pseudomonas fluorescens from Pfenex USA.

TT was procured from Clostridium Tetani (Harvard No 49205) obtained from Central research Institute (CRI),National Control Authority, Kasauli, Himachal Pradesh, India. Central research Institute (CRI) procured this strain from NVI, Netherland.
It is understood by a person skilled in the art, the sample to be tested may comprise of monovalent or multivalent conjugate preparation wherein multivalent polysaccharide-protein conjugate can either comprise of polysaccharide from same bacterial species or polysaccharide from atleast two different bacterial species.
According to a second embodiment of the present disclosure, the total protein concentration in a monovalent or multivalent conjugate preparation having same type of carrier protein was determined by BCA assay comprising of following steps:
• The Monovalent or Multivalent Protein – Polysaccharide conjugate preparation was taken as Test Sample in a 1 ml microfuge tube.
• A series of protein working standard concentrations were prepared in a 1 ml microfuge tube by serially diluting a known concentration of unconjugated carrier protein to be quantified in the test sample.
• Volume of all working standards was made upto 250 µL with Milli-Q Water (MQW).
• 96 well plate incubator was set to 37 °C.
• 25 µL of each working standard and test samples were pipetted out in 96 well plates in duplicate.
• To this 200 µL of reagent C was added and tapped gently on the sides of the plate to mix the content thoroughly.
• The plate was incubated at 37 °C for 30 minutes.
• Post incubation the plate was cooled to room temperature (RT).
• Post incubation the absorbance was measured at or near 562 nm on a plate reader.
• The average 562nm absorbance reading of the Blank standard replicates was subtracted from the 562nm reading of all other individual standard and test sample replicates.
• A standard curve was prepared by plotting the average Blank-corrected 562nm reading for each working standard vs. its concentration in µg/mL.
• The amount of total protein was quantified by comparing the area of the test sample with the area of the standard curve plotted against the concentration (µg/mL) generated using protein working standards.
• This total carrier protein concentration was used to determine the final percentage of unconjugated or free carrier protein in the conjugate sample.

