Brief Description:
Vaccines that contain protein covalently linked to
carbohydrate have proven remarkably successful in
inducing an immune response to the carbohydrate moiety.
Examples of such vaccines, known as "conjugates" are
available for Haemophilus influenzae type b (e.g.,
ActHib, Hiberix), Neisseria meningitidis types A C W and
Y (e.g., Menactra) and S. pneumoniae (e.g., Prevnar,
Synflorix). In order for the protein to be linked to the
carbohydrate, the latter generally needs to be activated
so that it can be reacted with the protein, either
directly or via a spacer (Dick, W.E. Jr and Beurret, M.
Glyco-conjugates of bacterial carbohydrate antigens. A
survey and consideration of design and preparation
factors. In: Conjugate Vaccines (Eds Cruse, J.M. and
Lewis, R.E.).
It has been reported previously that the polysaccharideprotein
conjugate purification procedures should remove
residual reagents used for conjugation and the removal of
reagents and reaction by-products such as cyanide, EDAC
and others depending on the conjugation chemistry, should
be confirmed by suitable tests or by validation of the
purification process wherein the acceptable limits should
be approved by the NRA. Refer Section A.3.3.1
WHO/BS/06.2041 2006 “Recommendations to assure the
quality, safety and efficacy of group A meningococcal
conjugate vaccines”
Regulatory authorities do consider potential toxicity of
the identified exogenous process related impurities and
accordingly require that the same should be considerably
lower than the 1.5 μg/dose .Refer EMEA Guideline(EMEA
3
/CHMP /QWP/251344/2006) on ‘The Limits of Genotoxic
Impurities’.
Improved methods for cyanating polysaccharides use 1 -
cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP)
(Lees, A., Producing immunogenic constructs using soluble
carbohydrates activated via organic cyanylating reagents.
U.S. Patent Nos. 5,651,971; 5,693,326; and 5,849,301).
GSK’s Synflorix is an approved 10-valent pneumococcal
polysaccharide-protein conjugate vaccine prepared by
using CDAP based cyanylation conjugation
chemistry.Further GSK’s Nimenrix is a pipeline
multivalent meningococcal polysaccharide-protein
conjugate vaccine prepared by using CDAP based
cyanylation conjugation chemistry. The activation of the
polysaccharide with CDAP introduces a cyanate group in
the polysaccharide and DMAP(4-dimethylamino-pyridine) is
liberated. It is known Synflorix contains residual DMAP
at a level of 65ng per dose.Residual DMAP results in
degradation of polysaccharide .Further DMAP has a rat
LD50 (oral)of 140 mg/kg .
To address above concerns of DMAP and subsequent queries
raised by regulatory agencies , GSK has previously
disclosed in-house assays to quantify residual DMAP
content in CDAP conjugation chemistry based
polysaccharide-protein conjugate vaccines (Synflorix,
Nimenrix)as well as utilized diafiltration for removal of
DMAP.Refer WO00/56359 and WO2007000342.
An isocratic reverse phase HPLC method for the analysis
of 4-Dimethylaminopyridine (DMAP)has been reported
earlier.Refer application notes on DMAP- From MicrSolv.
4
Polysaccharide- protein conjugate vaccines prepared
using 1-Cyano-4- pyrrolidinopyridinium tetrafluorborate
(CPPT)based cyanylation conjugation chemistry are still
in development phase and yet to be approved by regulatory
agencies.During conjugation process 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate gets hydrolysed to
form a reaction by-product“4-Pyrrolidinopyridine (4-
PPY)”.
Pipeline Multivalent conjugate vaccines(5-valent)may
contain residual 4-PPY at a level of 25-30ng per
dose.Further 4-PPY has a rat LD50 (oral) of 176 mg/kg.
To address above concerns of 4-PPY and queries that could
be raised by regulatory agencies , there is an urgent
need to develop an assay to quantify residual 4-PPY
content in CPPT conjugation chemistry based
polysaccharide-protein conjugate vaccines.Particularly,
quantification of 4-PPY would be necessary for pipeline
multivalent meningococcal polysaccharide protein
conjugate vaccine compositions comprising capsular
saccharide from serogroups X and capsular saccharide from
A, C, W135 and Y prepared by using 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate based cyanylation
conjugation chemistry.Refer WO2013/114268.
Capillary electrophoresis (CE) combines the
quantification and handling benefits of HPLC with the
separating power of conventional electrophoretic
techniques. As compared with HPLC, CE offers an important
advantage, namely a wide variation of analytical
conditions.
Free solution capillary electrophoresis, also called
capillary zone electrophoresis (CZE), is the most common
5
mode of capillary electroseparation, because it is
performed in a capillary filled only with an electrolyte
solution at a selected pH value and ionic composition.
CZE is applicable to a wide range of charged substances,
including pharmaceuticals, amino acids, peptides, and
proteins. The separation in CZE is based on the
differences in the electrophoretic mobilities, resulting
in different velocities of migration of ionic species in
the electrophoretic buffer contained in the capillary.
Neutral analytes can also be separated by a variation of
CZE termed micellar electrokinetic capillary
chromatography(MEKC). In this technique a surfactant,
usually sodium dodecyl sulfate, is added to the solution
of the neutral analytes. As the sodium dodecyl sulfate is
negatively charged, it moves in the opposite direction to
the electro-osmotic flow. Neutral analytes are swept
along with the electro-osmotic flow and are separated
based on their partitioning between the surfactant and
the surrounding aqueous phase. This technique was
introduced by Terabe (Anal. Chem. vol.57, pp. 834-841
(1985) and vol. 56, pp. 111-113, (1984))and have been
extended by Sepaniak, Fujiwara, Honda, and others to
pharmaceutical preparations Refer William E. Werner et al
“Size-Dependent Separation of Proteins Denatured in SDS
by Capillary Electrophoresis Using a Replaceable Sieving
Matrix” Analytical Biochemistry 212 (1993) 253-258.
Lamb DH et al discloses determination of free carrier
protein in protein-polysaccharide conjugate vaccines by
micellar electrokinetic chromatography.Refer J Chromatogr
A. 2000 Oct 13; 894(1-2):311-8.However it does not
disclose methods to quantify residuals(4PPY,DMAP)in
polysaccharide-protein conjugate vaccines prepared by
6
cyanylation conjugation chemistry.
Existing methods for quantification of residuals(4PPY and
DMAP) are not suitable for in-process analysis and suffer
from following setbacks i)do not provide separate peaks
for residuals(4PPY/DMAP) and polysaccharide-protein
conjugate in a given sample ii)require complex sample
preparation iii)long analysis time iv) additional time
for column stabilization and v) are imprecise due to
possibility of interference of sample matrix and
appearance of ghost peaks.
Present invention eliminates the foregoing shortcomings
of the prior art methods and thereby provides a rapid and
reproducible micellar electrokinetic capillary
chromatography based method for quantifying residual 4-
pyrrolidinopyridine(4-PPY) and 4-dimethylaminopyridine(
DMAP)in polysaccharide- protein conjugate vaccines
prepared by cyanylation conjugation chemistry using CPPT
and CDAP respectively.
Summary of Invention:
The present invention arises from the surprising finding
that it is possible to quantify 4-PPY and DMAP in
polysaccharide- protein conjugate vaccine formulations
and polysaccharide-protein conjugate bulk by utilizing a
novel micellar electrokinetic capillary chromatography
based assay.
Instant micellar electrokinetic capillary chromatography
based assay for 4PPY or DMAP quantification in
polysaccharide-protein conjugate samples has following
characteristics:
7
1.Said method is applicable for quantification of 4-PPY
or DMAP in any polysaccharide- protein conjugates(having
capsular polysaccharides from Streptococcus Pneumoniae,
Neisseria meningitidis , Haemophilus influenzae)prepared
by using CPPT or CDAP conjugation chemistry respectively.
2.It can be utilized for 4PPY or DMAP quantification for
in-process(bulk polysaccharide-protein conjugate) as well
as final polysaccharide-protein conjugate vaccine
formulation.
3.Provides separate peaks for residuals(4PPY/DMAP) and
polysaccharide-protein conjugate in a given sample
4.It can quantify 4-PPY and DMAP in any given sample
wherein 0.03125 μg/ml is the lowest detectable limit
observed.
5.It utilizes an under-vacuum/pressure mode of injection
at pressure of about 0.3psi to 0.5psi for a duration
between 20 and 25 seconds ,capillary temperature between
20 and 25oC, SDS as surfactant at a concentration between
80mM and 120mM,Borate buffer with pH between 9 to 9.5
having a concentration between 10mM and 50 mM.
6. Using 0.9% NaCl as sample dilution buffer for DMAP and
10mM Tris having pH 7.2 as sample dilution buffer for
4PPY instead of WFI to maintain stability of conjugate in
sample.
7.It is a rapid method with a total duration of 45
minutes.
8
8.It does not require any tedious sample preparation.
9.None of the sample matrix interferes with the 4-PPY
peak.
10.It shows good precision,linearity and better
reproducibility in migration times and peak areas.
Description of the figures
Figure 1:Spectral scan of 4PPY (10μg/ml) showed max.
Absorbance at 280nm.
Figure 2:4PPY standard 500ng/mL observed at migration
time 37.896 using CE-MEKC method.
Figure 3:MenA -TT Conjugate was found to be more
purified with Respect to 4PPY
Figure 4:The chromatogram showed different peaks for MenY
-CRM Conjugate and 4PPY (Migration Time 38.112)
Figure 5The chromatogram showed different peaks for
PNU23F-CRM Conjugate and 4PPY (Migration Time 37.496)
Figure 6:The chromatogram showed different peaks for HIB
-CRM Conjugate and 4PPY (Migration Time 37.104)
Figure 7:The chromatogram showed different peaks for
Multivalent Conjugates and 4PPY (Migration Time 38.938)
.
9
Figure 8:Spectral scan of DMAP (10μg/ml) showed max
Absorbance at 280nm
Figure 9:DMAP standard 500ng/mL observed at migration
time 38.133 using CE-MEKC method
Figure 10:The chromatogram showed different Migration
Time PNU 9V -CRM Conjugate and DMAP (Migration Time
37.833)
Figure 11:The chromatogram showed different Migration
Time for HIB -TT Conjugate and DMAP (Migration Time
39.030)
Figure 12:The chromatogram showed different Migration
Time for MenY Conjugate and DMAP (Migration Time
38.867)
Figure 13: In MenA -TT Conjugate, DMAP was found to be
Less than detection limit
Detailed Description
An important aspect of the instant invention is that said
4-PPY and DMAP quantification assay comprises of
following steps:
1. Preparation of working standard 100 μg/mL
(4PPY/DMAP).
2. Arranging set standards from 31.25 ng/mL to 500
ng/mL.
