DESC:FIELD
The present disclosure relates to estimation of carrier proteins, used in conjugate vaccines. The present disclosure particularly relates to quantification of unconjugated carrier protein in carbohydrate-protein conjugate vaccine.
DEFINITIONS:
As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which it is used indicate otherwise.
The term, “Carrier protein” as used herein refers to an integral membrane proteins; that is, they exist within and span the membrane across which they transport substances.
The term, “Affinity chromatography” as used herein refers to a method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid.
The term, “Affinity chromatography matrix” as used herein refers to a substrate for separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid.
The term, “Anti-carbohydrate antibodies” as used herein refers to antibodies that target glycans and are continuously produced without apparent external antigen stimulation.
The term, “Flow-through” as used herein refers to a fraction/elute collected by flow-through chromatography. Flow-Through Chromatography is used for isolation of proteins.
The term, “Bead based competitive inhibition assay” as used herein refers to an assay wherein protein A is bound to antibodies for detection of free or unconjugated protein A.
The term, “Suspension array system, (or SAT)” as used herein refers to a high throughput, large-scale, and multiplexed screening platform used in molecular biology. SAT has been widely applied to genomic and proteomic research.
The term, “Flow cytometry” as used herein refers to a technology that is used to analyze the physical and chemical characteristics of particles in a fluid as it passes through at least one laser.
The term, “Protein A” as used herein refers to a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus.
The term, “Protein G” as used herein refers to a bacterial cell wall protein isolated from group G Streptococci. DNA sequencing of native Protein G identifies two IgG-binding domains and sites for albumin and cell surface binding.
The term, “Protein L” as used herein refers to an immunoglobulin-binding protein (35.8kDa) that originates from the bacteria Peptostreptococcus magnus. Protein L binds specifically to the variable domain of Ig kappa light chain, as a consequence Protein L has the capacity to purify kappa light containing IgA antibodies.
The term, “ELISA (enzyme-linked immunosorbent assay)” as used herein refers to a test that uses antibodies and color change to identify a substance.
The term, “Sepharose” as used herein refers to is a crosslinked, beaded-form of agarose, a polysaccharide polymer material extracted from seaweed.
The term, “Agarose” as used herein refers to a polysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose.
The term, “Luminex assay” as used herein refers to an assay wherein the sample is added to a mixture of color-coded beads, pre-coated with analyte-specific capture antibodies. The antibodies bind to the analytes of interest. Biotinylated detection antibodies specific to the analytes of interest are added and form an antibody-antigen sandwich.
The term “multiplexed BBCIA” as used herein refers to a multiplexed Bead based competitive inhibition assay (BBCIA).
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 (meningococcal) 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, & 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 such as 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.
In the article, Vaccine. 2011 Jul 12;29(31):4881-90, titled “comparative effects of carrier proteins on vaccine-induced immune response” by Knuf M. et al; mention that to increase the coverage of these vaccines across serotypes for a bacterium, conjugated polysaccharide from various serotypes is added to vaccine composition, inadvertently increasing the concentration of carrier protein. It is observed that co-administration of conjugate vaccines bearing the same carrier protein, reduces the immune response (due to immune interference) against polysaccharides conjugated with that particular protein. Also, it has been observed that the combination of polysaccharide from a particular serotype with a specific carrier protein shows better efficacy which was suggested in the article titled, “Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines” by Tontini M. et al; Vaccine. 2013 Oct 1; 31(42):4827-33.
To circumvent the phenomenon of negative interference in multivalent vaccines composed of polysaccharide-protein conjugates and to provide better immune response, several vaccine manufacturers are developing vaccines comprising multiple carrier proteins conjugated to different polysaccharides so that the maximum load of each of the carrier proteins is not reached.
Synflorix®, a licensed 10 valent Pneumococcal polysaccharide conjugate vaccine comprises Ps (polysaccharide) conjugated to three different carrier proteins. Polysaccharide from serotype 1, 4, 5, 6B, 7F, 9V, 14 and 23F are conjugated to protein D (derived from non-type able Haemophilus influenzae) carrier protein; Polysaccharide from serotype 18C is conjugated to tetanus toxoid carrier protein; and polysaccharide from serotype 19F is conjugated to diphtheria toxoid carrier protein. A 11 valent Pneumococcal conjugate vaccine comprising Ps conjugated to at least two different carrier proteins have also been reported in the article titled, “Booster response to the tetanus and diphtheria toxoid carriers of 11-valent pneumococcal conjugate vaccine in adults and toddler” by Rose-Marie Olandera et al 2002; s; Vaccine 20; 336-341 wherein the polysaccharide is selected from l, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F and carrier proteins are Diphtheria toxoid and tetanus toxoid. Also, a pentavalent Meningococcal conjugate vaccine comprising Ps conjugated to two different carrier proteins is being developed, wherein polysaccharide from serotype A and X are individually conjugated to tetanus toxoid and polysaccharide from serotype C,Y,W are individually conjugated to CRM197 have been mentioned in the article titled, “Immunogenicity of the Meningitis Vaccine Project’s pentavalent MenACWYX polysaccharide conjugate vaccine MCV-5” by S S Pisal et al; Abstract ID: 035; 20th International Pathogenic Neisseria Conference, 4th-9th September 2016. Manchester, United Kingdom.
Further, it is known in the art that 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. According to the WHO’s recommendations in 54th meeting of the WHO Expert Committee on Biological Standardization, 17-21 November 2003, 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. Similarly, WHO’s Recommendations mentioned in WHO Expert Committee on Biological Standardization Fifty-seventh report; WHO Technical Report Series No. 962, 2011, suggested 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 HPLC, ion exchange chromatography & capillary electrophoresis and indicate the amounts of group A meningococcal polysaccharide and carrier protein contained in each single human dose labeled on the carton or the leaflet accompanying the container.
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 in the reference 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 formulations.
Techniques for the estimation of protein concentration in a sample are very well known in the art. The physico-chemical techniques like Lowry method, Bradford method, Biuret method, Micro 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.
CN101000351 discloses use of Lowry method and high efficiency gel filtration chromatography for determining the content of “free protein” in a bacterial capsular polysaccharide protein conjugate vaccine.
The use of HPLC-SEC to estimate the amount of “unconjugated protein” in conjugate samples has been previously reported wherein the percentage of “unconjugated protein” (CRM197) was calculated dividing the amount of “free protein” (CRM197) by the total amount of protein quantified in the sample by micro BCA. The study was reported by Francesca Micoli et al 2013; PNAS, vol. 110, no. 47, 19077–19082 & Francesca Micoli et al 2012; PLOS, Volume 7, Issue 11, e47039.
EP2659912 discloses a method of separating the free or unconjugated carrier protein from a mixture of polysaccharide antigen-carrier protein conjugate, by contacting the mixture with hydroxyapatite resin and collecting the polysaccharide antigen-carrier protein conjugates in the supernatant. The unconjugated carrier protein (CRM197) bound to the resin may optionally be eluted from the hydroxyapatite resin and re-used in a conjugation reaction.
Use of SDS-PAGE method for detecting free Carrier Protein (CRM197) in the PnPS-CRM conjugates has been previously reported by Turner AEB, et al Synthetic and Systems Biotechnology (2017). However, the above method does not adequately resolve higher molecular weight Proteins. Use of micellar electrokinetic chromatography (MEKC) for detection and quantitation of free diphtheria toxoid carrier protein in the presence of toxoid–polysaccharide conjugate vaccines (Men X-DT) has been previously disclosed by Lamb et. al. 2000; Journal of Chromatography, 311–318.
US6284250 discloses use of solid , restricted access media material (porous silica particles) procedure for removing unconjugated or free protein from a liquid mixture that includes a protein-polysaccharide conjugate and/or a polysaccharide component.
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 in the article by Hyun Sung Kim et al 2006.
EP2999709 discloses use of strong anion exchange chromatography followed by fluorescence detection to determine free protein in a polysaccharide conjugate vaccine.
WO2015068129 and WO2007071707 disclose the use of HPLC-SEC followed by UV detection for determining the content of free residual protein. The percentage of unconjugated protein was calculated dividing the amount of free protein by the total amount of protein quantified in the sample by micro BCA or Lowry Method.
Previously reported methods have been utilized either to determine the concentration of “total protein” or “free protein” or “removal of free protein”. Methods like MEKC, Micro-BCA are less sensitive, cannot differentiate between two different types of carrier proteins. Also, CZE/HPLC-SEC/Gel filtration/HIC may result in flow through having protein as well as Ps-Pr conjugate.
The methods mentioned in the aforementioned prior art documents do not discuss the determination of the individual concentration of one or more types of free carrier proteins used in the final conjugate vaccine composition/conjugate bulks.
Therefore, there remains a significant need of a rapid and convenient assay for estimation of individual concentration of free carrier proteins used in a monovalent/multivalent carbohydrate-protein conjugate vaccine composition/conjugate bulks wherein the composition comprises of one or more type of carrier protein(s).
OBJECTS
Some of the 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 to detect individual concentrations of multiple carrier proteins.
Still another object of the present disclosure is to provide a simple, rapid and cost-effective method for detecting individual concentration of multiple carrier proteins.
Yet another object of the present disclosure is to provide a method for detecting individual concentration of multiple carrier proteins, which generates reproducible results.
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 relates to a rapid and convenient method for quantification of the concentration of “free/unconjugated carrier protein” in a monovalent or multivalent carbohydrate protein conjugate vaccine composition/conjugate bulks wherein the composition comprises one or more types of carrier protein.
The method comprises coupling an anti-carbohydrate antibody to affinity chromatography matrix immobilized on a solid support; subjecting said vaccine to affinity chromatography matrix; wherein conjugated and unconjugated polysaccharide binds to respective anti – carbohydrate antibodies; collecting flow-through comprising unconjugated carrier protein; and quantifying individual concentration of unconjugated carrier protein(s) by bead based competitive inhibition assay using suspension array system. The assay is applicable for estimating free / unconjugated protein in “conjugate bulks” as well as “final conjugate vaccine bulk”.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure-1 illustrates the graphical representation of recovery of MnPs in samples treated with Protein A/ Protein A resin-Men MAb;
Figure-2 illustrates the graphical representation of recovery of Mening conjugate in samples treated with Protein A/ Protein A resin-Men MAb;
Figure-3 illustrates the graphical representation of recovery of CRM/TT in Mixture of Mening conjugates treated with Protein A/ Protein A resin-Men MAb;
Figure-4 illustrates the graphical representation of recovery of CRM/TT Mixture after treatment with Protein A/ Protein A resin-Men Mab;
Figure-5 illustrates the graphical representation of estimation of CRM/TT after adsorption of variable concentration of the meningococcal conjugate mixture with Protein A-MAb resin; and
Figures 6a and 6b illustrate the standard curves of CRM and TT in Multiplexed BBCIA.
DETAILED DESCRIPTION
The present disclosure relates to a novel and efficient method of estimation of individual concentration of “free / unconjugated carrier protein” in a multivalent carbohydrate-protein conjugate vaccine composition/conjugate bulks wherein the composition comprises one or more types of carrier proteins. As envisaged in the present disclosure, the first step involves the separation of free or unconjugated carrier protein in the supernatant via polysaccharide/carbohydrate specific antibody tagged to protein A resin following an affinity chromatography, further followed by determination of the concentration of free / unconjugated carrier protein(s) from flow through by multiplex bead based competitive inhibition assay. The method of the present disclosure overcomes the limitations of physico-chemical techniques, other chromatographic techniques, and detergent precipitation for protein quantification and helps to determine individual concentration of one or more type of “free/unconjugated carrier protein(s)” in final vaccine formulation/conjugate bulks.
In an aspect of the present disclosure, the method for quantification of an unconjugated carrier protein in a carbohydrate-protein conjugate vaccine comprises coupling an anti-carbohydrate antibody to affinity chromatography matrix immobilized on a solid support; subjecting the vaccine to affinity chromatography matrix, wherein conjugated and unconjugated polysaccharide binds to respective anti – carbohydrate antibodies; collecting flow-through comprising unconjugated carrier protein; and quantifying individual concentration of unconjugated carrier protein(s) by bead based competitive inhibition assay using suspension array system. The assay is applicable for estimating free / unconjugated protein in “conjugate bulks” as well as “final conjugate vaccine bulk”
In accordance with the present disclosure, the method can estimate the individual concentration of a free carrier protein in a monovalent or multivalent carbohydrate protein conjugate vaccine composition, wherein one or more carbohydrate(s) are conjugated to one or more types of carrier protein(s) in a vaccine or in-process sample. In an embodiment, the carbohydrate is selected from the group consisting of the natural carbohydrate, synthetic carbohydrate, oligosaccharide, polysaccharide, and combination thereof. In accordance with the present disclosure, the natural carbohydrates are capsular polysaccharide(s) A,C,W,Y, X purified from Bacteria, Neisseria meningitidis.