According to a first aspect of the second embodiment, the method described above was optimized to determine the total carrier protein concentration in a monovalent or multivalent conjugate preparation wherein the composition comprises one or more different types of bacterial capsular polysaccharides conjugated to a same type of carrier protein.
According to a second aspect of the second embodiment, the method described above was optimized to determine the total carrier protein concentration in a monovalent meningococcal conjugate preparation (wherein, Men capsular polysaccharide serogroup C conjugated to carrier protein CRM¬¬197; Men capsular polysaccharide serogroup Y conjugated to carrier protein CRM¬¬197; Men capsular polysaccharide serogroup W conjugated to carrier protein CRM¬¬197).
According to a third aspect of the second embodiment, the method described above can be optimized to determine the total carrier protein concentration in a multivalent meningococcal conjugate preparation consisting of more than one polysaccharide (Wherein Men-C Men-Y and Men-W conjugated to CRM¬¬197).
According to a fourth aspect of the second embodiment, the method described above was optimized to determine the total carrier protein concentration in a monovalent or multivalent polysaccharide-protein conjugate preparation wherein the composition comprises use of recombinant CRM197 expressed in Pseudomonas fluorescens, Corynebacterium diphtheriae, Escherichia coli, or Bacillus subtilis. Yet according to a preferred aspect, the composition comprises use of recombinant CRM197 expressed in Pseudomonas fluorescens.
According to a fifth aspect of the second embodiment, the method described above was optimized to determine the total carrier protein concentration in a monovalent or multivalent conjugate preparation wherein, i) Lower limit of quantification (LLOQ) Set: for recombinant CRM197 was 200µg/ml and ii) upper limit of quantification (ULOQ) for recombinant CRM197 was 2000µg/ml iii) The correlation coefficient was greater than 0.990.
The protocol as mentioned in the second embodiment of the present disclosure can be modified as per requirement by the person skilled in the art.
According to a third embodiment of the present disclosure, the concentration of unconjugated or free carrier protein in a monovalent or multivalent polysaccharide - protein conjugate preparation was quantified by HPLC-SEC.
Size exclusion chromatography (SEC), otherwise known as gel filtration or gel permeation chromatography, relies on the penetration of macromolecules in a mobile phase into the pores of stationary phase particles. Differential penetration of the macromolecules is a function of the hydrodynamic volume of the particles. Size exclusion media exclude larger molecules from the interior of the particles while the smaller molecules are accessible to this volume. The order of elution can be predicted by the size of the protein as a linear relationship exists between elution volume and the log of the molecular weight of the protein being eluted.
When assessing the eluate from the high performance size exclusion chromatography (HPLC-SEC) by spectroscopy. Typically, such spectroscopy will result in the generation of a graphical representation of peaks via a computer monitor or printout. The assessment of the eluate by spectroscopy allows for evaluating the eluate by one or more of these forms: 1) Allows for quantifying the free or unconjugated carrier protein by determining the area of the carrier protein containing peak or peaks, 2) Allows for quantifying the free or unconjugated polysaccharide by determining the area of the polysaccharide containing peak or peaks, 3) Allows for assessing the quality of the bacterial polysaccharide protein conjugate bulk by assessing the symmetry of the conjugate bulk containing peak or peaks, and/or 4) Allows for assessing the purity of the bacterial polysaccharide protein conjugate bulk sample by dividing the area of the bacterial polysaccharide protein conjugate bulk containing peaks by the area of all peaks of the graphical representation.
The assay was carried out as per following protocol:
• The Monovalent or Multivalent Protein – Polysaccharide conjugate bulk was taken as a Test Sample appropriately diluted to a suitable concentration based on the total protein concentration obtained by BCA method.
• A series of protein working standard concentrations were prepared in a 1 ml microfuge tube by serially diluting a known concentration of unconjugated carrier protein to be quantified in the test sample.
• Apart from this a Marker Mix sample and a spike sample was prepared which would work as internal control standard.
• The Blank containing MQW or PBS (pH 7.4), protein working standards, Test sample/s, Marker Mix and Spike were loaded in subsequent order onto a chromatography column comprising the silica resin fully balanced by mobile phase.
• The assessment of the eluate by spectroscopy resulted in the generation of a graphical representation of peaks via a computer monitor.
• The data was processed for the peak of interest i.e. standard and free protein peak in case of sample is marked and integrated to get the area.
• Input the values of area in the Excel Spreadsheet for the standard and the linearity is plot (Straight line equation). (Standard curve)
• The concentration of free or unconjugated carrier protein was quantified by comparing the peak area of the test sample with the peak area of the standard curve plotted against the concentration (µg/mL) generated using protein working standards.
According to a first aspect of the third embodiment, the size exclusion chromatographic resin used in high performance size exclusion chromatography include but are not limited to Phenomenex Yarra 3µm SEC-2000, Phenomenex Yarra 3µm SEC-3000, Phenomenex Yarra 3µm SEC-4000, Phenomenex Bio-Sep-SEC-S2000, Phenomenex Bio-Sep-SEC-S3000, Phenomenex Bio-Sep-SEC-S4000, Shodex™, HiPrep SEPHACRYL S100 HR, HiPrep SEPHACRYL S-200 HR, HiPrep SEPHACRYL S-300 HR, TSKgel SuperSW2000, TSKgel SuperSW3000, TSKgel UP-SW3000, TSKgel G5000PWXL, TSKgel G3000SWXL,TSKgel Ultra SW Aggregate. Yet the preferred size exclusion chromatography resin used in high performance size exclusion chromatography is the Phenomenex Yarra 3µm SEC-2000. Yet another preferred size exclusion chromatography resin used in high performance size exclusion chromatography is TSKgel Ultra SW Aggregate.