3. Diluting sample 0.1 to 1mg/mL on basis of
Polysaccharide concentration.
4. Preparing spike of 125ng/mL each 4PPY & DMAP.
10
5. Transferring sample and standards in PCR vial.
6. Washing capillary using instrument wash programme.
7. Running separation method at 17kv voltage for 45 min
using MEKC buffer (280nm).
8. Checking for correlation coefficient and % spike
recovery
A second aspect of the instant invention is that said
running solution can comprise of a surfactant selected
from but not limited to sodium dodecyl sulfate
(SDS),cetyltrimethylammonium bromide (CTAB) and sodium
tetradecyl sulfate (STS).
A third aspect of the instant invention is that said
micellar phase can be modified by adding a second
surfactant to form a mixed micelle or by selecting a
different counter ion.
A fourth aspect of the instant invention is that said
running solution can comprise of a buffer selected from
but not limited to Borate , Phosphate,Good’s and Tris
buffer.
Preferably the running solution comprises of 80mM to
about 120 mM of SDS, and 10mM to about 50 mM borate at pH
9.3.
Further the separation voltage can be between 15kV and
20kV.
Yet another aspect of the instant invention is that
aqueous phase can be modified for manipulating
selectivity by using additives selected from a group of
but not limited to cyclodextrins (CDs), ion-pair
11
reagents, urea, organic solvents and metal salts.
Further as per the instant invention said micellar
electrokinetic capillary chromatography can employ
organic modifiers that reduce electro-osmotic flow and
thereby increase peak capacity of the separation that can
be selected from but not limited to methanol, ethanol,
propan-2-ol,butan-1-ol, butan-2-ol, acetone, methyl ethyl
ketone and acetonitrile.
The cyanylation conjugation reaction by-products or
residuals (4-PPY, DMAP) in a sample present at a
concentration from about 0.03125 μg/ml to about 1μg/ml
can be detected by instant micellar electrokinetic
capillary chromatography based assay .
According to a preferred embodiment of instant invention
said micellar electrokinetic capillary chromatography
based assay for 4PPY or DMAP quantification in
polysaccharide-protein conjugate samples can employ
following operating conditions:
1. Sample dilution buffer: For DMAP - 0.9% NaCl and for
4PPY -10mM Tris, pH 7.2
2. Running solution:SDS - 100mM in Borate buffer -
25mM, pH 9.2
3. Capillary: 75 μm I.D. Total length 70 cm
4. Applied voltage:17kV (current 130μA)
5. Capillary Cartridge Temperature : 20°C
6. Sample solvent: water
7. Sample concentration: 0.1 to 1 mg/ ml of Ps of
conjugate .
8. Injection end:Positive end
9. Injection volume/duration: 0.5psi for 27
sec(Injection under vacuum mode/pressure mode)
12
10. Detection: 280 nm (PDA)
Detection methods which may be readily apparent include
ultraviolet and visible light absorption, electrochemical
detection, fluorescence and mass spectroscopy.
Another aspect of the invention is that above method for
4PPY and DMAP quantification can be applicable to all
polysaccharide-protein conjugate vaccines wherein said
polysaccharide is a bacterial capsular polysaccharide
selected from the group consisting of Neisseria
meningitidis groups A ,C , W135 Y and X, Haemophilus
influenzae,Group A Streptococcus, Group B Streptococcus,
Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6A, 6B,
7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,
19F, 20, 22F, 23F, 33F,Escherichia coli, Francisella
tularensis, Klebsiella, Moraxella catarrhalis,
Porphyromonas gingivalis, Pseudomonas aeruginosa,
Burkholderia cepacia, Salmonella typhi, Salmonella
typhimurium, Salmonella paratyphi, Shigella dysenteriae,
Shigella flexneri, Shigella sonnei, Vibrio
cholera,Enterococcus faecalis, Enterococcus
faecium,Mycobacterium tuberculosis, Staphylococcus aureus
and Staphylococcus epidermidis.
The carrier protein of said polysaccharide-protein
conjugates can be selected from a group of but not
limited to CRM 197,diphtheria toxoid,tetanus toxoid,
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 adhesin protein (PsaA),pneumococcal PhtD,
13
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),particularly CRM197.
Preferably the above method for 4PPY quantification can
be applicable to a multivalent meningococcal
polysaccharide-protein conjugate composition that can
comprise of (a)a conjugate of (i) the capsular
saccharide of serogroup A N meningitidis and (ii)tetanus
toxoid; (b) a conjugate of (i) capsular saccharide of
serogroup C N meningitidis and (ii)CRM197; (c) a
conjugate of (i) capsular saccharide of serogroup Y N.
meningitidis and (ii) CRM197;(d) a conjugate of (i)
capsular saccharide of serogroup W135 N.meningitidis and
(ii)CRM197; and (d) a conjugate of (i) capsular
saccharide of serogroup X N meningitidis and (ii)tetanus
toxoid.
One important aspect of the instant invention is that
above method for DMAP quantification can be applicable to
an immunogenic composition comprising a multivalent
polysaccharide-protein conjugate mixture consisting of
capsular polysaccharides from 12 F and 15B and atleast
one additional polysaccharide selected from 1, 2, 3, 4,
5, 6A, 6B,7F,9A,9V,9N,10A, 11A, 14,17F, 18C, 19A , 19F,
20, 22F,23F ,33F and 45.
Further the above method for DMAP quantification can be
applicable to a multivalent pneumococcal polysaccharideprotein
conjugate composition that can comprise of
14
capsular polysaccharides from serogroups 1 , 4, 5, 6A,
6B, 9V, 14,19A , 19F & 23F conjugated individually to
CRM197.
Examples
I) Preparation of MenA-TT Conjugate (CPPT Conjugation
chemistry)
Polysaccharide preparation:
MenA-TT conjugate was prepared by cyanylation and
carbodiimide chemistry. The native polysaccharide was
sized reduced by mechanical means. The sized reduced PS
(100-120kD on SEC-HPLC) was diafiltered & concentrated on
10kD cuttoff membrane. To 6mg/ml PS, freshly prepared
solution of CPPT(dissolved 114 mg/ml in acetonitrile)
added the pH was shifted to 9.5 with NaOH solution and
hold for upto 5 min. Adipic acid dihydrazide (ADH)
dissolved 100mg/ml in water was added to the PS solution.
The reaction was stirred for 1 hrs at 250C. The reaction
was quenched by addition of excess glycine solution. The
reaction mixture was diafiltered on 10kD TFF in MES
buffer to remove residuals and unreacted components. The
sample was stored at 2-80C until use. The PS content was
measured by phosphorous assay and extent of
derivatization was measured by TNBS assay.
Carrier Protein (TT) Preparation:
Tetanus toxoid was chromatographically purified to remove
aggregates that might cause adverse effect in the
conjugation process. Total TT load was 5-10% v/v of
packed column (toyopearl HW-65F, Tosoh biosciences) with
30cm/hr linear flow rate. The fractions were collected on
the basis of chromatogram obtained on UV detector of the
15
chromatography system. The fractions were pooled on the
basis of aggregate removal seen on SEC-HPLC. The
collected fractions were subjected to concentrationdiafiltration
on 30kD TFF in MES buffer. The protein
content was measured by Lowry assay.
Conjugation of Men A to TT:
The ADH-derivatized PS, purified TT and EDAC (1-ethyl-3-
(3-dimethyl-aminopropyl) carbodiimide) were mixed the
ratio of 1:0.8:1.5 by weight under stirring condition.
The reaction was monitored on HPLC for protein
conversion. The reaction was quenched after 3 hours by
raising pH with 10mM PB+EDTA pH 8.0 buffer.
Conjugate Purification:
The conjugate was purified on 300kD cut off membrane by
passing 30 volumes of 100mM PBS and followed by 30
volume10mM Tris pH 7.2. The sucrose-citrate stabilizer
was added to bulk conjugate and stored below -200C until
use. The purified bulk conjugate was analyzed for total
and unbound (free) PS, Total and unbound(free) protein
and residual 4-PPY in the conjugate bulk.
Table 1
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Men A-TT
Purified
Conjugate
1.1 2.23 4.10 2.27
II) Preparation of MenY-CRM197 Conjugate (CPPT
Conjugation chemistry)
16
Polysaccharide preparation:
Men Y PS sizing was carried out by chemical means . The
chemically sized PS was targeted to achieve size of 75-
100kDa on SEC-HPLC and then the polysaccharide was
diafiltered on 10kD cuttoff membrane.
Carrier Protein (CRM197) Preparation:
The carrier Protein derivatization process was same as
described above for MenC-CRM conjugate.
Conjugation of Men Y to CRM197:
MenY PS was mixed with CPPT (113 mg/ml in acetonitrile) ,
pH adjusted to 9.5 immediately after addition and then
with ADH-derivatized CRM197 in the ratio of 1:1.5:1.25 by
weight under stirring condition. The reaction was
monitored on HPLC. The reaction was quenched by addition
of excess glycine solution.
Conjugate Purification:
The conjugate was purified on 300kD cut off membrane by
passing 30 volumes of 100mM PBS and followed by 10
volume10mM Tris pH 7.2 and then 20 volumes of 10mM Tris
on 100kD cut off membrane. The sucrose-citrate stabilizer
mixture was added to the bulk conjugate and stored below
-200C until use.
The purified bulk conjugate was analyzed for total and
unbound (free) PS, Total and unbound(free) protein and
residual 4-PPY in the conjugate bulk.
Table 2
17
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Men Y-CRM197
Purified
Conjugate
1.46 3.59 <4.43 <4.09
III) Preparation of Pneumococcal conjugate 23F-CRM197-ADH
Conjugate (CPPT Conjugation chemistry)
Polysaccharide preparation:
In case of Pneumococcal 23F Ps, sizing was carried out by
mechanical means and sizing was continued until the PS
size was reached to 150-170 kD on SEC-HPLC and then the
polysaccharide was diafiltered-concentrated on 10kD
cuttoff membrane.
Carrier Protein (CRM197) Preparation:
In order to conjugate Pneumococcal 23F Ps to CRM197, the
carrier protein was derivatized by ADH linker prior to
conjugation.
The native CRM197 was allowed to react with ADH linker in
presence of EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)
carbodiimide) in MES buffer. The reaction was then
quenched by increasing pH by sodium carbonate solution
under stirring for 15min. The ADH-derivatized CRM197 was
then diafiltered on 10kD cuttoff membrane using 10mM
carbonate buffer pH 9.5 by passing about 35 volumes of
buffer. The sample was submitted to content analysis and
extent of derivatization.