Neisseria meningitidis A M1027 is procured from Synco Biopartners, Netherlands,
Neisseria meningitidis C C11(60E) is procured from CBER/DA, USA,
Neisseria meningitidis W S877is procured from CBER/DA, USA,
Neisseria meningitidis Y M10659 is procured from CDC, USA, and
Neisseria meningitidis C 8210 is procured from CBER/DA, USA.
The monovalent or multivalent polysaccharide protein conjugate bulk/vaccine composition comprises one or more polysaccharide selected from the group consisting of gram positive or gram-negative bacteria, wherein the polysaccharide can be with or without phosphodiester bond.
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, 35B, 38, 45 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 one or more carrier protein selected from the group consisting of, but not limited to, CRM197, diphtheria toxoid, Tetanus toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, 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, 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).
In an embodiment, multivalent conjugate(s) polysaccharide protein conjugate bulk/vaccine composition comprises various combinations of afore-mentioned polysaccharides conjugated to afore-mentioned carrier proteins.
In accordance with the present disclosure, the anti-carbohydrate antibodies are developed in-house at Serum Institute, Pune, Maharashtra, India. In an embodiment of the present disclosure, the monoclonal antibodies against polysaccharide of interest are produced using a cell hybridoma technique. In an embodiment, BALB/c (female) mice are immunized with 2µg of Neisseria Meningococcal (Men) Serogroup A – protein conjugate of interest along with adjuvant on day 1, 14 and 28. Mice are bled on day 35 to determine humoral immune response against Men A polysaccharide and mouse giving good IgG titer against Men A polysaccharide are identified. Splenocytes immunized with Men A conjugate are isolated from the identified mouse and fused with SP2/O-Ag14 cells (mouse myeloma cells) using polyethylene glycol (PEG). Fused cells are selected using HAT (Hypoxanthine Aminopterin Thymidine) medium over a period of 15 days. The supernatant of the fused cells are screened for the presence of anti-Men A antibody by in-house analytical methods like bead based assay and ELISA to check the presence of antibodies against Men A polysaccharide. Limiting dilution (LD1) of the positive clones is performed to select the stable hybrid clones secreting antibodies against Men A polysaccharide. Propagate this Single anti- Men A polysaccharide MAb secreting clone in 6 well plates. Further, second limiting dilution (LD2) was performed and selected clones are propagated in 2 liters media for the production of MAbs.
In another embodiment, the Anti-Men A polysaccharide MAbs are purified by affinity chromatography using Akta Explorer system. Protein A resin having high affinity for IgG is used for packing the column. The column is equilibrated with the sodium phosphate buffer and then the clarified harvest is loaded onto the column. Post loading, the column can be washed using wash buffer (phosphate buffered saline with 500mM NaCl) to remove any nonspecific binding of proteins present in the harvest. The MAbs are eluted using a low pH buffer (Sodium citrate buffer pH 3.5). Further, 0.01 % of tween 80 is added to the collected elute. The pH of elute is adjusted to pH 6.5 using 3N NaOH and filtered through 0.2 micron filter until further use. Dimers and Trimers are removed by passing elute through cation exchange (CE) chromatography (Fracto gel). Fractions collected after CE chromatography is analyzed for MAb purity (% Monomer). Fractions with higher purity (% Monomer) are pooled and stored at -20°C.
In accordance with the present disclosure, the method can be used for preparation of monoclonal antibodies against other Meningococcal serogroup B, C, D, W135, X, Y, Z, 29E capsular polysaccharides.
In one embodiment, the method can be used for preparation of monoclonal antibodies against groups consisting of other gram positive or gram-negative bacteria.
In accordance with the present disclosure, the method can be used for preparation of monoclonal antibodies against carrier protein selected from CRM197, tetanus toxoid, 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).
In accordance with the present disclosure, CRM197 is procured from Recombinant Strain CS463-003 (MB101) of Pseudomonas fluorescens from Pfenex USA.
In accordance with the present disclosure, TT is 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.
In an embodiment of the present disclosure, the polyclonal antibodies are against CRM197. CRM197 polyclonal antibodies are generated by immunizing BALB/c (female) mouse with 50µg of CRM197 along with adjuvant on day 1, 14 and 28. The mouse is bled on day 35 to determine humoral response against CRM197. The mouse with good humoral response is selected and terminally bled to isolate the anti-sera. The isolated anti-sera are checked for reactivity with CRM197 and cross reactivity with other carrier proteins for multiplex assay.
In an embodiment, the polyclonal antibodies against afore-mentioned carrier proteins can also be generated by the method described herein above.
In accordance with the present disclosure, anti - polysaccharide MAbs are coupled to Protein A resin by a specific method described below and thereby resulting in efficient binding to a Polysaccharide epitope from the conjugate bulk/vaccine and isolation of free or unconjugated carrier proteins.
In the preferred embodiment, the method for quantification of unconjugated carrier protein in a carbohydrate protein conjugate vaccine comprises coupling of anti – polysaccharide monoclonal antibodies (MABs) to Protein A resin and isolation of free / unconjugated carrier protein from the conjugate bulk/vaccine comprises following steps:
• Adding 5 ml of protein A slurry in a 15ml tube (glass vial) and Subjecting to centrifugation @ 1600 rpm for 1 min to obtain a 2.5ml of protein A bed.
• Discarding the supernatant and re-suspending the protein A resin in 25ml of Phosphate buffer (pH 7.0) in 50 ml centrifuge tube. The protein A resin tube is further subjected to vortexing followed by centrifugation @1600 rpm for 1 min. Repeat the step to ensure removal of traces of ethanol.
• The supernatant is discarded and equal volume of phosphate buffer is added to the protein A bed to re-suspend the pellet.
• Aliquoting 600µl of re-suspended resin in 1 ml centrifuge tube and further subjecting it to centrifugation @1600 rpm for 1 min to obtain 300µl of protein A resin bed.
• The supernatant is discarded and 500µl of anti-polysaccharide monoclonal antibodies (MAbs) are added to protein A resin bed. The volume of Protein A Bed is proportionally increased with valency of serogroup in vaccine/bulk.
• MAbs with protein A resin are subjected to rotation at room temperature using rotator for 30 min.
• After 30 minutes, the MAbs – protein A resin mixture are subjected to centrifugation @ 1600 rpm for 1 min and supernatant is discarded.
• The pellet is further subjected to washing to remove any nonspecific binding by adding 1ml of 1x PBS (phosphate buffered saline) to the pellet. The re-suspended pellet is further subjected to centrifugation @ 1600 rpm for 1 min and the supernatant is discarded.
• Conjugate bulk /vaccine in the range of 1-4µg/ml (Polysaccharide content) are added to the MAbs – protein A resin bed followed by rotation at room temperature using rotator for 30 min.
• After 30 minutes of incubation, the tubes are subjected to centrifugation @ 1600 rpm for 1 min and the supernatant having the free protein is collected in a separate tube.
• The pellet tubes are washed for presence of any loosely bound free protein by adding 1ml of Luminex assay buffer to the tube followed by incubating the tubes for 30 minutes @ room temperature and further subjecting it to centrifugation @ 1600 rpm for 1 min. Collect the supernatant in the same tubes having the earlier supernatant.
• The above supernatant is analyzed further to determine the individual concentration of free carrier protein 1 and carrier protein 2 present in the supernatant using Bead based competitive inhibition assay.
In accordance with the present disclosure, the method does not show any non-specific binding (Blank resin, Flow through analysis does not show any conjugate or free polysaccharide) in respect of the multiple parameters tested as mentioned below:
1. Nonspecific binding of Ps to Protein A resin
2. Nonspecific binding of conjugate Ps to protein A resin
3. Nonspecific binding of Carrier Protein to protein A resin
4. Nonspecific binding of Carrier Protein to antibody coupled protein A resin
In accordance with the present disclosure, the concentration of anti-polysaccharide antibody bound to protein A resin have been optimized to ensure Polysaccharide (Ps) -Protein (Pr) conjugate and free Ps gets bound thereby resulting in flow through with free/unconjugated protein content devoid of Ps-Pr or Ps content.
In accordance with the present disclosure, the Protein A resin can be selected from the group consisting of MabSelect SuRe™, MabSelect, MabSelect SuRe LX, MabSelect Xtra, rProtein A Sepharose Fast Flow, Poros® MabCapture A, Amsphere™ Protein A JWT203, ProSep HC, ProSep Ultra, and ProSep Ultra Plus. In one embodiment of the present disclosure the Protein A affinity chromatography resin/matrix is MabSelect SuRe™, which is procured from GE, India.
In an embodiment of the present disclosure, Sodium Chloride (NaCl) salt solution (150mM), 0.2% Bovine Serum Albumin (BSA), and 0.1% tween 20 can be used in the method that contributes to the stability and promotion of the hydrophobic interaction between antibody and Protein A ligand thereby enhancing the retention of the antibody of interest on the Protein A resin as well as reducing non-specific binding.
In accordance with the present disclosure a bead based competitive inhibition assay for estimation of free carrier protein in a monovalent or multivalent conjugate bulk/vaccine composition comprising one or more types of carrier proteins is disclosed.
In an embodiment, the carrier proteins are coupled to polystyrene beads using amine coupling kit (Biorad) procedure. The coupling procedure is applicable to covalently couple the water-soluble proteins ranging in size from 6 to 150 kDa via carboxyl groups on the surface of polystyrene beads. The coupling procedure is a two-step carbodiimide reaction. Before coupling to protein, the carboxyl groups on the surface of polystyrene beads are activated with EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) a carbodiimide derivative. EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) reacts with carboxyl groups on the surface of the polystyrene beads to form an active O-acylisourea intermediate. This unstable intermediate is stabilized in aqueous solutions using S-NHS (N-hydroxysulfosuccinimide). EDC couples S-NHS to the carboxyl group resulting in an S-NHS activated site. Both the carbodiimide's, O-acylisourea intermediate and the S-NHS-ester formed are amine-reactive; however, S-NHS-ester has much greater stability at the physiological pH. These intermediates then react with primary amines of proteins to form amide bonds. The intermediate is hydrolyzed if it fails to react with an amine and the carboxyl is regenerated, releasing an N-unsubstituted urea.
In an exemplary embodiment, the bead activation and coupling of the carrier protein to polystyrene bead are explained in detail as:
Bead activation:
• Allow the vial of COOH beads to come to room temperature for approximately 5 min., vortex and sonicate the beads for approximately 1-2 min in-order to avoid aggregation and to monodisperse them.
• Take 100 µl of mono dispersed COOH beads (1.25 x 106 beads) in coupling reaction tube provided with the kit.
• Centrifuge the beads at 14000 rpm per g for 5 min. carefully remove and discard the supernatant from the bead pellet.
• Add 100 µl of bead wash buffer from the kit. Vortex and sonicate for 10 sec. Centrifuge the beads at 14000 x g for 5 min and carefully remove and discard the supernatant from the bead pellet.
• Re-suspend the bead pellet in 80 µl of bead activation buffer from the kit. Vortex the beads for 30 sec, and sonicate the beads by bath sonication for 30 sec.
• Prepare 100 µl each of 50 mg/ml of EDC and S-NHS by adding accurately weighed 5 mg each of EDC and S-NHS in 100 µl of bead activation buffer in two different tubes, immediately prior to their use.
• Add 10 µl of freshly made 50 mg/ml EDC in the bead suspension closely followed by 10 µl of freshly made 50 mg/ml of S-NHS.
• Vortex the beads at high speed for 30 sec. Cover coupling reaction tube with aluminium foil and keep the tube on rotospin for 20 min at RT with 30 rpm for 1X reaction.
• Add 150 µl of PBS, pH 7.4 from the kit and vortex the activated beads at high speed for 10 sec. Centrifuge the beads at 14000 x g for 5 min and carefully remove and discard the supernatant form the pellet. Repeat this step once.
• Re-suspend the activated beads in 100 µl PBS. Vortex the beads at medium speed for 30 sec and sonicate by bath sonication for 15 sec.
Carrier Protein coupling to activated beads:
• Add carrier protein (approximately 5 to 15 µg based on optimization experiments) to the tube of activated beads.
• Adjust the final volume to 500 µl with PBS, pH 7.4. Cover the tube with aluminium foil and agitate the beads on rotospin at RT for 2 hr with 30 rpm for 1X reaction.
• Centrifuge the coupled beads at 14000 x g for 5 min., carefully remove, and discard the supernatant. Wash the beads with 500 µl of PBS, pH 7.4 by centrifuging at 14000 x g for 5 min.
• Carefully remove and discard the supernatant. Re-suspend the coupled beads with 250 µl of blocking buffer from the kit.
• Vortex the beads at medium speed for 15 sec. Cover the tube with aluminium foil and agitate the beads on rotospin at RT for 30 min. with 30 rpm.
• Centrifuge the beads at 14000 x g for 5 min., carefully remove, and discard the supernatant.
• Wash the coupled beads with 500 µl of storage buffer from the kit. Centrifuge the beads at 16500 x g for 7 min., carefully remove, and discard the supernatant.
• Re-suspend the coupled beads in 150 µl of storage buffer by vortexing approximately for 1 min.
• Determine Bead concentration using haemocytometer.
In one embodiment of the present disclosure, CRM197 carrier protein is coupled to polystyrene beads using amine coupling kit (Biorad) procedure, as described above.
In another embodiment, Tetanus Toxoid carrier protein is coupled to polystyrene beads using amine coupling kit (Biorad) procedure, as described above.
In accordance with the present disclosure the individual concentrations of free carrier protein in a monovalent or multivalent polysaccharide - protein conjugate bulk/vaccine composition are determined, as per protocol mentioned below. In one embodiment the assay is carried out as per the following:
? Take the flow through as Test Sample in a 1 ml microfuge tube.
? Take a carrier protein as working standard in 1 ml of microfuge tube adjusted to a suitable concentration using luminex assay buffer.
? Prepare the series of working standard from S1 to S7 (Carrier Protein in the first column of 96-well titer plate using luminex assay buffer by serial 2 fold dilution as per the following table 1. Discard 125µl from the last well after mixing. Final total volume of each well should be 125µl.
Table 1:
Vaccine formulation Standard Working standard (µL) Luminex assay buffer (µL)