According to a second aspect of the third embodiment, the size exclusion chromatographic resin used may be described in terms of pore size, wherein the pore size of the resin selected is in the range of 5 nm - 100 nm. Yet the preferred pore size of the size exclusion chromatographic resin selected is about 14.5 nm.
According to a third aspect of the third embodiment, the mobile phase buffer used in the process is selected from phosphate buffer saline, Tris, MES, HEPES, citrate or combination thereof having a pH in the range 5.0 to 7.5. According to one of the preferred aspect, the mobile phase buffer used in the process is a phosphate buffer saline having a pH in the range 5.0 to 7.5. Yet another preferred aspect, wherein the mobile phase buffer is preferably a phosphate buffer saline having a pH 7.4.
According to a fourth aspect of the third embodiment, the marker mix is selected from Salmon DNA, Thyroglobulin, BSA, Carbonic Anhydrase and Tyrosine having concentration in range of 0.2 – 1 mg/ml. Yet the preferred marker mix is selected from Salmon DNA, Thyroglobulin, BSA, Carbonic Anhydrase and Tyrosine having concentration of 0.5 mg/ml.
According to a fifth aspect of the third embodiment, the loading of samples can be performed at a flow rate in the range 0.2 – 1 ml/minute. In yet another aspect, the loading of samples can be performed at flow rate in the range 0.2 – 0.8 ml/minute, 0.2 – 0.7 ml/minute, 0.2 – 0.6 ml/minute, 0.2 – 0.5 ml/minute, or 0.2 – 0.4 ml/minute. According to one of the preferred aspect, the loading of samples is performed at a flow rate in the range 0.2 – 0.4 ml/minute.
According to a sixth aspect of the third embodiment, preferred temperature of auto sampler may be between 22°C and 32°C. Yet the preferred auto sampler temperature is 30°C.
According to a seventh aspect of the third embodiment, the method is a high performance size exclusion chromatography method (HPLC-SEC) in which the sample is delivered through a column resin and wherein the column is run at a temperature in between 25°C - 32°C. Yet the preferred column temperature is 30°C.
According to an eighth aspect of the third embodiment, the injection volume of the sample can be in the range 100µl - 200µl. Yet the preferred injection volume of the sample was 100µl.
According to a ninth aspect of the third embodiment, when assessing the eluate of a monovalent or multivalent protein-polysaccharide conjugate bulk sample subjected to high performance size exclusion chromatography, any form of spectroscopy is contemplated. Such forms of spectroscopy include, but are not limited to: refractive index detectors, wavelength absorbance detectors, diode array detectors, chiral detectors, chemiluminescence detectors, circular dichroism detectors, light scattering detectors and fluorescence emission detectors. Yet the preferred spectroscopy method is wavelength absorbance, refractive index, or diode array detectors. According to one of the preferred aspect, the detection is carried out at a wavelength in the range of 190 to 800 nm.
According to a tenth aspect of the third embodiment, the process described above was optimized to determine the free or unconjugated carrier protein concentration in a monovalent or multivalent conjugate bulk wherein the composition comprises one or more different types of bacterial capsular polysaccharides conjugated to a same type of carrier protein.
According to a eleventh aspect of the third embodiment, the process described above was optimized to determine the free or unconjugated carrier protein concentration in a monovalent meningococcal conjugate bulk (wherein, Men capsular polysaccharide serogroup C, conjugated to carrier protein CRM¬¬197; Men capsular polysaccharide serogroup Y, conjugated to carrier protein CRM¬-197; Men capsular polysaccharide serogroup W, conjugated to carrier protein CRM¬¬197).
According to a preferred aspect of the third embodiment, the process described above was optimized to determine the free or unconjugated carrier protein concentration in a multivalent meningococcal conjugate bulk consisting of more than one polysaccharide (Wherein Men-C Men-Y and Men-W conjugated to CRM¬¬197).
Yet another preferred aspect of the third embodiment, the method described above was optimized to determine the free or unconjugated carrier protein concentration in a monovalent or multivalent polysaccharide-protein conjugate bulk wherein the composition comprises use of recombinant CRM197 expressed in Pseudomonas fluorescens, Corynebacterium diphtheriae, Escherichia coli, or Bacillus subtilis. Yet according to a preferred aspect, the composition comprises use of recombinant CRM197 expressed in Pseudomonas fluorescens.
According to the fourth embodiment of the present disclosure, the percentage of unconjugated or free protein in the monovalent or multivalent conjugate bulk was calculated by dividing the amount of free protein by the total amount of protein quantified in the test sample.
TECHNICAL ADVANCEMENTS
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of an assay that estimates the free carrier protein content in a monovalent conjugate bulk. When compared to other methods such as MEKC, CZE & SDS-PAGE, the method of the present disclosure provides the following advantages:
• The method is simple;
• Reduced turnaround time per sample (Only 75 Minutes)
• No sample pre-treatment was required prior to analysis (no sample filtration, no sample digestion, no heating, no voltage based separation, no specific pH requirement)
• The method demands simple buffer i.e. PBS pH 7.4;
• Low sample volume required for analysis;
• Keeps the same work flow as ELISA;
• Since the method doesn’t require any kind of sample pre-treatment prior to analysis and its stability is not affected at any stage of method the percentage recovery of the protein at the end of the method is quite high and hence the method provides accurate results consistently.
• The method has an advantage wherein the molecular weight of the free protein and the bacterial capsular polysaccharide conjugate has a large difference, hence the separation of free protein and bacterial polysaccharide protein conjugate by using size exclusion high performance liquid chromatography method, provided well separated peaks. This is essential for reliable quantitation. The carrier protein with the known concentration is used as a standard, so that the free protein content in the bacterial polysaccharide conjugate can be accurately determined.
• The presence of high concentration of excipient doesn’t affect with the determination of free or unconjugated protein content in a conjugate bulk, hence doesn’t affect the final outcome of the results.

EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
The instant method was designed to estimate the individual concentration of unconjugated protein in the following Carbohydrate-Protein conjugate vaccine composition:
Table 1- Contents of a single 5-Dose Vaccine vial of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197) (freeze-dried)
Component Quantity
Men A PS* 30 ?g
Men C PS# 30 ?g
Men Y PS# 30 ?g
Men W PS# 30 ?g
Men X PS* 30 ?g
Sucrose 15 mg
Sodium citrate (dihydrate) 2.5 mg
Tris (Trometamol) 0.61 mg

* - Conjugated to Tetanus Toxoid (TT); # - Conjugated to CRM197
Table 2- Contents of a single 1-Dose Vaccine vial of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197) (freeze-dried)
Component Quantity
Men A PS* 6 ?g
Men C PS# 6 ?g
Men Y PS# 6 ?g
Men W PS# 6 ?g
Men X PS* 6 ?g
Sucrose 15 mg
Sodium citrate (dihydrate) 2.5 mg
Tris (Trometamol) 1.21 mg
* - Conjugated to Tetanus Toxoid (TT); # - Conjugated to CRM197

Table 3- Contents of Monovalent meningococcal conjugate sample
Component Quantity
Polysaccharide (PS)* Estimated Concentration
Protein (CRM197) Estimated concentration
Sucrose 3 % (~ 30mg/ml)
Sodium citrate (dihydrate) 0.5% (~ 5mg/ml)
Tris (Trometamol) 10 mM

* - Men A PS, Men C PS, Men Y PS, Men W PS, Men X PS
Example 2
Estimation of total recombinant CRM197 protein concentration (C1) in a monovalent meningococcal conjugate sample (wherein, Men capsular polysaccharide serogroup C conjugated to carrier protein CRM¬¬197; Men capsular polysaccharide serogroup Y conjugated to carrier protein CRM¬¬197; Men capsular polysaccharide serogroup W conjugated to carrier protein CRM¬¬197) by Micro-BCA Protein assay (Thermo Scientific) comprising of following steps:
a) The Monovalent meningococcal conjugate sample was taken as Test Sample in a 1 ml microfuge tube.
b) A series of CRM197 protein working standard concentrations from 200ug/ml to 2000ug/ml were prepared in a 1 ml microfuge tube by serially diluting a known concentration of unconjugated CRM197 carrier protein to be quantified in the test sample.
c) Volume of all working standards was made upto 250 µL with Milli-Q Water (MQW).
d) 96 well plate incubator was set to 37 °C.
e) 25 µL of each working standard and test samples were pipetted out in 96 well plates in duplicate.
f) To this 200 µL of reagent C was added and tapped gently on the sides of the plate to mix the content thoroughly.
g) The plate was incubated at 37 °C for 30 minutes.
h) Post incubation the plate was cooled to room temperature (RT).
i) Post incubation the absorbance was measured at or near 562 nm on a plate reader.
j) The average 562nm absorbance reading of the Blank standard replicates was subtracted from the 562nm reading of all other individual standard and test sample replicates.
k) A standard curve was prepared by plotting the average Blank-corrected 562nm reading for each working standard vs. its concentration in µg/mL.
l) The amount of total CRM197 protein was quantified by comparing the area of the test sample with the area of the standard curve plotted against the concentration (µg/mL) generated using protein working standards.
m) Based on the total protein concentration (C1) obtained the sample was diluted with physiological saline to obtain 1000 µg/ml of total CRM197 protein concentration (C2)
n) For example: If the total protein concentration obtained by BCA test is 1.5 mg/mL. Take 667 µL of conjugate bulk sample and make it up to 1 ml with by addition 333 µL of mobile phase PBS to obtain final concentration of 1 mg/ml diluted sample (sample 667 µL + 333 µL mobile phase = 1 mg/mL) which is further used for the free protein analysis.
o) Hence there is no fixed dilution for particular sample but depends onto the concentration of total protein present to get the final 1mg/mL dilution.
i) Lower limit of quantification (LLOQ) Set: for recombinant CRM197 was 200µg/ml and
ii) ii) upper limit of quantification (ULOQ) for recombinant CRM197 was 2000µg/ml
iii) iii) The correlation coefficient was greater than 0.990
Table 4 - Total Protein Concentration (C1) by Micro-BCA
Sr.No Serogroup Batch No. Total Protein concentration (mg/mL)
1 Men C 541G14002 1.81
2 Men C 541G14003 2.11
3 Men C 541G15001 1.39
4 Men Y 544G14001 3.59
5 Men Y 544G14002 3.06
6 Men Y 544G15001 1.58
7 Men W 547G14001 2.54
8 Men W 547G14002 1.97
9 Men W 547G15001 1.38

Table 5 - Table of dilution to obtain 1 mg/ml of total protein concentration (C2)
Sr.No. Sero group Batch No. Total Protein conc. (mg/mL)

(CB) Dilutions
µL of CB µL of PBS Protein Conc. (mg/mL)
1 Men C 541G14002 1.81 552 448

1 mg/mL
2 Men C 541G14003 2.11 474 526
3 Men C 541G15001 1.39 719 281
4 Men Y 544G14001 3.59 279 721
5 Men Y 544G14002 3.06 327 673
6 Men Y 544G15001 1.58 633 367
7 Men W 547G14001 2.54 394 606
8 Men W 547G14002 1.97 508 492
9 Men W 547G15001 1.38 725 275