18
Conjugation of Pneumococcal 23F-CRM197:
Pneumococcal 23F PS was mixed with CPPT( in acetonitrile)
and then ADH-derivatized CRM197 in the ratio of
1:1.25:1.25 by weight under stirring condition. The
reaction was monitored on HPLC. The reaction was quenched
after 3 hours by addition of excess glycine solution.
Conjugate Purification:
The crude conjugate was purified on 300kD cut off
membrane by passing 30 volumes of 10mM PBS and followed
by 20 volume10mM Tris pH 7.2. The purified bulk conjugate
was analyzed for total and unbound (free) PS, total and
unbound(free) protein and residual 4-PPY in the conjugate
bulk.
Table 3
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Pneumococcal
23F-CRM197
Conjugate
0.87 1.58 1.67 2.11
IV) Preparation of Pneumococcal (9V- CRM
197),Meningococcal(Men A-TT Conjugate ,Men Y-CRM197
Conjugate) & Hib-TT conjugates by CDAP conjugation
chemistry
Polysaccharides were conjugated to carrier by using CDAP
conjugation method disclosed in Lees et al (Vaccine 26:
190-198, 1996)
19
Table 4: Estimation of 4-PPY in polysaccharide protein
conjugate vaccines
Table 5: Estimation of DMAP in polysaccharide protein
conjugate vaccines
Spi
ke
Rec
overy (DMAP & 4PPY) for all above samples was found to be
between 80-120%. Further MEKC-CE method was found to be
sensitive to capillary temperature variation.Temperature
of about 20oC was found to maintain the viscosity of the
buffer thereby ensuring optimal electrophoretic and EOF
velocities ultimately resulting into optimal migration
time.
Samples 4-PPY (ng/μg of Ps)
Men ATT
Conjugate
Below Detection limit
Men Y-CRM197 Conjugate 0.58
Hib-
CRM197 Conjugate
1.01
PN23FCRM
197 Conjugate
1.61
Samples DMAP (ng/μg of Ps)
Men ATT
Conjugate
Below Detection
limit
Men Y-CRM197 Conjugate 0.71
Hib-
TT Conjugate
0.59
Pneumococcal 9VCRM
197 Conjugate
1.01
20
Further said MEKC method for assaying 4-PPY & DMAP in
samples elucidates substantial improvement in
reproducibility in quantitative analysis, including
migration time and peak height or peak area.MEKC-CE
method provides also a number of advantages over other
analytical techniques: the rapid development of the
analysis method, its analysis speed, reduced consumable
and solvent expenses, low sample amount, simplicity of
operations, and a greater possibility of implementation
of a single set of method conditions for the analysis of
several different samples.
It is to be understood that typical methods of Micellar
electrokinetic capillary chromatography(MEKC)technology
have been applied, and that a person skilled in the art
of MEKC is well aware of minor adjustments that would not
alter the results to be obtained, such as different
measurements at other suitable wavelengths or by the use
of other column material that would not severely alter
the results obtained by the invention.In view of the many
possible embodiments to which the principles of the
disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only
preferred examples of the invention and should not be
taken as limiting the scope of the invention.
21
We Claim:
1. An improved method for assaying a sample suspected
to contain cyanylation conjugation reaction byproducts
of 1-Cyano-4- pyrrolidinopyridinium
tetrafluorborate (CPPT)or 1 -cyano-4-
dimethylaminopyridine tetrafluoroborate
(CDAP),characterized in that the quantification is
performed by a micellar electrokinetic capillary
chromatography method .
2. A method according to claim 1, wherein the
cyanylation conjugation reaction by-products are 4-
pyrrolidinopyridine(4-PPY) for CPPT and 4-
dimethylaminopyridine(DMAP) for CDAP.
3. A method according to claim 1, wherein the method
provides improved peak resolution characterized in
that separate peaks for cyanylation conjugation
reaction by-products and polysaccharide-protein
conjugate can be obtained in a given sample.
4. A method according to claim 1, wherein the MEKC
based method can comprise of following steps:
a) Preparing working standard 100 μg/mL (4PPY or DMAP);
b) Arranging set standards from 31.25 ng/mL to 500
ng/mL;
c) Diluting sample 0.1 to 1mg/mL on basis of
Polysaccharide concentration;
d) Preparing spike of 125ng/mL each for 4PPY and DMAP;
e) Transferring sample and standards in PCR vial;
f) Washing capillary using instrument wash programme ;
g) Running separation method at 17kv voltage for 45 min
using MEKC buffer (280nm);and
22
h) Checking for correlation coefficient and % spike
recovery.
5. A method according to claim 4, wherein the sample
dilution buffer for DMAP is 0.9% NaCl and for 4PPY
is 10mM Tris, pH 7.2.
6. A method according to claim 4, wherein sample
concentration can be between 0.1 to 1 mg/ ml of Ps
of conjugate.
7. A method according to claim 4,wherein sample
injection is carried out under pressure mode at
0.5psi for about 27 sec.
8. A method according to claim 4, wherein the running
solution can comprise of i) a surfactant selected
from a group of sodium dodecyl sulfate
(SDS),cetyltrimethylammonium bromide (CTAB) and
sodium tetradecyl sulfate (STS) and ii)a buffer
selected from a group of Borate ,phosphate ,Good’s
and tris (hydroxymethyl) aminomethane (Tris)buffer.
9. A method according to claim 8, wherein the running
solution comprises of i) SDS having concentration
between 80 mM to about 120 mM and ii) borate having
concentration between 10mM to about 50 mM at pH 9.3.
10. A method according to claim 4, wherein said aqueous
phase can be modified for manipulating selectivity
by using additives selected from a group of
cyclodextrins (CDs), ion-pair reagents, urea,
organic solvents and metal salts.
23
11. A method according to claim 4,wherein said micellar
phase can be modified by adding a second surfactant
to form a mixed micelle or by selecting a different
counter ion.
12.A method according to claim 3, wherein said micellar
electrokinetic capillary chromatography can employ
organic modifiers that reduce electro-osmotic flow
and thereby increase peak capacity of the separation
that can be selected from a group of methanol,
ethanol, propan-2-ol,butan-1-ol, butan-2-ol,
acetone, methyl ethyl ketone and acetonitrile.
13. A method according to claim 4, wherein capillary is
a 75 μm I.D.having total length of about 70 cm.
14. A method according to claim 4, wherein applied
voltage is 17kV (current 130μA).
15.A method according to claim 4, wherein capillary
cartridge temperature is between 15°C and about
20°C.
16. A method according to claim 4, wherein detection
methods can be selected from ultraviolet and visible
light absorption, electrochemical detection,
fluorescence and mass spectroscopy, characterized in
that the detection is carried at a wavelength of
280nm.
17.The method of any preceding claim wherein the
capsular polysaccharide is a polysaccharide selected
from a Meningococcal polysaccharide, a Pneumococcal
polysaccharide, a Haemophilus influenzae type B
24
polysaccharide, a Vi polysaccharide of Salmonella
typhi, or a group B Streptococcus polysaccharide.
18.The method of any preceding claim wherein the
polysaccharide can be selected from Meningococcal
group A polysaccharide, a Meningococcal group C
polysaccharide, a Meningococcal group W-135
polysaccharide, a Meningococcal group Y and a
Meningococcal group X.
19. The method of any preceding claim wherein the
carrier protein is a protein selected from the group
consisting of tetanus toxoid (TT), fragment C of TT,
diphtheria toxoid, CRM197, Pneumolysin, protein D,
PhtD, PhtDE,OMPC and N19.
20. The method of any preceding claim,wherein the sample
being assayed for 4-PPY comprises of atleast one
conjugate selected from (a)a conjugate of (i) the
capsular saccharide of serogroup A N meningitidis
and (ii)tetanus toxoid; (b) a conjugate of (i)
capsular saccharide of serogroup C N meningitidis
and (ii)CRM197; (c) a conjugate of (i) capsular
saccharide of serogroup Y N. meningitidis and (ii)
CRM197;(d) a conjugate of (i) capsular saccharide of
serogroup W135 N.meningitidis and (ii)CRM197; and
(d) a conjugate of (i) capsular saccharide of
serogroup X N meningitidis and (ii)tetanus toxoid.
21. The method of any preceding claim wherein the sample
being assayed for DMAP comprises of atleast one
polysaccharide-protein conjugate with polysaccharide
selected from group of Streptococcus pneumoniae type
1, 2, 3, 4, 5, 6A, 6B,7F,8, 9A,9V,9N,10A, 11A
25
,12F,14,15B, 17F, 18C, 19A , 19F, 20, 22F,23F ,33F
and 45.
Dated this 20th day of May 2014
Of Anand and Anand Advocates
Agent for the Applicant
26
ABSTRACT
MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY BASED
ASSAY FOR QUANTIFYING RESIDUAL 4- PYRROLIDINOPYRIDINE (4-
PPY) AND 4- DIMETHYLAMINOPYRIDINE (DMAP)IN POLYSACCHARIDE
- PROTEIN CONJUGATE VACCINES
The present invention relates to a rapid and reproducible
micellar electrokinetic capillary chromatography based
method for quantifying residual 4-pyrrolidinopyridine(4-
PPY)and4-dimethylaminopyridine(DMAP) in polysaccharideprotein
conjugate vaccines prepared by cyanylation
conjugation chemistry using 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate (CPPT) and 1 -
cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP)
respectively.Said method provides separate peaks for
residuals(4PPY/DMAP) and polysaccharide-protein conjugate
in a given sample,does not require any tedious sample
preparation ,is applicable for quantification of 4-PPY
and DMAP in any polysaccharide-protein conjugate bulk or
vaccine formulation samples and is highly
reproducible.Further good precision and linearity of
instant method can be attributed to under-pressure mode
of injection, injection volume,injection duration,
concentration of surfactant and concentration of buffer.

FORM-2
THE PATENT ACT,1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10;rule 13)
" MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY BASED ASSAY FOR QUANTIFYING RESIDUAL 4-
PYRROLIDINOPYRIDINE (4-PPY) AND 4- DIMETHYLAMINOPYRIDINE (DMAP)IN POLYSACCHARIDE - PROTEIN
CONJUGATE VACCINES "
SERUM INSTITUTE OF INDIA LTD., an Indian Institute, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028,
Maharashtra, India.
The following specification particularly describes the invention and the manner in which it is to be performed:
2
Brief Description:
Vaccines that contain protein covalently linked to
carbohydrate have proven remarkably successful in
inducing an immune response to the carbohydrate moiety.
Examples of such vaccines, known as "conjugates" are
available for Haemophilus influenzae type b (e.g.,
ActHib, Hiberix), Neisseria meningitidis types A C W and
Y (e.g., Menactra) and S. pneumoniae (e.g., Prevnar,
Synflorix). In order for the protein to be linked to the
carbohydrate, the latter generally needs to be activated
so that it can be reacted with the protein, either
directly or via a spacer (Dick, W.E. Jr and Beurret, M.