S1 250 µL working stock solution -
S2 125 µL of S1 solution
125
S3 125 µL of S2 solution
125
S4 125 µL of S3 solution
125
S5 125 µL of S4 solution
125
S6 125 µL of S5 solution 125
S7 125 µL of S6 solution 125
• Transfer 100 µl of the standards to respective adjacent columns in the same plate from the above series of dilution.
• Take a mixture of suitably diluted antibodies adjusted to a suitable concentration against carrier protein-1 and carrier protein-2 in luminex assay buffer. Add 100 µl of antibody solution in to each respective well of standards and test sample.
• Incubate the plate at 37°C for 1 hour with shaking at 150 rpm for pre-adsorption.
• Prewet the required wells of multiscreen filter plate with 100 µl of luminex assay buffer for 1-2 minutes and aspirate the plate using vacuum manifold.
• Prepare the mixture of required volume of Carrier protein-1 and Carrier protein-2 coupled beads in luminex assay buffer to obtain ~4000 beads of each carrier protein per 50µl of final suspension. Add 4000 beads per well per 50 µl in each required well of filter plate. Aspirate the filter plate using vacuum manifold. (Caution: Beads are photosensitive, utmost care should be taken to avoid exposure of samples to direct light).
• Add 50 µl of pre-incubated standards and test samples from dilution plate in duplicates on filter plate containing beads with positive and negative control as per assay plate template planned. Incubate the plate at 37°C for 1 hour with shaking at 150 rpm in dark.
• Wash the filter plate with 100µl of luminex assay buffer followed by aspirating it on vacuum manifold. This step should be performed 3 times.
• Prepare the mixture of required volume of Anti-M-PE with a final dilution of 1:250 and add 50 µl in each well containing standards and test samples. Incubate the plate at 37°C for 30 minutes with shaking at 150 rpm in dark.
• Wash the plate three times following the procedure mentioned in bead washing step.
• Add 100 µl of luminex assay buffer and read the plate on Bio-Plex 200 system.
In accordance with the present disclosure, the fluorescence is measured through the use of a flow cytometer or a bead array reader. In an embodiment, a BioPlex-100, a BioPlex-200, a Luminex-100, a Luminex-200 or MagPix bead array reader is used.
The individual concentrations of the free carrier protein in a monovalent or multivalent protein – polysaccharide conjugate bulk/vaccine composition can be determined wherein the composition comprises one or more different types of bacterial capsular polysaccharides conjugated to two or more different types of carrier proteins.
The individual concentrations of carrier protein CRM197 and TT are determined in a monovalent meningococcal conjugate bulk wherein, capsular polysaccharide serogroup C, Y, W; conjugated to carrier protein CRM¬¬197 and (capsular polysaccharide A & X conjugated to carrier protein TT.
The individual concentrations of carrier protein CRM197 and TT are determined in a multivalent meningococcal conjugate vaccine (Capsular Polysaccharide Serogroup A, C, Y, W and X; conjugated to Carrier protein TT and CRM197).
According to an embodiment of the present disclosure, the multivalent meningococcal conjugate vaccine consists of polysaccharide from Men-A and Men-X conjugated to TT, whereas the polysaccharide from Men-C, Men-W and Men-Y are conjugated to CRM¬¬197. The concentration of the individual carrier protein is determined using following protocol.
The working standards are prepared in a 1ml microfuge tube by suitably diluting the carrier protein native TT & Derivatized CRM197 with Luminex assay buffer to achieve final concentration of 1 µg/mL of CRM content and 1 µg/mL of TT content. Further dilutions are carried out as per following table 2:
Table 2:
Vaccine Formulation Standard Working standard (µL) Luminex assay buffer(µL) Concentration (µg/mL)
CRM TT
S1 250 µL working stock - 1 1
S2 125 µL of S1 solution 125 0.5 0.5
S3 125 µL of S2 solution 125 0.25 0.25
S4 125 µL of S3 solution 125 0.125 0.125
S5 125 µL of S4 solution 125 0.0625 0.0625
S6 125 µL of S5 solution 125 0.03125 0.03125
S7 125 µL of S6 solution 125 0.015625
• Transfer 100 µl of the standards to respective adjacent columns in the same plate from the above series of dilution.
• Take a mixture of 1:20000 diluted anti CRM mice sera and 1:50000 dilution of anti TT MAb in luminex assay buffer. Add 100 µl of antibody solution in to each respective well of standards and test sample.
• Incubate the plate at 37°C for 1 hour with shaking at 150 rpm for pre-adsorption.
• Prewet the required wells of multiscreen filter plate with 100 µl of luminex assay buffer for 1-2 minutes and aspirate the plate using Vacuum manifold.
• Prepare the mixture of required volume of CRM and TT coupled beads in luminex assay buffer to obtain ~4000 beads of each carrier protein per 50µl of final suspension. Add 4000 beads per well per 50µl in each required well of filter plate. Aspirate the filter plate using Vacuum manifold. (Caution: Beads are photosensitive, utmost care should be taken to avoid exposure of samples to direct light).
• Add 50µl of pre-incubated standards and test samples from dilution plate in duplicates on filter plate containing beads with positive and negative control as per assay plate template planned. Incubate the plate at 37°C for 1 hour with shaking at 150 rpm in dark.
• Wash the plate with 100ul of luminex assay buffer followed by aspirating it on vacuum manifold. This step should be performed 3 times.
• Prepare the mixture of required volume of Anti-M-PE with a final dilution of 1:250 and add 50 µl in each well containing standards and samples. Incubate the plate at 37°C for 30 minutes with shaking at 150 rpm in dark.
• Wash the plate three times following the procedure mentioned in bead washing step.
• Add 100ul of luminex assay buffer and read the plate on Bio-Plex 200 system.
The method described above is optimized for detection and quantification of two or more free carrier protein in a conjugate bulk/vaccine composition wherein, i) Lower limit of detection (LLOD) for CRM197 was 7.8 ng/ml, for TT is 15.6 ng/ml, ii) Lower limit of quantification (LLOQ) Set: for CRM197 is 15.6 ng/ml, for TT is 31.25 ng/ml and iii) upper limit of quantification (ULOQ) for CRM197 is 1000 ng/ml, for TT is 1000ng/ml. The method of the present disclosure enables to detect individual concentration of multiple carrier proteins used in the final conjugate bulk/vaccine composition and 90% saving in reagent costs hence economical. The method of the present disclosure enables multiplexing capability to perform multiple experiments simultaneously instead of sequentially and hence the turnaround time is reduced from 21 to 8 hours.
In accordance with the present disclosure, the readings from suspension array system i.e. Bioplex-200 system are plotted between concentrations on X axis and Fluorescence intensity on Y axis. Using this as standard curve, the individual concentrations of the carrier proteins in a monovalent or multivalent conjugate bulk/vaccine composition are determined and given in the figured 1 to 6.
The present disclosure is further described in light of the following experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. The following experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial scale.
EXPERIMENTAL DETAILS:
Example 1: Isolation of free/unconjugated carrier protein from a pentavalent meningococcal polysaccharide protein conjugate vaccine (Men ACWYX)
Vaccine sample: Meningococcal ACWYX Conjugate Vaccine
[Men-A and Men-X polysaccharide conjugated to TT; and Men-C, Men-W and Men-Y polysaccharide conjugated to CRM197].
Free/unconjugated carrier protein from a pentavalent meningococcal polysaccharide protein conjugate vaccine was isolated as follows:
a) 5ml of protein A slurry was taken for 2.5ml of protein A bed in 15ml tube and was centrifuged @ 1600 rpm for 1 min. The supernatant was discarded and re-suspended protein A resin was added in 25ml of Phosphate buffer (pH 7.0) in 50ml centrifuge tube.
b) Vortexed protein A resin was centrifuged @1600 rpm for 1 min. Repeated the first step to ensure removal of traces of ethanol. Protein A resin was procured from Mabselect, SuReTm, GE, India
c) Discarded the supernatant and measured the volume of protein A bed. Added equal volume of phosphate buffer to re-suspend the pellet.
d) Aliquot 600µl of re-suspended resin was taken in 5 ml centrifuge tube to obtain 300µl of protein A resin bed. Centrifuged @1600 rpm for 1 min.
e) Discarded the supernatant and added 500µl of Monoclonal antibody (Respective MAbs in case of conjugate bulk/mixture of MAbs (Men A, Men C, Men Y, Men W and Men X in case of pentavalent meningococcal conjugate vaccine). Increased the volume of Protein A Bed proportionally with valency of serogroup in vaccine/bulk.
f) Incubated MAbs with protein A resin for 30 min with rotation using rotator.
g) After 30 minutes, centrifuged the tubes @ 1600 rpm for 1 min. The supernatant was discarded.
h) Added 1ml of 1x PBS (phosphate buffered saline) for washing to remove nonspecific binding. Centrifuged the tubes @ 1600 rpm for 1 min. The supernatant was discarded.
i) Added conjugate bulk (mening)/vaccine in the range of 1-4µg/ml (Ps content). Incubated the tubes for 30 minutes with rotation.
j) Centrifuged the tubes @ 1600 rpm for 1 min. The supernatant was collected in separate tube. Added 1ml of Luminex assay buffer for washing loosely bound protein (CRM/TT). Kept the tubes for 30 minutes @ room temperature.
k) Centrifuged the tubes @ 1600 rpm for 1 min. Collected the supernatant / Flow through in separate tubes.
l) Flow through obtained after antigen incubation with antibody coupled protein A resin along with Luminex assay buffer was analyzed for free CRM/TT using Bead based competitive inhibition assay.
Observations: ELISA was performed to detect presence of unbound polysaccharide and polysaccharide-protein conjugate in flow through. No unbound polysaccharide and polysaccharide-protein conjugate in flow through was detected.
Example 2: Estimation of individual concentration of unconjugated carrier protein i.e. CRM197 or TT in Meningococcal ACWYX Conjugate Vaccine by Monoplexed BBCIA
Samples for free or unconjugated Tetanus toxoid (TT) analysis were obtained by incubating Men A and Men X conjugate bulks with Anti Men A mAb and anti Men X mAb coupled Protein A resin respectively. The flow through obtained after incubation was analyzed by Monoplexed BBCIA for free/unconjugated TT using following protocol:
a. Native TT/ derivatized CRM were taken as working standard in 1 ml of microfuge tube. Diluted it to the final concentration of 1 µg/mL using luminex assay buffer
b. Prepared the series of standard from S1 to S7 for CRM or S1 to S6 for TT in the first column of 96-well titer plate using luminex assay buffer by serial 2 fold dilution as per the following table-3. Discarded 125µl from the last well after mixing. Final total volume of each well was maintained to be 125µl.