Example 3
The concentration (C3) of unconjugated or free CRM197 carrier protein in a monovalent Meningococcal polysaccharide - protein conjugate sample was quantified by HPLC-SEC.
The assay was carried out as per following protocol:
a) The Monovalent Meningococcal polysaccharide-protein conjugate preparation was taken as a Test Sample appropriately diluted to obtain 1 mg/ml of total protein concentration (C2) based on the total protein concentration obtained by Micro-BCA method.
b) A series of protein working standard concentrations were prepared in a 1 ml microfuge tube by serially diluting a known concentration of unconjugated CRM197 (No - IHWS/0.4mg/ml CRM/16/01) carrier protein to be quantified in the test sample.
c) Apart from this a Marker Mix sample was prepared by adding approximately of 0.5 mg/mL each of Salmon DNA, Thyroglobulin, BSA, Carbonic Anhydrase and Tyrosine in MQW and a spike sample (1000 µl of 1mg/ml Sample + 125µl of 0.4mg/ml CRM Standard) was prepared which would work as internal control standard.
d) The Blank containing MQW or PBS (pH 7.4), protein working standards, Test sample/s, Marker Mix and Spike were loaded in subsequent order onto a chromatography column comprising the silica resin fully balanced by mobile phase(PBS pH 7.4).
e) The assessment of the eluate by spectroscopy resulted in the generation of a graphical representation of peaks via a computer monitor.
f) The data was processed for the peak of interest i.e. standard and free protein peak in case of sample is marked and integrated to get the area.
g) Input the values of area in the Excel Spreadsheet for the standard and the linearity is plot (Straight line equation). (Standard curve)
h) The concentration of free or unconjugated CRM197 carrier protein was quantified by comparing the peak area of the test sample with the peak area of the standard curve plotted against the concentration (µg/mL) generated using protein working standards.
i) The operational conditions of SE-HPLC optimized for estimation of unconjugated recombinant CRM197 protein concentration (C3) in a monovalent meningococcal conjugate sample (wherein, Men capsular polysaccharide serogroup C conjugated to carrier protein CRM¬¬197; Men capsular polysaccharide serogroup Y conjugated to carrier protein CRM¬¬197; Men capsular polysaccharide serogroup W conjugated to carrier protein CRM¬¬197) were as mentioned below:
Condition Set 1: Based on SEC Yarra 2000 column
• Chromatography system: High Performance Size Exclusion Chromatography System (Waters/Thermo scientific)
• Column: SEC Yarra 2000
• Column temperature : upto 30°C
• Auto sampler Temperature: Upto 30°C
• Flow rate: 0.4 mL/min
• Injection Volume: 100 µL
• Run Time: Upto 60 Minutes
• Detection: PDA/UV.
• Wavelength: 280nm
• Column Storage: 0.02% sodium azide
• Sample Preparation: Dilute sample in suitable volume of mobile phase to get approximately 1.0 mg/mL.

Condition Set 2: Tosoh TSKgel Ultra SW Aggregate
• Chromatography system: High Performance Size Exclusion Chromatography System (Waters/Thermo scientific)
• Column: Tosoh TSKgel Ultra SW Aggregate
• Column temperature : 25°C
• Auto sampler Temperature: 25°C
• Flow rate: 0.4 mL/min
• Injection Volume: 100 µL
• Run Time: 100 Minutes
• Detection: PDA/UV.
• Wavelength: 280nm
• Column Storage: 0.02% sodium azide.
• Sample Preparation: Dilute sample in suitable volume of mobile phase to get approximately 1.0 mg/mL.
% free protein = Free protein conc. (C3) X Dilution factor x 100
Total Protein conc. (C1)
Table 6: Comparison of column details is provided below
Name Tosoh TSKgel Ultra SW Aggregate Phenomenex Yarra Sec 2000
Resin Silica gel based Silica
Dimension 7.8 mm ID X 30 Cm 7.8 mm ID X 30 Cm
Particle size 3 µm 3 µm
Separation range 10,000 Dalton to 2000000 Dalton 1000 Dalton -150000 Dalton