Glyco-conjugates of bacterial carbohydrate antigens. A
survey and consideration of design and preparation
factors. In: Conjugate Vaccines (Eds Cruse, J.M. and
Lewis, R.E.).
It has been reported previously that the polysaccharideprotein
conjugate purification procedures should remove
residual reagents used for conjugation and the removal of
reagents and reaction by-products such as cyanide, EDAC
and others depending on the conjugation chemistry, should
be confirmed by suitable tests or by validation of the
purification process wherein the acceptable limits should
be approved by the NRA. Refer Section A.3.3.1
WHO/BS/06.2041 2006 “Recommendations to assure the
quality, safety and efficacy of group A meningococcal
conjugate vaccines”
Regulatory authorities do consider potential toxicity of
the identified exogenous process related impurities and
accordingly require that the same should be considerably
lower than the 1.5 μg/dose .Refer EMEA Guideline(EMEA
3
/CHMP /QWP/251344/2006) on ‘The Limits of Genotoxic
Impurities’.
Improved methods for cyanating polysaccharides use 1 -
cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP)
(Lees, A., Producing immunogenic constructs using soluble
carbohydrates activated via organic cyanylating reagents.
U.S. Patent Nos. 5,651,971; 5,693,326; and 5,849,301).
GSK’s Synflorix is an approved 10-valent pneumococcal
polysaccharide-protein conjugate vaccine prepared by
using CDAP based cyanylation conjugation
chemistry.Further GSK’s Nimenrix is a pipeline
multivalent meningococcal polysaccharide-protein
conjugate vaccine prepared by using CDAP based
cyanylation conjugation chemistry. The activation of the
polysaccharide with CDAP introduces a cyanate group in
the polysaccharide and DMAP(4-dimethylamino-pyridine) is
liberated. It is known Synflorix contains residual DMAP
at a level of 65ng per dose.Residual DMAP results in
degradation of polysaccharide .Further DMAP has a rat
LD50 (oral)of 140 mg/kg .
To address above concerns of DMAP and subsequent queries
raised by regulatory agencies , GSK has previously
disclosed in-house assays to quantify residual DMAP
content in CDAP conjugation chemistry based
polysaccharide-protein conjugate vaccines (Synflorix,
Nimenrix)as well as utilized diafiltration for removal of
DMAP.Refer WO00/56359 and WO2007000342.
An isocratic reverse phase HPLC method for the analysis
of 4-Dimethylaminopyridine (DMAP)has been reported
earlier.Refer application notes on DMAP- From MicrSolv.
4
Polysaccharide- protein conjugate vaccines prepared
using 1-Cyano-4- pyrrolidinopyridinium tetrafluorborate
(CPPT)based cyanylation conjugation chemistry are still
in development phase and yet to be approved by regulatory
agencies.During conjugation process 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate gets hydrolysed to
form a reaction by-product“4-Pyrrolidinopyridine (4-
PPY)”.
Pipeline Multivalent conjugate vaccines(5-valent)may
contain residual 4-PPY at a level of 25-30ng per
dose.Further 4-PPY has a rat LD50 (oral) of 176 mg/kg.
To address above concerns of 4-PPY and queries that could
be raised by regulatory agencies , there is an urgent
need to develop an assay to quantify residual 4-PPY
content in CPPT conjugation chemistry based
polysaccharide-protein conjugate vaccines.Particularly,
quantification of 4-PPY would be necessary for pipeline
multivalent meningococcal polysaccharide protein
conjugate vaccine compositions comprising capsular
saccharide from serogroups X and capsular saccharide from
A, C, W135 and Y prepared by using 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate based cyanylation
conjugation chemistry.Refer WO2013/114268.
Capillary electrophoresis (CE) combines the
quantification and handling benefits of HPLC with the
separating power of conventional electrophoretic
techniques. As compared with HPLC, CE offers an important
advantage, namely a wide variation of analytical
conditions.
Free solution capillary electrophoresis, also called
capillary zone electrophoresis (CZE), is the most common
5
mode of capillary electroseparation, because it is
performed in a capillary filled only with an electrolyte
solution at a selected pH value and ionic composition.
CZE is applicable to a wide range of charged substances,
including pharmaceuticals, amino acids, peptides, and
proteins. The separation in CZE is based on the
differences in the electrophoretic mobilities, resulting
in different velocities of migration of ionic species in
the electrophoretic buffer contained in the capillary.
Neutral analytes can also be separated by a variation of
CZE termed micellar electrokinetic capillary
chromatography(MEKC). In this technique a surfactant,
usually sodium dodecyl sulfate, is added to the solution
of the neutral analytes. As the sodium dodecyl sulfate is
negatively charged, it moves in the opposite direction to
the electro-osmotic flow. Neutral analytes are swept
along with the electro-osmotic flow and are separated
based on their partitioning between the surfactant and
the surrounding aqueous phase. This technique was
introduced by Terabe (Anal. Chem. vol.57, pp. 834-841
(1985) and vol. 56, pp. 111-113, (1984))and have been
extended by Sepaniak, Fujiwara, Honda, and others to
pharmaceutical preparations Refer William E. Werner et al
“Size-Dependent Separation of Proteins Denatured in SDS
by Capillary Electrophoresis Using a Replaceable Sieving
Matrix” Analytical Biochemistry 212 (1993) 253-258.
Lamb DH et al discloses determination of free carrier
protein in protein-polysaccharide conjugate vaccines by
micellar electrokinetic chromatography.Refer J Chromatogr
A. 2000 Oct 13; 894(1-2):311-8.However it does not
disclose methods to quantify residuals(4PPY,DMAP)in
polysaccharide-protein conjugate vaccines prepared by
6
cyanylation conjugation chemistry.
Existing methods for quantification of residuals(4PPY and
DMAP) are not suitable for in-process analysis and suffer
from following setbacks i)do not provide separate peaks
for residuals(4PPY/DMAP) and polysaccharide-protein
conjugate in a given sample ii)require complex sample
preparation iii)long analysis time iv) additional time
for column stabilization and v) are imprecise due to
possibility of interference of sample matrix and
appearance of ghost peaks.
Present invention eliminates the foregoing shortcomings
of the prior art methods and thereby provides a rapid and
reproducible micellar electrokinetic capillary
chromatography based method for quantifying residual 4-
pyrrolidinopyridine(4-PPY) and 4-dimethylaminopyridine(
DMAP)in polysaccharide- protein conjugate vaccines
prepared by cyanylation conjugation chemistry using CPPT
and CDAP respectively.
Summary of Invention:
The present invention arises from the surprising finding
that it is possible to quantify 4-PPY and DMAP in
polysaccharide- protein conjugate vaccine formulations
and polysaccharide-protein conjugate bulk by utilizing a
novel micellar electrokinetic capillary chromatography
based assay.
Instant micellar electrokinetic capillary chromatography
based assay for 4PPY or DMAP quantification in
polysaccharide-protein conjugate samples has following
characteristics:
7
1.Said method is applicable for quantification of 4-PPY
or DMAP in any polysaccharide- protein conjugates(having
capsular polysaccharides from Streptococcus Pneumoniae,
Neisseria meningitidis , Haemophilus influenzae)prepared
by using CPPT or CDAP conjugation chemistry respectively.
2.It can be utilized for 4PPY or DMAP quantification for
in-process(bulk polysaccharide-protein conjugate) as well
as final polysaccharide-protein conjugate vaccine
formulation.
3.Provides separate peaks for residuals(4PPY/DMAP) and
polysaccharide-protein conjugate in a given sample
4.It can quantify 4-PPY and DMAP in any given sample
wherein 0.03125 μg/ml is the lowest detectable limit
observed.
5.It utilizes an under-vacuum/pressure mode of injection
at pressure of about 0.3psi to 0.5psi for a duration
between 20 and 25 seconds ,capillary temperature between
20 and 25oC, SDS as surfactant at a concentration between
80mM and 120mM,Borate buffer with pH between 9 to 9.5
having a concentration between 10mM and 50 mM.
6. Using 0.9% NaCl as sample dilution buffer for DMAP and
10mM Tris having pH 7.2 as sample dilution buffer for
4PPY instead of WFI to maintain stability of conjugate in
sample.
7.It is a rapid method with a total duration of 45
minutes.
8
8.It does not require any tedious sample preparation.
9.None of the sample matrix interferes with the 4-PPY
peak.
10.It shows good precision,linearity and better
reproducibility in migration times and peak areas.
Description of the figures
Figure 1:Spectral scan of 4PPY (10μg/ml) showed max.
Absorbance at 280nm.
Figure 2:4PPY standard 500ng/mL observed at migration
time 37.896 using CE-MEKC method.
Figure 3:MenA -TT Conjugate was found to be more
purified with Respect to 4PPY
Figure 4:The chromatogram showed different peaks for MenY
-CRM Conjugate and 4PPY (Migration Time 38.112)
Figure 5The chromatogram showed different peaks for
PNU23F-CRM Conjugate and 4PPY (Migration Time 37.496)
Figure 6:The chromatogram showed different peaks for HIB
-CRM Conjugate and 4PPY (Migration Time 37.104)
Figure 7:The chromatogram showed different peaks for
Multivalent Conjugates and 4PPY (Migration Time 38.938)
.
9
Figure 8:Spectral scan of DMAP (10μg/ml) showed max
Absorbance at 280nm
Figure 9:DMAP standard 500ng/mL observed at migration
time 38.133 using CE-MEKC method
Figure 10:The chromatogram showed different Migration
Time PNU 9V -CRM Conjugate and DMAP (Migration Time
37.833)
Figure 11:The chromatogram showed different Migration
Time for HIB -TT Conjugate and DMAP (Migration Time
39.030)
Figure 12:The chromatogram showed different Migration
Time for MenY Conjugate and DMAP (Migration Time
38.867)
Figure 13: In MenA -TT Conjugate, DMAP was found to be
Less than detection limit
Detailed Description
An important aspect of the instant invention is that said
4-PPY and DMAP quantification assay comprises of
following steps:
1. Preparation of working standard 100 μg/mL
(4PPY/DMAP).
2. Arranging set standards from 31.25 ng/mL to 500
ng/mL.
3. Diluting sample 0.1 to 1mg/mL on basis of
Polysaccharide concentration.
4. Preparing spike of 125ng/mL each 4PPY & DMAP.
10
5. Transferring sample and standards in PCR vial.
6. Washing capillary using instrument wash programme.
7. Running separation method at 17kv voltage for 45 min
using MEKC buffer (280nm).