Table 3:
Standards Working standard (µL) Luminex assay
buffer (µL) Concentration (µg/mL)
CRM TT
S1 250 µL working stock solution
- 1 1
S2 125 µL of S1 solution
125 0.5 0.5
S3 125 µL of S2 solution
125 0.25 0.25
S4 125 µL of S3 solution
125 0.125 0.125
S5 125 µL of S4 solution
125 0.0625 0.0625
S6 125 µL of S5 solution 125 0.03125 0.03125
S7 125 µL of S6 solution 125 0.015625

c. Transferred 100 µl of the standards to respective adjacent columns in the same plate from the above series of dilution.
d. Took a mixture of optimum dilution of anti CRM mice sera/anti TT MAb in luminex assay buffer. Added 100 µl of antibody solution in to each respective well of standards and sample.
e. Incubated the plate at 37°C for 1 hour with shaking at 150 rpm for pre-adsorption.
f. Prewetted the required wells of multiscreen filter plate with 100 µl of luminex assay buffer for 1-2 minutes and aspirated the plate using Vacuum manifold.
g. Prepared required volume of CRM/TT coupled beads in luminex assay buffer to obtain ~4000 beads of carrier protein per 50µl of final suspension. Added 4000 beads per well per 50µl in each required well of filter plate. Aspirated the filter plate using Vacuum manifold. (Caution: Beads are photosensitive, utmost care should be taken to avoid exposure of samples to direct light).
h. Added 50µl of pre-incubated standards/samples from dilution plate in duplicates on filter plate containing beads with positive and negative control as per assay plate template planned. Incubated the plate at 37°C for 1 hour with shaking at 150 rpm in dark.
i. Washed the plate with 100 µl of luminex assay buffer followed by aspirating it on vacuum manifold. This step was performed 3 times.
j. Prepared the mixture of required volume of Anti-M-PE with a final dilution of 1:250 and add 50 µl in each well containing standards and samples. Incubated the plate at 37°C for 30 minutes with shaking at 150 rpm in dark.
k. Washed the plate three times following the procedure mentioned in bead washing step.
l. Added 100 µl of luminex assay buffer and read the plate on Bio-Plex 200 system.
Calculation of % unconjugated carrier protein was done using following formula and the result obtained is summarized in table 4.
% unconjugated carrier protein = Obtained concentration of
unconjugated carrier protein X 100
Total concentration of carrier protein taken for free protein isolation
Table 4:
Samples % Free Protein % Recovery
Men A – Sample 1 Non detectable
Men A – Sample 1 + 5% TT 112
Men A – Sample 1 + 1ug TT 100
Men X – Sample 2 Non detectable
Men X – Sample 2+ 5% TT 77
Men X – Sample 2+ 1ug TT 94