Example 4
A) Method validation: Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. (Impurities/degradants in our case is free protein/free Ps and matrix is sucrose, Tris and sodium citrate)
The method under discussion is demonstrated for the estimation of free protein in Men C, Men Y and Men W -CRM197 conjugates. The conjugate bulk matrix contains sucrose, Tris and sodium citrate. In this study during the method development, the sample matrix components i.e. sucrose, Tris and sodium citrate assessed at the optimized set method conditions. Native as well as activated CRM197 is used as positive control. The retention time of positive control i.e. native/activated CRM197 is obtained at ~ 20.19 minutes. No peak observed at the retention time of positive control in case of Sucrose, Tris and sodium citrate (Matrix) confirms the method is specific.
In addition, the conjugate bulks analyzed in this method are present in their final matrix and accuracy studies are performed by spiking the sample with known amount of analyte; recovery of the known amount of analyte further supports the specific quantitation of free protein.
Determination of free protein is done using UV/PDA detector. For the product related impurity like free polysaccharide, the choice of detector is RI and not UV/PDA. Hence it is not expected to show any response in UV. Further the absence of peak at the retention time of CRM197 in RI confirms it.
This confirms the specificity of the method for the estimation of free protein in Men C, Men Y and Men W CRM197 Conjugates.
Figures 1A, 1B, 1C, 1D & 1E provides specificity data
B) Method validation: Linearity
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.
The experiment was performed to assess the linearity and range of 1 mg/mL Native CRM197 standard used for the quantitation of free protein.
The standard used for the estimation of free protein was from 3.125 µg/mL to 100 µg/mL to assess the linearity.
Multiple assays were run over the period of time during the product development to establish the linearity and range.
Table 7: The area data of individual standard along with correlation coefficient is presented below.
Std conc. (µg/mL) Assay 1 Assay 2 Assay 3 Assay 4
Area RT Area RT Area RT Area RT
3.125 21184 16.15 8360 20.19
6.25 87643 20.10 52737 16.08 15346 20.18
12.5 181855 20.11 96519 20.20 89370 16.13 33239 20.19
25 322769 20.13 261406 20.10 157598 16.10 63374 20.19
50 637931 20.13 611055 20.20 329098 16.12 132107 20.19
75 934534 20.14 903898 20.20 483185 16.13 199362 20.19
100 1256078 20.13 1238723 20.20 643088 16.16 271004 20.19
R2 0.999 0.999 0.999 0.999

The obtained R2 (correlation coefficient) values confirms the method is linear from standard 3.125 µg/mL to 100 µg/mL for the estimation of free protein in Men C, Men Y and Men W CRM197 Conjugates.
Figures 2: Standard Curve Plot
C) Method validation: Limit of Quantitation for CRM197
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.
The method is able to quantitate the sample at 0.32% also which prove the sensitivity of the method.
Lower limit of detection (LLOD): 1µg/ml,
Lower limit of quantification (LLOQ): 3µg/ml
Upper limit of quantification (ULOQ): 100µg/ml.
Table 8: Limit of Quantitation
Details Standard Response
Standard (µg/ml) Area
10 % 100 271004
7.5 % 75 199362
5 % 50 132107
2.5 % 25 63374
1.25 % 12.5 33239
0.63 % 6.3 15346
0.32%(LOQ) 3.2 8360

D) Method validation: Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision & reproducibility.
Intermediate precision
Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. These experiments are compiled to check the variation obtained by different analyst, different days, and different batches. (I.e. intermediate precision and reproducibility).Three batches each of Men C-CRM, Men Y-CRM and Men W-CRM monovalent conjugate bulk analyzed for free protein estimation. Each batch of all the three serogroups (Men C, Men Y and Men W) analyzed for free protein at least three times during the method development studies.
Table 9: Initial analysis of all the 09 batches of Men C-CRM, Men Y-CRM and Men W-CRM Conjugate bulk
Free protein (%)
Men C % Free Pr Men Y % Free Pr Men W % Free Pr
541G14002 < 1 544G14001 Not detectable 547G14001 Not detectable
541G14003 < 1 544G14002 Not detectable 547G14002 Not detectable
541G15001 < 1 544G15001 Not detectable 547G15001 Not detectable

Table 10: Analysis of Men-conjugate bulk
Men C-CRM Conjugate – Each batch analyzed 03 times.
Men Y-CRM Conjugate – Each batch analyzed 03 times.
Men W-CRM Conjugate – Each batch analyzed 03 times.
Details B.No. Assay 1 Assay 2 Assay 3
Men C 541G14002 < 1 < 1 < 1
541G14003 < 1 < 1 < 1
541G15001 < 1 < 1 < 1
Men Y 544G14001 Not detectable Not detectable Not detectable
541G14002 Not detectable Not detectable Not detectable
541G15001 Not detectable Not detectable Not detectable
Men W 547G14001 Not detectable Not detectable Not detectable
547G14002 Not detectable Not detectable Not detectable
547G15001 Not detectable Not detectable Not detectable

Note: For Men Y and W since the samples are not detectable for free protein, during the development all the samples were spiked with 5% spike and analyzed for recoveries which was obtained between 80-120%. This indicates and supports the precision of the method.
E) Method validation: Accuracy
The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness.
In accuracy studies, for all the three serogroup, the monovalent conjugate bulk is spiked with 5% (i.e. 50µg in 1000 µg/mL of sample) of native CRM and analyzed for free protein estimation. The recoveries obtained for the serogroup was in the range of 80 to 120%.All the experiment performed during the development studies had 5% spike as a spike control and showed recovery in between 80 to 120%.Also, The concentration of spike and sample + spike was calculated to assess the accuracy in the samples where free protein obtained is not detectable.
Table 11: Analysis of Men-conjugate bulk
Free protein percentage (%)
Men C Percentage Recovery Men Y Percentage Recovery Men W Percentage Recovery
541G14002 94 544G14001 102 547G14001 104
541G14003 96 544G14002 103 547G14002 105
541G15001 99 544G15001 100 547G15001 105