8. Checking for correlation coefficient and % spike
recovery
A second aspect of the instant invention is that said
running solution can comprise of a surfactant selected
from but not limited to sodium dodecyl sulfate
(SDS),cetyltrimethylammonium bromide (CTAB) and sodium
tetradecyl sulfate (STS).
A third aspect of the instant invention is that said
micellar phase can be modified by adding a second
surfactant to form a mixed micelle or by selecting a
different counter ion.
A fourth aspect of the instant invention is that said
running solution can comprise of a buffer selected from
but not limited to Borate , Phosphate,Good’s and Tris
buffer.
Preferably the running solution comprises of 80mM to
about 120 mM of SDS, and 10mM to about 50 mM borate at pH
9.3.
Further the separation voltage can be between 15kV and
20kV.
Yet another aspect of the instant invention is that
aqueous phase can be modified for manipulating
selectivity by using additives selected from a group of
but not limited to cyclodextrins (CDs), ion-pair
11
reagents, urea, organic solvents and metal salts.
Further as per the instant invention said micellar
electrokinetic capillary chromatography can employ
organic modifiers that reduce electro-osmotic flow and
thereby increase peak capacity of the separation that can
be selected from but not limited to methanol, ethanol,
propan-2-ol,butan-1-ol, butan-2-ol, acetone, methyl ethyl
ketone and acetonitrile.
The cyanylation conjugation reaction by-products or
residuals (4-PPY, DMAP) in a sample present at a
concentration from about 0.03125 μg/ml to about 1μg/ml
can be detected by instant micellar electrokinetic
capillary chromatography based assay .
According to a preferred embodiment of instant invention
said micellar electrokinetic capillary chromatography
based assay for 4PPY or DMAP quantification in
polysaccharide-protein conjugate samples can employ
following operating conditions:
1. Sample dilution buffer: For DMAP - 0.9% NaCl and for
4PPY -10mM Tris, pH 7.2
2. Running solution:SDS - 100mM in Borate buffer -
25mM, pH 9.2
3. Capillary: 75 μm I.D. Total length 70 cm
4. Applied voltage:17kV (current 130μA)
5. Capillary Cartridge Temperature : 20°C
6. Sample solvent: water
7. Sample concentration: 0.1 to 1 mg/ ml of Ps of
conjugate .
8. Injection end:Positive end
9. Injection volume/duration: 0.5psi for 27
sec(Injection under vacuum mode/pressure mode)
12
10. Detection: 280 nm (PDA)
Detection methods which may be readily apparent include
ultraviolet and visible light absorption, electrochemical
detection, fluorescence and mass spectroscopy.
Another aspect of the invention is that above method for
4PPY and DMAP quantification can be applicable to all
polysaccharide-protein conjugate vaccines wherein said
polysaccharide is a bacterial capsular polysaccharide
selected from the group consisting of Neisseria
meningitidis groups A ,C , W135 Y and X, Haemophilus
influenzae,Group A Streptococcus, Group B Streptococcus,
Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6A, 6B,
7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,
19F, 20, 22F, 23F, 33F,Escherichia coli, Francisella
tularensis, Klebsiella, Moraxella catarrhalis,
Porphyromonas gingivalis, Pseudomonas aeruginosa,
Burkholderia cepacia, Salmonella typhi, Salmonella
typhimurium, Salmonella paratyphi, Shigella dysenteriae,
Shigella flexneri, Shigella sonnei, Vibrio
cholera,Enterococcus faecalis, Enterococcus
faecium,Mycobacterium tuberculosis, Staphylococcus aureus
and Staphylococcus epidermidis.
The carrier protein of said polysaccharide-protein
conjugates can be selected from a group of but not
limited to CRM 197,diphtheria toxoid,tetanus toxoid,
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 adhesin protein (PsaA),pneumococcal PhtD,
13
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),particularly CRM197.
Preferably the above method for 4PPY quantification can
be applicable to a multivalent meningococcal
polysaccharide-protein conjugate composition that can
comprise of (a)a conjugate of (i) the capsular
saccharide of serogroup A N meningitidis and (ii)tetanus
toxoid; (b) a conjugate of (i) capsular saccharide of
serogroup C N meningitidis and (ii)CRM197; (c) a
conjugate of (i) capsular saccharide of serogroup Y N.
meningitidis and (ii) CRM197;(d) a conjugate of (i)
capsular saccharide of serogroup W135 N.meningitidis and
(ii)CRM197; and (d) a conjugate of (i) capsular
saccharide of serogroup X N meningitidis and (ii)tetanus
toxoid.
One important aspect of the instant invention is that
above method for DMAP quantification can be applicable to
an immunogenic composition comprising a multivalent
polysaccharide-protein conjugate mixture consisting of
capsular polysaccharides from 12 F and 15B and atleast
one additional polysaccharide selected from 1, 2, 3, 4,
5, 6A, 6B,7F,9A,9V,9N,10A, 11A, 14,17F, 18C, 19A , 19F,
20, 22F,23F ,33F and 45.
Further the above method for DMAP quantification can be
applicable to a multivalent pneumococcal polysaccharideprotein
conjugate composition that can comprise of
14
capsular polysaccharides from serogroups 1 , 4, 5, 6A,
6B, 9V, 14,19A , 19F & 23F conjugated individually to
CRM197.
Examples
I) Preparation of MenA-TT Conjugate (CPPT Conjugation
chemistry)
Polysaccharide preparation:
MenA-TT conjugate was prepared by cyanylation and
carbodiimide chemistry. The native polysaccharide was
sized reduced by mechanical means. The sized reduced PS
(100-120kD on SEC-HPLC) was diafiltered & concentrated on
10kD cuttoff membrane. To 6mg/ml PS, freshly prepared
solution of CPPT(dissolved 114 mg/ml in acetonitrile)
added the pH was shifted to 9.5 with NaOH solution and
hold for upto 5 min. Adipic acid dihydrazide (ADH)
dissolved 100mg/ml in water was added to the PS solution.
The reaction was stirred for 1 hrs at 250C. The reaction
was quenched by addition of excess glycine solution. The
reaction mixture was diafiltered on 10kD TFF in MES
buffer to remove residuals and unreacted components. The
sample was stored at 2-80C until use. The PS content was
measured by phosphorous assay and extent of
derivatization was measured by TNBS assay.
Carrier Protein (TT) Preparation:
Tetanus toxoid was chromatographically purified to remove
aggregates that might cause adverse effect in the
conjugation process. Total TT load was 5-10% v/v of
packed column (toyopearl HW-65F, Tosoh biosciences) with
30cm/hr linear flow rate. The fractions were collected on
the basis of chromatogram obtained on UV detector of the
15
chromatography system. The fractions were pooled on the
basis of aggregate removal seen on SEC-HPLC. The
collected fractions were subjected to concentrationdiafiltration
on 30kD TFF in MES buffer. The protein
content was measured by Lowry assay.
Conjugation of Men A to TT:
The ADH-derivatized PS, purified TT and EDAC (1-ethyl-3-
(3-dimethyl-aminopropyl) carbodiimide) were mixed the
ratio of 1:0.8:1.5 by weight under stirring condition.
The reaction was monitored on HPLC for protein
conversion. The reaction was quenched after 3 hours by
raising pH with 10mM PB+EDTA pH 8.0 buffer.
Conjugate Purification:
The conjugate was purified on 300kD cut off membrane by
passing 30 volumes of 100mM PBS and followed by 30
volume10mM Tris pH 7.2. The sucrose-citrate stabilizer
was added to bulk conjugate and stored below -200C until
use. The purified bulk conjugate was analyzed for total
and unbound (free) PS, Total and unbound(free) protein
and residual 4-PPY in the conjugate bulk.
Table 1
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Men A-TT
Purified
Conjugate
1.1 2.23 4.10 2.27
II) Preparation of MenY-CRM197 Conjugate (CPPT
Conjugation chemistry)
16
Polysaccharide preparation:
Men Y PS sizing was carried out by chemical means . The
chemically sized PS was targeted to achieve size of 75-
100kDa on SEC-HPLC and then the polysaccharide was
diafiltered on 10kD cuttoff membrane.
Carrier Protein (CRM197) Preparation:
The carrier Protein derivatization process was same as
described above for MenC-CRM conjugate.
Conjugation of Men Y to CRM197:
MenY PS was mixed with CPPT (113 mg/ml in acetonitrile) ,
pH adjusted to 9.5 immediately after addition and then
with ADH-derivatized CRM197 in the ratio of 1:1.5:1.25 by
weight under stirring condition. The reaction was
monitored on HPLC. The reaction was quenched by addition
of excess glycine solution.
Conjugate Purification:
The conjugate was purified on 300kD cut off membrane by
passing 30 volumes of 100mM PBS and followed by 10
volume10mM Tris pH 7.2 and then 20 volumes of 10mM Tris
on 100kD cut off membrane. The sucrose-citrate stabilizer
mixture was added to the bulk conjugate and stored below
-200C until use.
The purified bulk conjugate was analyzed for total and
unbound (free) PS, Total and unbound(free) protein and
residual 4-PPY in the conjugate bulk.
Table 2
17
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Men Y-CRM197
Purified
Conjugate
1.46 3.59 <4.43 <4.09
III) Preparation of Pneumococcal conjugate 23F-CRM197-ADH
Conjugate (CPPT Conjugation chemistry)
Polysaccharide preparation:
In case of Pneumococcal 23F Ps, sizing was carried out by
mechanical means and sizing was continued until the PS
size was reached to 150-170 kD on SEC-HPLC and then the
polysaccharide was diafiltered-concentrated on 10kD
cuttoff membrane.
Carrier Protein (CRM197) Preparation:
In order to conjugate Pneumococcal 23F Ps to CRM197, the
carrier protein was derivatized by ADH linker prior to
conjugation.
The native CRM197 was allowed to react with ADH linker in
presence of EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)
carbodiimide) in MES buffer. The reaction was then
quenched by increasing pH by sodium carbonate solution
under stirring for 15min. The ADH-derivatized CRM197 was
then diafiltered on 10kD cuttoff membrane using 10mM
carbonate buffer pH 9.5 by passing about 35 volumes of
buffer. The sample was submitted to content analysis and
extent of derivatization.
18
Conjugation of Pneumococcal 23F-CRM197:
Pneumococcal 23F PS was mixed with CPPT( in acetonitrile)
and then ADH-derivatized CRM197 in the ratio of
1:1.25:1.25 by weight under stirring condition. The
reaction was monitored on HPLC. The reaction was quenched
after 3 hours by addition of excess glycine solution.
Conjugate Purification:
The crude conjugate was purified on 300kD cut off
membrane by passing 30 volumes of 10mM PBS and followed
by 20 volume10mM Tris pH 7.2. The purified bulk conjugate
was analyzed for total and unbound (free) PS, total and
unbound(free) protein and residual 4-PPY in the conjugate
bulk.