Inference:
No free TT was observed in Men A conjugate bulks (Sample 1) and Men X conjugate bulks (Sample 2). Accuracy of assay was assessed by spiking conjugate bulks with TT equivalent to (5% TT in given conjugate bulks & 1µg TT). Recoveries of spiked conjugate bulks were found to be in between 75-125%.
Example 3: Estimation of individual concentration of unconjugated carrier protein i.e. CRM197 or TT in Multivalent Meningococcal Conjugate Vaccine by Multiplexed BBCIA
Isolation of free or unconjugated carrier protein in Multivalent Meningococcal Conjugate Vaccine was done using protocol as mentioned in Example 1; employing respective anti- Meningococcal Polysaccharide mAbs coupled to protein A resin. The flow through obtained after incubation was analyzed by Multiplexed BBCIA for free/unconjugated CRM & TT using following protocol:
a. Taken Native TT and derivatized CRM as working standard in 1 ml of microfuge tube. Diluted it to the final concentration of 1 µg/mL using luminex assay buffer.
b. Prepared the series of standard from S1 to S7 for CRM and S1 to S6 for TT in the first column of 96-well titer plate using luminex assay buffer by serial 2 fold dilution as per the following table-5. Discarded 125µl from the last well after mixing. Final total volume of each well was maintained at 125µl.
Table 5:
Standards Working standard (µL) Luminex assay
buffer (µL) Concentration (µg/mL)
CRM TT
S1 250 µL working stock solution
- 1 1
S2 125 µL of S1 solution
125 0.5 0.5
S3 125 µL of S2 solution
125 0.25 0.25
S4 125 µL of S3 solution
125 0.125 0.125
S5 125 µL of S4 solution
125 0.0625 0.0625
S6 125 µL of S5 solution 125 0.03125 0.03125
S7 125 µL of S6 solution 125 0.015625