Example 5
Method assurance data wherein conjugated protein and unconjugated protein is differentiated
The method is explored to separate out free protein from monovalent Men CYW-CRM conjugate bulks. The polysaccharide (100-150 KDa) is conjugated to recombinant CRM197 (58 KDa) to generate intended polysaccharide protein conjugate molecules which is bigger in size as compare to its starting material i.e. CRM197. In this method the molecules in solution are separated by their size. The conjugates molecules i.e. Ps + Pr are expected to elute out first followed by small molecules i.e. free CRM.
The big conjugate bulk molecules i.e. conjugated protein gets eluted at ~ 13 minutes as compare to that of the unconjugated protein molecule (Free protein) which gets eluted at ~ 20 minutes. This separation indicates the conjugated protein and unconjugated protein is well differentiated from each other. Additionally, the sample was spiked with known amount of unconjugated protein and analyzed which also clearly indicate the differentiation and separation of conjugated protein and unconjugated protein. The graphical presentation provided below further proves the same.
Figures 3A, 3B, 4A, 4B, 5A, 5B: Method assurance
,CLAIMS:We Claim:
1. A method for evaluating a carbohydrate-protein conjugate sample, comprising:
a) Subjecting the carbohydrate-protein conjugate sample to Micro-bicinchoninic acid protein assay to determine the total protein concentration (C1)
b) Based on the total protein concentration (C1) the carbohydrate-protein conjugate sample was appropriately diluted to obtain 50 - 1000 µg/ml of total protein concentration (C2)
c) loading the sample of (step b) onto a high performance size exclusion chromatographic column thereby producing an eluate;
d) subjecting the eluate to spectroscopy; and
e) evaluating the eluate by one or more of the following:
i) quantifying the unconjugated protein concentration (C3) by determining the area of the unconjugated protein containing peak or peaks,
ii) assessing the quality of the carbohydrate-protein conjugate by assessing the symmetry of the carbohydrate-protein conjugate containing peak or peaks
iii) assessing the purity of the carbohydrate-protein conjugate by dividing the area of the carbohydrate-protein conjugate containing peaks by the area of all peaks of the graphical representation
2. The method according to claim 1, wherein the carbohydrate is a natural or synthetic carbohydrate, polysaccharide (Ps), oligosaccharide (Os), or combination thereof.
3. The method according to claim 2, wherein the carbohydrate-protein conjugate sample to be evaluated comprises polysaccharide selected from the group of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Vibrio cholerae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Hemophilus influenzae, Escherichia coli, Shigella spp., Ehrlichia spp., and Rickettsia spp. Polysaccharides of Streptococcus pneumoniae type 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19F, 19A, 20, 22F, 23B, 23F, 24F, 33F, 35B, 38 and 45. Polysaccharides of Meningococcal serogroup A, B, C, D, W135, X, Y, Z, 29E, H. influenzae type b.
4. The method according to claim 1, wherein the carbohydrate-protein conjugate sample to be evaluated comprises unconjugated protein and/or conjugated protein selected from the group of CRM197, diphtheria toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11 , protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD)
5. The method according to claim 1, wherein the high performance size exclusion chromatographic column has a pore size of about 5 nm to 100 nm.
6. The method according to any preceding claim, wherein the high performance size exclusion chromatographic column is any one of the following: Phenomenex Yarra 3µm SEC-2000, Phenomenex Yarra 3µm SEC-3000, Phenomenex Yarra 3µm SEC-4000, Phenomenex Bio-Sep-SEC-S2000, Phenomenex Bio-Sep-SEC-S3000, Phenomenex Bio-Sep-SEC-S4000, Shodex™, HiPrep SEPHACRYL S100 HR, HiPrep SEPHACRYL S-200 HR, HiPrep SEPHACRYL S-300 HR, TSKgel SuperSW2000, TSKgel SuperSW3000, TSKgel UP-SW3000, TSKgel G5000PWXL, TSKgel G3000SWXL,TSKgel UltraSW Aggregate.
7. The method according to claim 1, wherein the spectroscopy is wavelength absorbance.
8. The method according to claim 7, wherein the wavelength is about 190 to 800 nm.
9. The method according to claim 1, wherein the spectroscopy is refractive index.
10. The method according to claim 1, wherein the spectroscopy is diode array.
11. The method according to claim 1, wherein the spectroscopy is fluorescence emission.
12. A method for evaluating a bacterial polysaccharide-protein conjugate sample, comprising:
a) Subjecting the bacterial polysaccharide-protein conjugate sample to Micro-bicinchoninic acid protein assay to determine the total CRM197 protein concentration (C1)
b) Based on the total CRM197 protein concentration (C1) the bacterial polysaccharide-protein conjugate sample was appropriately diluted to obtain 50 - 1000 µg/ml of total CRM197 protein concentration (C2)
c) Loading the sample of (step b) onto a high performance size exclusion chromatographic column thereby producing an eluate;
d) subjecting the eluate to spectroscopy;
e) evaluating the eluate by one or more of the following:
i) quantifying the unconjugated CRM197 protein concentration (C3) by determining the area of the unconjugated CRM197 protein containing peak or peaks,
ii) assessing the quality of the bacterial polysaccharide-protein conjugate by assessing the symmetry of the bacterial polysaccharide-protein conjugate containing peak or peaks
iii) assessing the purity of the bacterial polysaccharide-protein conjugate by dividing the area of the bacterial polysaccharide-protein conjugate containing peaks by the area of all peaks of the graphical representation