Table 3
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Pneumococcal
23F-CRM197
Conjugate
0.87 1.58 1.67 2.11
IV) Preparation of Pneumococcal (9V- CRM
197),Meningococcal(Men A-TT Conjugate ,Men Y-CRM197
Conjugate) & Hib-TT conjugates by CDAP conjugation
chemistry
Polysaccharides were conjugated to carrier by using CDAP
conjugation method disclosed in Lees et al (Vaccine 26:
190-198, 1996)
19
Table 4: Estimation of 4-PPY in polysaccharide protein
conjugate vaccines
Table 5: Estimation of DMAP in polysaccharide protein
conjugate vaccines
Spi
ke
Rec
overy (DMAP & 4PPY) for all above samples was found to be
between 80-120%. Further MEKC-CE method was found to be
sensitive to capillary temperature variation.Temperature
of about 20oC was found to maintain the viscosity of the
buffer thereby ensuring optimal electrophoretic and EOF
velocities ultimately resulting into optimal migration
time.
Samples 4-PPY (ng/μg of Ps)
Men ATT
Conjugate
Below Detection limit
Men Y-CRM197 Conjugate 0.58
Hib-
CRM197 Conjugate
1.01
PN23FCRM
197 Conjugate
1.61
Samples DMAP (ng/μg of Ps)
Men ATT
Conjugate
Below Detection
limit
Men Y-CRM197 Conjugate 0.71
Hib-
TT Conjugate
0.59
Pneumococcal 9VCRM
197 Conjugate
1.01
20
Further said MEKC method for assaying 4-PPY & DMAP in
samples elucidates substantial improvement in
reproducibility in quantitative analysis, including
migration time and peak height or peak area.MEKC-CE
method provides also a number of advantages over other
analytical techniques: the rapid development of the
analysis method, its analysis speed, reduced consumable
and solvent expenses, low sample amount, simplicity of
operations, and a greater possibility of implementation
of a single set of method conditions for the analysis of
several different samples.
It is to be understood that typical methods of Micellar
electrokinetic capillary chromatography(MEKC)technology
have been applied, and that a person skilled in the art
of MEKC is well aware of minor adjustments that would not
alter the results to be obtained, such as different
measurements at other suitable wavelengths or by the use
of other column material that would not severely alter
the results obtained by the invention.In view of the many
possible embodiments to which the principles of the
disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only
preferred examples of the invention and should not be
taken as limiting the scope of the invention.
21
We Claim:
1. An improved method for assaying a sample suspected
to contain cyanylation conjugation reaction byproducts
of 1-Cyano-4- pyrrolidinopyridinium
tetrafluorborate (CPPT)or 1 -cyano-4-
dimethylaminopyridine tetrafluoroborate
(CDAP),characterized in that the quantification is
performed by a micellar electrokinetic capillary
chromatography method .
2. A method according to claim 1, wherein the
cyanylation conjugation reaction by-products are 4-
pyrrolidinopyridine(4-PPY) for CPPT and 4-
dimethylaminopyridine(DMAP) for CDAP.
3. A method according to claim 1, wherein the method
provides improved peak resolution characterized in
that separate peaks for cyanylation conjugation
reaction by-products and polysaccharide-protein
conjugate can be obtained in a given sample.
4. A method according to claim 1, wherein the MEKC
based method can comprise of following steps:
a) Preparing working standard 100 μg/mL (4PPY or DMAP);
b) Arranging set standards from 31.25 ng/mL to 500
ng/mL;
c) Diluting sample 0.1 to 1mg/mL on basis of
Polysaccharide concentration;
d) Preparing spike of 125ng/mL each for 4PPY and DMAP;
e) Transferring sample and standards in PCR vial;
f) Washing capillary using instrument wash programme ;
g) Running separation method at 17kv voltage for 45 min
using MEKC buffer (280nm);and
22
h) Checking for correlation coefficient and % spike
recovery.
5. A method according to claim 4, wherein the sample
dilution buffer for DMAP is 0.9% NaCl and for 4PPY
is 10mM Tris, pH 7.2.
6. A method according to claim 4, wherein sample
concentration can be between 0.1 to 1 mg/ ml of Ps
of conjugate.
7. A method according to claim 4,wherein sample
injection is carried out under pressure mode at
0.5psi for about 27 sec.
8. A method according to claim 4, wherein the running
solution can comprise of i) a surfactant selected
from a group of sodium dodecyl sulfate
(SDS),cetyltrimethylammonium bromide (CTAB) and
sodium tetradecyl sulfate (STS) and ii)a buffer
selected from a group of Borate ,phosphate ,Good’s
and tris (hydroxymethyl) aminomethane (Tris)buffer.
9. A method according to claim 8, wherein the running
solution comprises of i) SDS having concentration
between 80 mM to about 120 mM and ii) borate having
concentration between 10mM to about 50 mM at pH 9.3.
10. A method according to claim 4, wherein said aqueous
phase can be modified for manipulating selectivity
by using additives selected from a group of
cyclodextrins (CDs), ion-pair reagents, urea,
organic solvents and metal salts.
23
11. A method according to claim 4,wherein said micellar
phase can be modified by adding a second surfactant
to form a mixed micelle or by selecting a different
counter ion.
12.A method according to claim 3, wherein said micellar
electrokinetic capillary chromatography can employ
organic modifiers that reduce electro-osmotic flow
and thereby increase peak capacity of the separation
that can be selected from a group of methanol,
ethanol, propan-2-ol,butan-1-ol, butan-2-ol,
acetone, methyl ethyl ketone and acetonitrile.
13. A method according to claim 4, wherein capillary is
a 75 μm I.D.having total length of about 70 cm.
14. A method according to claim 4, wherein applied
voltage is 17kV (current 130μA).
15.A method according to claim 4, wherein capillary
cartridge temperature is between 15°C and about
20°C.
16. A method according to claim 4, wherein detection
methods can be selected from ultraviolet and visible
light absorption, electrochemical detection,
fluorescence and mass spectroscopy, characterized in
that the detection is carried at a wavelength of
280nm.
17.The method of any preceding claim wherein the
capsular polysaccharide is a polysaccharide selected
from a Meningococcal polysaccharide, a Pneumococcal
polysaccharide, a Haemophilus influenzae type B
24
polysaccharide, a Vi polysaccharide of Salmonella
typhi, or a group B Streptococcus polysaccharide.
18.The method of any preceding claim wherein the
polysaccharide can be selected from Meningococcal
group A polysaccharide, a Meningococcal group C
polysaccharide, a Meningococcal group W-135
polysaccharide, a Meningococcal group Y and a
Meningococcal group X.
19. The method of any preceding claim wherein the
carrier protein is a protein selected from the group
consisting of tetanus toxoid (TT), fragment C of TT,
diphtheria toxoid, CRM197, Pneumolysin, protein D,
PhtD, PhtDE,OMPC and N19.
20. The method of any preceding claim,wherein the sample
being assayed for 4-PPY comprises of atleast one
conjugate selected from (a)a conjugate of (i) the
capsular saccharide of serogroup A N meningitidis
and (ii)tetanus toxoid; (b) a conjugate of (i)
capsular saccharide of serogroup C N meningitidis
and (ii)CRM197; (c) a conjugate of (i) capsular
saccharide of serogroup Y N. meningitidis and (ii)
CRM197;(d) a conjugate of (i) capsular saccharide of
serogroup W135 N.meningitidis and (ii)CRM197; and
(d) a conjugate of (i) capsular saccharide of
serogroup X N meningitidis and (ii)tetanus toxoid.
21. The method of any preceding claim wherein the sample
being assayed for DMAP comprises of atleast one
polysaccharide-protein conjugate with polysaccharide
selected from group of Streptococcus pneumoniae type
1, 2, 3, 4, 5, 6A, 6B,7F,8, 9A,9V,9N,10A, 11A
25
,12F,14,15B, 17F, 18C, 19A , 19F, 20, 22F,23F ,33F
and 45.
Dated this 20th day of May 2014
Of Anand and Anand Advocates
Agent for the Applicant
26
ABSTRACT
MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY BASED
ASSAY FOR QUANTIFYING RESIDUAL 4- PYRROLIDINOPYRIDINE (4-
PPY) AND 4- DIMETHYLAMINOPYRIDINE (DMAP)IN POLYSACCHARIDE
- PROTEIN CONJUGATE VACCINES
The present invention relates to a rapid and reproducible
micellar electrokinetic capillary chromatography based
method for quantifying residual 4-pyrrolidinopyridine(4-
PPY)and4-dimethylaminopyridine(DMAP) in polysaccharideprotein
conjugate vaccines prepared by cyanylation
conjugation chemistry using 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate (CPPT) and 1 -
cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP)
respectively.Said method provides separate peaks for
residuals(4PPY/DMAP) and polysaccharide-protein conjugate
in a given sample,does not require any tedious sample
preparation ,is applicable for quantification of 4-PPY
and DMAP in any polysaccharide-protein conjugate bulk or
vaccine formulation samples and is highly
reproducible.Further good precision and linearity of
instant method can be attributed to under-pressure mode
of injection, injection volume,injection duration,
concentration of surfactant and concentration of buffer.

FORM-2
THE PATENT ACT,1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10;rule 13)
" MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY BASED ASSAY FOR QUANTIFYING RESIDUAL 4-
PYRROLIDINOPYRIDINE (4-PPY) AND 4- DIMETHYLAMINOPYRIDINE (DMAP)IN POLYSACCHARIDE - PROTEIN
CONJUGATE VACCINES "
SERUM INSTITUTE OF INDIA LTD., an Indian Institute, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028,
Maharashtra, India.
The following specification particularly describes the invention and the manner in which it is to be performed:
2
Brief Description:
Vaccines that contain protein covalently linked to
carbohydrate have proven remarkably successful in
inducing an immune response to the carbohydrate moiety.
Examples of such vaccines, known as "conjugates" are
available for Haemophilus influenzae type b (e.g.,
ActHib, Hiberix), Neisseria meningitidis types A C W and
Y (e.g., Menactra) and S. pneumoniae (e.g., Prevnar,
Synflorix). In order for the protein to be linked to the
carbohydrate, the latter generally needs to be activated
so that it can be reacted with the protein, either
directly or via a spacer (Dick, W.E. Jr and Beurret, M.
Glyco-conjugates of bacterial carbohydrate antigens. A
survey and consideration of design and preparation
factors. In: Conjugate Vaccines (Eds Cruse, J.M. and
Lewis, R.E.).