c. 100 µl of the standards was added to respective adjacent columns in the same plate from the above series of dilution.
d. Taken a mixture of optimum dilution of anti CRM mice sera and anti TT MAb in luminex assay buffer. Added 100 µl of antibody solution in to each respective well of standards and sample.
e. Incubated the plate at 37oC for 1 hour with shaking at 150 rpm for pre-adsorption.
f. Prewetted the required wells of multiscreen filter plate with 100 µl of luminex assay buffer for 1-2 minutes and aspirated the plate using Vacuum manifold.
g. Prepared the mixture of required volume of CRM and TT coupled beads in luminex assay buffer to obtain ~4000 beads of each carrier protein per 50µl of final suspension. Added 4000 beads per well per 50µl in each required well of filter plate. Aspirated the filter plate using Vacuum manifold. (Caution: Beads are photosensitive, utmost care should be taken to avoid exposure of samples to direct light).
h. Added 50µl of pre-incubated standards/samples from dilution plate in duplicates on filter plate containing beads with positive and negative control as per assay plate template planned. Incubated the plate at 37oC for 1 hour with shaking at 150 rpm in dark.
i. Washed the plate with 100 µl of luminex assay buffer followed by aspirating it on vacuum manifold. This step should be performed 3 times.
j. Prepared the mixture of required volume of Anti-M-PE with a final dilution of 1:250 and add 50 µl in each well containing standards and samples. Incubated the plate at 37oC for 30 minutes with shaking at 150 rpm in dark.
k. Washed the plate three times following the procedure mentioned in bead washing step.
l. Added 100 µl of luminex assay buffer and read the plate on Bio-Plex 200 system
Calculation of % unconjugated carrier protein was done using following formula and the result obtained is summarized in table 6.
% unconjugated carrier protein = Obtained concentration of
unconjugated carrier protein X 100
Total concentration of carrier protein taken for free protein isolation

Table 6: Free TT and CRM analysis in Men A, Men C, Men W, Men Y and Men X final conjugate bulks, Men A, Men X and Men W (10 min) Conjugates (incomplete reaction) and Final Vaccine
% Free Protein % Spiked Recovery
Free TT Analysis Men-A Conjugate <2
Men-A Conjugate + 1 µg TT 97
Men-A Conjugate (incomplete reaction) 16
Men-A Conjugate (incomplete reaction) + 1 µg TT 88
Men-X Conjugate <2
Men-X Conjugate + 1 µg TT 92
Men-X Conjugate (incomplete reaction) 21.8
Men-X Conjugate (incomplete reaction) + 1 µg TT 78
Multivalent Meningococcal Conjugate Vaccine (Final) <2
Multivalent Meningococcal Conjugate Vaccine (Final) + 1 µg TT 93