13. The method according to claim 12, wherein the bacterial polysaccharide-protein conjugate sample to be evaluated comprises polysaccharide selected from the group of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Vibrio cholerae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Hemophilus influenzae, Escherichia coli, Shigella spp., Ehrlichia spp., and Rickettsia spp. Polysaccharides of Streptococcus pneumoniae type 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19F, 19A, 20, 22F, 23B, 23F, 24F, 33F, 35B, 38 and 45. Polysaccharides of Meningococcal serogroup A, B, C, D, W135, X, Y, Z, 29E, H. influenzae type b.
14. The method according to claim 13, wherein the bacterial polysaccharide-protein conjugate sample to be evaluated comprises Neisseria meningitidis serogroup A polysaccharide derived from Neisseria meningitidis serogroup A strain M1027 procured from SynCoBiopartners (Netherlands)
15. The method according to claim 13, wherein the bacterial polysaccharide-protein conjugate sample to be evaluated comprises Neisseria meningitidis serogroup C polysaccharide derived from Neisseria meningitidis serogroup C strain C11(60E) procured from CBER/FDA, USA
16. The method according to claim 13, wherein the bacterial polysaccharide-protein conjugate sample to be evaluated comprises Neisseria meningitidis serogroup W polysaccharide derived from Neisseria meningitidis serogroup W strain S877 procured from CBER/FDA, USA
17. The method according to claim 13, wherein the bacterial polysaccharide-protein conjugate sample to be evaluated comprises Neisseria meningitidis serogroup Y polysaccharide derived from Neisseria meningitidis serogroup Y strain M10659 procured from CDC, USA
18. The method according to claim 13, wherein the bacterial polysaccharide-protein conjugate sample to be evaluated comprises Neisseria meningitidis serogroup X polysaccharide derived from Neisseria meningitidis serogroup X strain M8210 procured from CBER/FDA, USA
19. The method according to claim 12, wherein the said CRM197 protein is a recombinant CRM197 protein derived from strain CS463-003 (MB101) of Pseudomonas fluorescens procured from Pfenex USA.
20. The method according to claim 12, wherein the high performance size exclusion chromatographic column has a pore size of about 5 nm to 100 nm.
21. The method according to claim 20, wherein the high performance size exclusion chromatographic column is any one of the following: Phenomenex Yarra 3µm SEC-2000, Phenomenex Yarra 3µm SEC-3000, Phenomenex Yarra 3µm SEC-4000, Phenomenex Bio-Sep-SEC-S2000, Phenomenex Bio-Sep-SEC-S3000, Phenomenex Bio-Sep-SEC-S4000, Shodex™, HiPrep SEPHACRYL S100 HR, HiPrep SEPHACRYL S-200 HR, HiPrep SEPHACRYL S-300 HR, TSKgel SuperSW2000, TSKgel SuperSW3000, TSKgel UP-SW3000, TSKgel G5000PWXL, TSKgel G3000SWXL,TSKgel UltraSW Aggregate.
22. The method according to claim 21, wherein the high performance size exclusion chromatographic column is Phenomenex Yarra Sec 2000
23. The method according to claim 21, wherein the high performance size exclusion chromatographic column is TSKgel Ultra SW Aggregate
24. The method according to claim 12, wherein the spectroscopy is wavelength absorbance.
25. The method according to claim 24, wherein the wavelength is about 190 to 800 nm.
26. The method according to claim 25, wherein the wavelength is about 280 nm.
27. The method according to claim 12, wherein the spectroscopy is diode array.
28. The method according to claim 12, wherein the method is able to quantitate the unconjugated CRM197 protein concentration (C3) in the range of 3.125 µg/mL to 100 µg/mL.