It has been reported previously that the polysaccharideprotein
conjugate purification procedures should remove
residual reagents used for conjugation and the removal of
reagents and reaction by-products such as cyanide, EDAC
and others depending on the conjugation chemistry, should
be confirmed by suitable tests or by validation of the
purification process wherein the acceptable limits should
be approved by the NRA. Refer Section A.3.3.1
WHO/BS/06.2041 2006 “Recommendations to assure the
quality, safety and efficacy of group A meningococcal
conjugate vaccines”
Regulatory authorities do consider potential toxicity of
the identified exogenous process related impurities and
accordingly require that the same should be considerably
lower than the 1.5 μg/dose .Refer EMEA Guideline(EMEA
3
/CHMP /QWP/251344/2006) on ‘The Limits of Genotoxic
Impurities’.
Improved methods for cyanating polysaccharides use 1 -
cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP)
(Lees, A., Producing immunogenic constructs using soluble
carbohydrates activated via organic cyanylating reagents.
U.S. Patent Nos. 5,651,971; 5,693,326; and 5,849,301).
GSK’s Synflorix is an approved 10-valent pneumococcal
polysaccharide-protein conjugate vaccine prepared by
using CDAP based cyanylation conjugation
chemistry.Further GSK’s Nimenrix is a pipeline
multivalent meningococcal polysaccharide-protein
conjugate vaccine prepared by using CDAP based
cyanylation conjugation chemistry. The activation of the
polysaccharide with CDAP introduces a cyanate group in
the polysaccharide and DMAP(4-dimethylamino-pyridine) is
liberated. It is known Synflorix contains residual DMAP
at a level of 65ng per dose.Residual DMAP results in
degradation of polysaccharide .Further DMAP has a rat
LD50 (oral)of 140 mg/kg .
To address above concerns of DMAP and subsequent queries
raised by regulatory agencies , GSK has previously
disclosed in-house assays to quantify residual DMAP
content in CDAP conjugation chemistry based
polysaccharide-protein conjugate vaccines (Synflorix,
Nimenrix)as well as utilized diafiltration for removal of
DMAP.Refer WO00/56359 and WO2007000342.
An isocratic reverse phase HPLC method for the analysis
of 4-Dimethylaminopyridine (DMAP)has been reported
earlier.Refer application notes on DMAP- From MicrSolv.
4
Polysaccharide- protein conjugate vaccines prepared
using 1-Cyano-4- pyrrolidinopyridinium tetrafluorborate
(CPPT)based cyanylation conjugation chemistry are still
in development phase and yet to be approved by regulatory
agencies.During conjugation process 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate gets hydrolysed to
form a reaction by-product“4-Pyrrolidinopyridine (4-
PPY)”.
Pipeline Multivalent conjugate vaccines(5-valent)may
contain residual 4-PPY at a level of 25-30ng per
dose.Further 4-PPY has a rat LD50 (oral) of 176 mg/kg.
To address above concerns of 4-PPY and queries that could
be raised by regulatory agencies , there is an urgent
need to develop an assay to quantify residual 4-PPY
content in CPPT conjugation chemistry based
polysaccharide-protein conjugate vaccines.Particularly,
quantification of 4-PPY would be necessary for pipeline
multivalent meningococcal polysaccharide protein
conjugate vaccine compositions comprising capsular
saccharide from serogroups X and capsular saccharide from
A, C, W135 and Y prepared by using 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate based cyanylation
conjugation chemistry.Refer WO2013/114268.
Capillary electrophoresis (CE) combines the
quantification and handling benefits of HPLC with the
separating power of conventional electrophoretic
techniques. As compared with HPLC, CE offers an important
advantage, namely a wide variation of analytical
conditions.
Free solution capillary electrophoresis, also called
capillary zone electrophoresis (CZE), is the most common
5
mode of capillary electroseparation, because it is
performed in a capillary filled only with an electrolyte
solution at a selected pH value and ionic composition.
CZE is applicable to a wide range of charged substances,
including pharmaceuticals, amino acids, peptides, and
proteins. The separation in CZE is based on the
differences in the electrophoretic mobilities, resulting
in different velocities of migration of ionic species in
the electrophoretic buffer contained in the capillary.
Neutral analytes can also be separated by a variation of
CZE termed micellar electrokinetic capillary
chromatography(MEKC). In this technique a surfactant,
usually sodium dodecyl sulfate, is added to the solution
of the neutral analytes. As the sodium dodecyl sulfate is
negatively charged, it moves in the opposite direction to
the electro-osmotic flow. Neutral analytes are swept
along with the electro-osmotic flow and are separated
based on their partitioning between the surfactant and
the surrounding aqueous phase. This technique was
introduced by Terabe (Anal. Chem. vol.57, pp. 834-841
(1985) and vol. 56, pp. 111-113, (1984))and have been
extended by Sepaniak, Fujiwara, Honda, and others to
pharmaceutical preparations Refer William E. Werner et al
“Size-Dependent Separation of Proteins Denatured in SDS
by Capillary Electrophoresis Using a Replaceable Sieving
Matrix” Analytical Biochemistry 212 (1993) 253-258.
Lamb DH et al discloses determination of free carrier
protein in protein-polysaccharide conjugate vaccines by
micellar electrokinetic chromatography.Refer J Chromatogr
A. 2000 Oct 13; 894(1-2):311-8.However it does not
disclose methods to quantify residuals(4PPY,DMAP)in
polysaccharide-protein conjugate vaccines prepared by
6
cyanylation conjugation chemistry.
Existing methods for quantification of residuals(4PPY and
DMAP) are not suitable for in-process analysis and suffer
from following setbacks i)do not provide separate peaks
for residuals(4PPY/DMAP) and polysaccharide-protein
conjugate in a given sample ii)require complex sample
preparation iii)long analysis time iv) additional time
for column stabilization and v) are imprecise due to
possibility of interference of sample matrix and
appearance of ghost peaks.
Present invention eliminates the foregoing shortcomings
of the prior art methods and thereby provides a rapid and
reproducible micellar electrokinetic capillary
chromatography based method for quantifying residual 4-
pyrrolidinopyridine(4-PPY) and 4-dimethylaminopyridine(
DMAP)in polysaccharide- protein conjugate vaccines
prepared by cyanylation conjugation chemistry using CPPT
and CDAP respectively.
Summary of Invention:
The present invention arises from the surprising finding
that it is possible to quantify 4-PPY and DMAP in
polysaccharide- protein conjugate vaccine formulations
and polysaccharide-protein conjugate bulk by utilizing a
novel micellar electrokinetic capillary chromatography
based assay.
Instant micellar electrokinetic capillary chromatography
based assay for 4PPY or DMAP quantification in
polysaccharide-protein conjugate samples has following
characteristics:
7
1.Said method is applicable for quantification of 4-PPY
or DMAP in any polysaccharide- protein conjugates(having
capsular polysaccharides from Streptococcus Pneumoniae,
Neisseria meningitidis , Haemophilus influenzae)prepared
by using CPPT or CDAP conjugation chemistry respectively.
2.It can be utilized for 4PPY or DMAP quantification for
in-process(bulk polysaccharide-protein conjugate) as well
as final polysaccharide-protein conjugate vaccine
formulation.
3.Provides separate peaks for residuals(4PPY/DMAP) and
polysaccharide-protein conjugate in a given sample
4.It can quantify 4-PPY and DMAP in any given sample
wherein 0.03125 μg/ml is the lowest detectable limit
observed.
5.It utilizes an under-vacuum/pressure mode of injection
at pressure of about 0.3psi to 0.5psi for a duration
between 20 and 25 seconds ,capillary temperature between
20 and 25oC, SDS as surfactant at a concentration between
80mM and 120mM,Borate buffer with pH between 9 to 9.5
having a concentration between 10mM and 50 mM.
6. Using 0.9% NaCl as sample dilution buffer for DMAP and
10mM Tris having pH 7.2 as sample dilution buffer for
4PPY instead of WFI to maintain stability of conjugate in
sample.
7.It is a rapid method with a total duration of 45
minutes.
8
8.It does not require any tedious sample preparation.
9.None of the sample matrix interferes with the 4-PPY
peak.
10.It shows good precision,linearity and better
reproducibility in migration times and peak areas.
Description of the figures
Figure 1:Spectral scan of 4PPY (10μg/ml) showed max.
Absorbance at 280nm.
Figure 2:4PPY standard 500ng/mL observed at migration
time 37.896 using CE-MEKC method.
Figure 3:MenA -TT Conjugate was found to be more
purified with Respect to 4PPY
Figure 4:The chromatogram showed different peaks for MenY
-CRM Conjugate and 4PPY (Migration Time 38.112)
Figure 5The chromatogram showed different peaks for
PNU23F-CRM Conjugate and 4PPY (Migration Time 37.496)
Figure 6:The chromatogram showed different peaks for HIB
-CRM Conjugate and 4PPY (Migration Time 37.104)
Figure 7:The chromatogram showed different peaks for
Multivalent Conjugates and 4PPY (Migration Time 38.938)
.
9
Figure 8:Spectral scan of DMAP (10μg/ml) showed max
Absorbance at 280nm
Figure 9:DMAP standard 500ng/mL observed at migration
time 38.133 using CE-MEKC method
Figure 10:The chromatogram showed different Migration
Time PNU 9V -CRM Conjugate and DMAP (Migration Time
37.833)
Figure 11:The chromatogram showed different Migration
Time for HIB -TT Conjugate and DMAP (Migration Time
39.030)
Figure 12:The chromatogram showed different Migration
Time for MenY Conjugate and DMAP (Migration Time
38.867)
Figure 13: In MenA -TT Conjugate, DMAP was found to be
Less than detection limit
Detailed Description
An important aspect of the instant invention is that said
4-PPY and DMAP quantification assay comprises of
following steps:
1. Preparation of working standard 100 μg/mL
(4PPY/DMAP).
2. Arranging set standards from 31.25 ng/mL to 500
ng/mL.
3. Diluting sample 0.1 to 1mg/mL on basis of
Polysaccharide concentration.
4. Preparing spike of 125ng/mL each 4PPY & DMAP.
10
5. Transferring sample and standards in PCR vial.
6. Washing capillary using instrument wash programme.
7. Running separation method at 17kv voltage for 45 min
using MEKC buffer (280nm).
8. Checking for correlation coefficient and % spike
recovery
A second aspect of the instant invention is that said
running solution can comprise of a surfactant selected
from but not limited to sodium dodecyl sulfate
(SDS),cetyltrimethylammonium bromide (CTAB) and sodium
tetradecyl sulfate (STS).
A third aspect of the instant invention is that said
micellar phase can be modified by adding a second
surfactant to form a mixed micelle or by selecting a
different counter ion.
A fourth aspect of the instant invention is that said
running solution can comprise of a buffer selected from
but not limited to Borate , Phosphate,Good’s and Tris
buffer.