Free CRM197 Analysis Men-C Conjugate <2
Men-C Conjugate + 1 µg CRM197 123
Men-Y Conjugate <2
Men-Y Conjugate + 1 µg CRM197 112
Men-W Conjugate <2
Men-W Conjugate + 1 µg CRM197 102
Men-W Conjugate (incomplete reaction) 5
Men-W Conjugate (incomplete reaction) + 1 µg CRM197 79
Multivalent Meningococcal Conjugate Vaccine (Final) <2
Multivalent Meningococcal Conjugate Vaccine (Final) + 1 µg CRM197 106

Inference of Table 6:
No free CRM/TT was observed in Men A, Men C, Men W, Men Y and Men X final conjugate sample. Men A, Men X and Men W conjugate bulks (conjugation quenched - incomplete reaction) were found to contain free TT and free CRM, 16%, 21.8% and 5% respectively. All the conjugate bulks were spiked with 1µg TT. % Recovery of spiked conjugates was found to be in between 75-125%. This suggests that the method is accurate enough to estimate free carrier protein.
Example 4: Estimation of individual concentration of unconjugated carrier protein i.e. CRM197 or TT in Multivalent Pneumococcal Conjugate Vaccine by Multiplexed BBCIA
Isolation of free or unconjugated carrier protein in Multivalent Pneumococcal Conjugate Vaccine (PCV) was done using protocol as mentioned in Example 1; employing respective anti-Pneumococcal Polysaccharide mAbs coupled to protein A resin. The flow through obtained after incubation was analyzed by Multiplexed BBCIA for free/unconjugated CRM & TT using following protocol:
a. Taken Native TT and derivatized CRM as working standard in 1 ml of microfuge tube. Dilute it to the final concentration of 1 µg/mL using luminex assay buffer.
b. Prepared the series of standard from S1 to S7 for CRM and S1 to S6 for TT in the first column of 96-well titer plate using luminex assay buffer by serial 2 fold dilution as per the following table 7. Discarded 125µl from the last well after mixing. Final total volume of each well was maintained at 125µl.
Table 7: Sample Dilution
Standards Working standard (µL) Luminex assay
buffer (µL) Concentration (µg/mL)
CRM TT
S1 250 µL working stock solution
- 1 1
S2 125 µL of S1 solution
125 0.5 0.5
S3 125 µL of S2 solution
125 0.25 0.25
S4 125 µL of S3 solution
125 0.125 0.125
S5 125 µL of S4 solution
125 0.0625 0.0625
S6 125 µL of S5 solution 125 0.03125 0.03125
S7 125 µL of S6 solution 125 0.015625

c. 100 µl of the standards was added to respective adjacent columns in the same plate from the above series of dilution.
d. Taken a mixture of optimum dilution of anti CRM mice sera and anti TT MAb in luminex assay buffer. Added 100 µl of antibody solution in to each respective well of standards and sample.
e. Incubated the plate at 37oC for 1 hour with shaking at 150 rpm for pre-adsorption.
f. Prewetted the required wells of multiscreen filter plate with 100 µl of luminex assay buffer for 1-2 minutes and aspirate the plate using Vacuum manifold.
g. Prepared the mixture of required volume of CRM and TT coupled beads in luminex assay buffer to obtain ~4000 beads of each carrier protein per 50µl of final suspension. Added 4000 beads per well per 50µl in each required well of filter plate. Aspirated the filter plate using Vacuum manifold. (Caution: Beads are photosensitive, utmost care should be taken to avoid exposure of samples to direct light).
h. Added 50µl of pre-incubated standards/samples from dilution plate in duplicates on filter plate containing beads with positive and negative control as per assay plate template planned. Incubated the plate at 37oC for 1 hour with shaking at 150 rpm in dark.
i. Washed the plate with 100 µl of luminex assay buffer followed by aspirating it on vacuum manifold. This step should be performed 3 times.
j. Prepared the mixture of required volume of Anti-M-PE with a final dilution of 1:250 and add 50 µl in each well containing standards and samples. Incubated the plate at 37oC for 30 minutes with shaking at 150 rpm in dark.
k. Washed the plate three times following the procedure mentioned in bead washing step.
l. Added 100ul of luminex assay buffer and read the plate on Bio-Plex 200 system

Calculation of % unconjugated carrier protein was done using following formula and the result obtained is summarized in table 8.
% unconjugated carrier protein = Obtained concentration of
unconjugated carrier protein X 100
Total concentration of carrier protein taken for free protein isolation

Table 8:
Samples % Free TT % Recovery % Free CRM197 % Recovery
Multivalent PCV – Sample 1 Non detectable - <2% -
Multivalent PCV – Sample 1 + 1ug TT / CRM 197 75 89
Inference for Table 8:
No free TT was observed in multivalent PCVconjugate bulks (Sample 1) but < 2% unconjugated CRM197 was observed. Accuracy of assay was assessed by spiking PCV with TT and CRM197 (equivalent to 1µg TT/CRM197). Recoveries of spiked conjugate bulks were found to be in between 75-125%.
Example 5: Determination of nonspecific binding of Men Ps (in Men ACWYX Conjugate Vaccine) to Protein A resin
During method development, quantity of Men Ps/ Conjugate to be neutralized by anti Men Ab coupled protein A resin was optimized, so that all conjugate/free Men Ps can be removed from either Men ACWYX Conjugate Vaccine or individual meningococcal conjugate bulks.
2.5µg/ml of MnPs was incubated with protein A resin and anti Men MAb coupled protein A resin. Procedure as mentioned above in example 1 (free protein isolation) was performed to adsorb MnPs to Ab coupled protein A resin/Protein A resin alone. The supernatant was analyzed by BBCIA to estimate MnPs in the samples incubated with protein A resin and anti Men MAb coupled protein A resin, and the result obtained is illustrated in Figure-1.
Observation and Inference: In above experiment no MnPs were recovered in samples treated with Protein A resin coupled with anti Men MAbs in multiplexed BBCIA. The recovery of Men Ps in samples treated with only protein A resin showed recovery in between 75-125% indicated there is no nonspecific binding of Men Ps to protein A resin.
Example 6: Determination of nonspecific binding of Men ACWYX Conjugate to Protein A resin
2.5µg/ml of Men conjugate was incubated with protein A resin and anti Men MAb coupled protein A resin. Procedure as mentioned in example 1 (free Protein isolation) was performed to adsorb Mening conjugate to Ab coupled protein A resin and Protein A resin alone. The supernatant was analyzed by two different methods. First BBCIA (for MenPs-Conj estimation) to estimate MnPs and second BBCIA for estimation of free Protein (BBCIA method as mentioned above) in the samples incubated with protein A resin and anti Men MAb coupled protein A resin were performed, and the result obtained is illustrated in Figure-1 and Figure-2.
Observation and Inference: In above experiments no meningococcal conjugates and relative free protein (CRM/TT) were recovered in samples treated with Protein A resin coupled with anti Men MAbs by respective multiplexed BBCIAs (Figures 2 and 3). The recovery of Men conj and CRM/TT in samples treated with only protein A resin showed recovery in between 75-125% indicated there is no nonspecific binding of Men conjugate and relative carrier proteins to protein A resin.
Example 7: Determination of Nonspecific binding of CRM/TT to protein A resin and Protein A-MAbs resin
1µg/ml of CRM/TT mixture was incubated with protein A resin and anti Men MAb coupled protein A resin. Procedure as mentioned above (free Protein isolation) was performed to adsorb them to Ab coupled protein A resin/Protein A resin alone. The supernatant was analyzed by BBCIA to estimate carrier proteins in respective samples and the result obtained is illustrated in Figure-4 and table 9.
Table 9: Nonspecific binding of Carrier protein (CRM/TT) to Protein A resin with and without mAb.
Carrier protein Protein A Protein A-MAb
CRM 117 121
TT 89 84

Observation and Inference: In above experiment, recovery of CRM/TT after treatment with protein A resin/ Protein A resin coupled with Men MAbs was observed in between 75-125% indicated there is no non specific binding of CRM/TT to protein A resin as well as protein A resin coupled with Men MAbs.
Example 8: Study & Analysis on “availability of sufficient antibodies” for interacting with respective serogroup Ps or Ps-Pr conjugate.
Availability of sufficient antibodies for interacting with respective Men Conjugate was analyzed by allowing different concentration of Men conjugate mixture (1µg/ml to 5ug/ml) to Men MAb (500µl of respective MAb/Protein A resin) coupled protein A resin. Concentrations of CRM and TT were determined by BBCIA after treatment with Protein A-Men MAb as per above mentioned protocol (free protein isolation). The result obtained is illustrated in Figure-5 and table 10.
Table 10: Adsorption of Men CJ with Protein A-Men MAb at variable concentration
Men Conj Mix CRM TT
1 µg/ml 0 0
2 µg/ml 0 0
3 µg/ml 0 0
4 µg/ml 0 0
5 µg/ml 150 103

Observation and Inference: In above experiment meningococcal conjugate mixture was incubated with Protein A-Men MAb resin at variable concentration starting from 1µg/ml to 5ug/ml. Supernatant obtained after treatment was analyzed for total CRM and TT by BBCIA. CRM and TT were not observed to be at detectable level till 4µg/ml concentration of meningococcal conjugate mixture. This indicates that all the conjugate till 4µg/ml concentration was adsorbed with Men MAbs coupled to protein A resin which shows Men MAb taken for coupling is sufficient to neutralize meningococcal conjugate till 4µg/ml conc.
CRM and TT were found to be detected above 100 µg/ml when 5µg/ml of conjugate mixture was treated with Protein A-Men MAb. This indicated MAbs used to neutralize meningococcal conjugate mixture equal to 5 µg/ml was not sufficient when higher concentration of meningococcal conjugate mixture was used.
Example 9: Determination of standard range for Lower Limit of Detection; Lower Limit of Quantitation & Upper Limit of Quantitation for TT and CRM by multiplexed/monoplexed BBCIA
Different dilutions of native TT/derivatized CRM197 were assayed by multiplexed BBCIA to obtain standard range as mentioned in Example 2.The result obtained is illustrated in Figure 6a and Figure 6b.
Inference:
Lower limit of detection (LLOD): for CRM; 7.8ng/ml, for TT; 15.6ng/ml
Lower limit of quantification (LLOQ): for CRM; 15.6ng/ml, for TT; 31.25ng/ml
Upper limit of quantification (ULOQ): for CRM; 1000ng/ml, for TT; 1000ng/ml

TECHNICAL ADVANCEMENTS
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of an assay that estimates individual concentration of one or more carrier protein in a conjugate bulk/final vaccine composition. When compared to conventional ELISA, the method of the present disclosure provides the following advantages:
• Able to detect individual concentration of multiple carrier proteins used in the final conjugate bulk/vaccine composition;
• Reduced turnaround time from 21 to 8 hours;
• Upto 90% saving in reagent costs;
• Reduced sample volume required;
• Keeps the same work flow as ELISA;
• Multiplexing capability enables to perform multiple experiments simultaneously instead of sequentially; and
• Beads generate more reproducible results.
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.
The use of the expression “at least” 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 formulation 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 invention.
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 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.
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 illustrative of the disclosure and not as a limitation.

,CLAIMS:WE CLAIM:
1. A method for quantifying unconjugated carrier protein in a carbohydrate protein conjugate vaccine, said method comprising the following steps:
a. coupling an anti-carbohydrate antibody to affinity chromatography matrix immobilized on a solid support;
b. subjecting said vaccine to affinity chromatography matrix; wherein conjugated and unconjugated polysaccharide binds to respective anti – carbohydrate antibodies;
c. collecting flow-through comprising unconjugated carrier protein; and
d. quantifying individual concentration of unconjugated carrier protein(s) by bead based competitive inhibition assay using suspension array system.

2. The method as claimed in claim 1, wherein said carbohydrate is selected from a natural carbohydrate, synthetic carbohydrate, oligosaccharide, polysaccharide, and a combination thereof.

3. The method as claimed in claim 1, wherein said affinity chromatography matrix is selected from the group consisting of Protein A, Protein G and Protein L.

4. The method as claimed in claim 1, wherein said beads are selected from the group consisting of sepharose beads, agarose beads, polystyrene beads and polymeric microspheres.

5. The method as claimed in claim 1 or claim 2, wherein said carbohydrate protein conjugate vaccine comprises one or more polysaccharide(s) derived from gram positive bacteria and gram-negative bacteria.
6. The method as claimed in claim 5, wherein said polysaccharide of polysaccharide – protein conjugate vaccines is 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.

7. The method as claimed in claim 6, wherein said polysaccharide of Streptococcus pneumoniae is derived from the group consisting of Streptococcus pneumoniae serogroup 1, 2, 3, 4,5,6A, 6B, 7F, 8, 9A, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19F, 19A, 20, 22F, 23B, 23F, 24F, 33F, 35B, 38 AND 45.

8. The method as claimed in claim 6, wherein said polysaccharide of Neisseria meningitidis is derived from the group consisting of Neisseria meningitidis serogroup A, B, C, D, W135, X, Y, Z, and 29E.

9. The method as claimed in claim 1, wherein said carrier protein of carbohydrate – protein conjugate vaccine is selected from the group consisting of CRM197, diphtheria toxoid, Tetanus 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).

10. The method as claimed in claim 1, wherein said carrier protein of carbohydrate – protein conjugate vaccine is at least one selected from group consisting of CRM197, diphtheria toxoid, and Tetanus toxoid.

11. The method as claimed in claim 1 or claim 3, wherein said affinity chromatography matrix is a Protein A resin.

12. The method as claimed in claim 1, wherein said vaccine is a multivalent Meningococcal Conjugate Vaccine.

13. The method as claimed in claim 12, wherein said multivalent Meningococcal Conjugate Vaccine comprises polysaccharides derived from Neisseria Meningitidis serogroup A, C, W, X and Y; and carrier protein selected from CRM197, Diphtheria Toxoid and Tetanus Toxoid.

14. The method as claimed in claim 1, wherein said vaccine is a multivalent Pneumococcal Conjugate Vaccine.

15. The method as claimed in claim 14, wherein said multivalent Pneumococcal Conjugate Vaccine comprises polysaccharides derived from Streptococcus pneumoniae serogroup 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19F, 19A, 20, 22F, 23B, 23F, 24F, 33F, 35B, 38 and 45; and carrier protein selected from CRM197, Diphtheria Toxoid and Tetanus Toxoid.

16. The method as claimed in claim 1, wherein said quantitation is carried out using flow cytometry or a suspension array system.