Preferably the running solution comprises of 80mM to
about 120 mM of SDS, and 10mM to about 50 mM borate at pH
9.3.
Further the separation voltage can be between 15kV and
20kV.
Yet another aspect of the instant invention is that
aqueous phase can be modified for manipulating
selectivity by using additives selected from a group of
but not limited to cyclodextrins (CDs), ion-pair
11
reagents, urea, organic solvents and metal salts.
Further as per the instant invention said micellar
electrokinetic capillary chromatography can employ
organic modifiers that reduce electro-osmotic flow and
thereby increase peak capacity of the separation that can
be selected from but not limited to methanol, ethanol,
propan-2-ol,butan-1-ol, butan-2-ol, acetone, methyl ethyl
ketone and acetonitrile.
The cyanylation conjugation reaction by-products or
residuals (4-PPY, DMAP) in a sample present at a
concentration from about 0.03125 μg/ml to about 1μg/ml
can be detected by instant micellar electrokinetic
capillary chromatography based assay .
According to a preferred embodiment of instant invention
said micellar electrokinetic capillary chromatography
based assay for 4PPY or DMAP quantification in
polysaccharide-protein conjugate samples can employ
following operating conditions:
1. Sample dilution buffer: For DMAP - 0.9% NaCl and for
4PPY -10mM Tris, pH 7.2
2. Running solution:SDS - 100mM in Borate buffer -
25mM, pH 9.2
3. Capillary: 75 μm I.D. Total length 70 cm
4. Applied voltage:17kV (current 130μA)
5. Capillary Cartridge Temperature : 20°C
6. Sample solvent: water
7. Sample concentration: 0.1 to 1 mg/ ml of Ps of
conjugate .
8. Injection end:Positive end
9. Injection volume/duration: 0.5psi for 27
sec(Injection under vacuum mode/pressure mode)
12
10. Detection: 280 nm (PDA)
Detection methods which may be readily apparent include
ultraviolet and visible light absorption, electrochemical
detection, fluorescence and mass spectroscopy.
Another aspect of the invention is that above method for
4PPY and DMAP quantification can be applicable to all
polysaccharide-protein conjugate vaccines wherein said
polysaccharide is a bacterial capsular polysaccharide
selected from the group consisting of Neisseria
meningitidis groups A ,C , W135 Y and X, Haemophilus
influenzae,Group A Streptococcus, Group B Streptococcus,
Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6A, 6B,
7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,
19F, 20, 22F, 23F, 33F,Escherichia coli, Francisella
tularensis, Klebsiella, Moraxella catarrhalis,
Porphyromonas gingivalis, Pseudomonas aeruginosa,
Burkholderia cepacia, Salmonella typhi, Salmonella
typhimurium, Salmonella paratyphi, Shigella dysenteriae,
Shigella flexneri, Shigella sonnei, Vibrio
cholera,Enterococcus faecalis, Enterococcus
faecium,Mycobacterium tuberculosis, Staphylococcus aureus
and Staphylococcus epidermidis.
The carrier protein of said polysaccharide-protein
conjugates can be selected from a group of but not
limited to CRM 197,diphtheria toxoid,tetanus toxoid,
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 adhesin protein (PsaA),pneumococcal PhtD,
13
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),particularly CRM197.
Preferably the above method for 4PPY quantification can
be applicable to a multivalent meningococcal
polysaccharide-protein conjugate composition that can
comprise of (a)a conjugate of (i) the capsular
saccharide of serogroup A N meningitidis and (ii)tetanus
toxoid; (b) a conjugate of (i) capsular saccharide of
serogroup C N meningitidis and (ii)CRM197; (c) a
conjugate of (i) capsular saccharide of serogroup Y N.
meningitidis and (ii) CRM197;(d) a conjugate of (i)
capsular saccharide of serogroup W135 N.meningitidis and
(ii)CRM197; and (d) a conjugate of (i) capsular
saccharide of serogroup X N meningitidis and (ii)tetanus
toxoid.
One important aspect of the instant invention is that
above method for DMAP quantification can be applicable to
an immunogenic composition comprising a multivalent
polysaccharide-protein conjugate mixture consisting of
capsular polysaccharides from 12 F and 15B and atleast
one additional polysaccharide selected from 1, 2, 3, 4,
5, 6A, 6B,7F,9A,9V,9N,10A, 11A, 14,17F, 18C, 19A , 19F,
20, 22F,23F ,33F and 45.
Further the above method for DMAP quantification can be
applicable to a multivalent pneumococcal polysaccharideprotein
conjugate composition that can comprise of
14
capsular polysaccharides from serogroups 1 , 4, 5, 6A,
6B, 9V, 14,19A , 19F & 23F conjugated individually to
CRM197.
Examples
I) Preparation of MenA-TT Conjugate (CPPT Conjugation
chemistry)
Polysaccharide preparation:
MenA-TT conjugate was prepared by cyanylation and
carbodiimide chemistry. The native polysaccharide was
sized reduced by mechanical means. The sized reduced PS
(100-120kD on SEC-HPLC) was diafiltered & concentrated on
10kD cuttoff membrane. To 6mg/ml PS, freshly prepared
solution of CPPT(dissolved 114 mg/ml in acetonitrile)
added the pH was shifted to 9.5 with NaOH solution and
hold for upto 5 min. Adipic acid dihydrazide (ADH)
dissolved 100mg/ml in water was added to the PS solution.
The reaction was stirred for 1 hrs at 250C. The reaction
was quenched by addition of excess glycine solution. The
reaction mixture was diafiltered on 10kD TFF in MES
buffer to remove residuals and unreacted components. The
sample was stored at 2-80C until use. The PS content was
measured by phosphorous assay and extent of
derivatization was measured by TNBS assay.
Carrier Protein (TT) Preparation:
Tetanus toxoid was chromatographically purified to remove
aggregates that might cause adverse effect in the
conjugation process. Total TT load was 5-10% v/v of
packed column (toyopearl HW-65F, Tosoh biosciences) with
30cm/hr linear flow rate. The fractions were collected on
the basis of chromatogram obtained on UV detector of the
15
chromatography system. The fractions were pooled on the
basis of aggregate removal seen on SEC-HPLC. The
collected fractions were subjected to concentrationdiafiltration
on 30kD TFF in MES buffer. The protein
content was measured by Lowry assay.
Conjugation of Men A to TT:
The ADH-derivatized PS, purified TT and EDAC (1-ethyl-3-
(3-dimethyl-aminopropyl) carbodiimide) were mixed the
ratio of 1:0.8:1.5 by weight under stirring condition.
The reaction was monitored on HPLC for protein
conversion. The reaction was quenched after 3 hours by
raising pH with 10mM PB+EDTA pH 8.0 buffer.
Conjugate Purification:
The conjugate was purified on 300kD cut off membrane by
passing 30 volumes of 100mM PBS and followed by 30
volume10mM Tris pH 7.2. The sucrose-citrate stabilizer
was added to bulk conjugate and stored below -200C until
use. The purified bulk conjugate was analyzed for total
and unbound (free) PS, Total and unbound(free) protein
and residual 4-PPY in the conjugate bulk.
Table 1
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Men A-TT
Purified
Conjugate
1.1 2.23 4.10 2.27
II) Preparation of MenY-CRM197 Conjugate (CPPT
Conjugation chemistry)
16
Polysaccharide preparation:
Men Y PS sizing was carried out by chemical means . The
chemically sized PS was targeted to achieve size of 75-
100kDa on SEC-HPLC and then the polysaccharide was
diafiltered on 10kD cuttoff membrane.
Carrier Protein (CRM197) Preparation:
The carrier Protein derivatization process was same as
described above for MenC-CRM conjugate.
Conjugation of Men Y to CRM197:
MenY PS was mixed with CPPT (113 mg/ml in acetonitrile) ,
pH adjusted to 9.5 immediately after addition and then
with ADH-derivatized CRM197 in the ratio of 1:1.5:1.25 by
weight under stirring condition. The reaction was
monitored on HPLC. The reaction was quenched by addition
of excess glycine solution.
Conjugate Purification:
The conjugate was purified on 300kD cut off membrane by
passing 30 volumes of 100mM PBS and followed by 10
volume10mM Tris pH 7.2 and then 20 volumes of 10mM Tris
on 100kD cut off membrane. The sucrose-citrate stabilizer
mixture was added to the bulk conjugate and stored below
-200C until use.
The purified bulk conjugate was analyzed for total and
unbound (free) PS, Total and unbound(free) protein and
residual 4-PPY in the conjugate bulk.
Table 2
17
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Men Y-CRM197
Purified
Conjugate
1.46 3.59 <4.43 <4.09
III) Preparation of Pneumococcal conjugate 23F-CRM197-ADH
Conjugate (CPPT Conjugation chemistry)
Polysaccharide preparation:
In case of Pneumococcal 23F Ps, sizing was carried out by
mechanical means and sizing was continued until the PS
size was reached to 150-170 kD on SEC-HPLC and then the
polysaccharide was diafiltered-concentrated on 10kD
cuttoff membrane.
Carrier Protein (CRM197) Preparation:
In order to conjugate Pneumococcal 23F Ps to CRM197, the
carrier protein was derivatized by ADH linker prior to
conjugation.
The native CRM197 was allowed to react with ADH linker in
presence of EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)
carbodiimide) in MES buffer. The reaction was then
quenched by increasing pH by sodium carbonate solution
under stirring for 15min. The ADH-derivatized CRM197 was
then diafiltered on 10kD cuttoff membrane using 10mM
carbonate buffer pH 9.5 by passing about 35 volumes of
buffer. The sample was submitted to content analysis and
extent of derivatization.
18
Conjugation of Pneumococcal 23F-CRM197:
Pneumococcal 23F PS was mixed with CPPT( in acetonitrile)
and then ADH-derivatized CRM197 in the ratio of
1:1.25:1.25 by weight under stirring condition. The
reaction was monitored on HPLC. The reaction was quenched
after 3 hours by addition of excess glycine solution.
Conjugate Purification:
The crude conjugate was purified on 300kD cut off
membrane by passing 30 volumes of 10mM PBS and followed
by 20 volume10mM Tris pH 7.2. The purified bulk conjugate
was analyzed for total and unbound (free) PS, total and
unbound(free) protein and residual 4-PPY in the conjugate
bulk.
Table 3
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Pneumococcal
23F-CRM197
Conjugate
0.87 1.58 1.67 2.11
IV) Preparation of Pneumococcal (9V- CRM
197),Meningococcal(Men A-TT Conjugate ,Men Y-CRM197
Conjugate) & Hib-TT conjugates by CDAP conjugation
chemistry
Polysaccharides were conjugated to carrier by using CDAP
conjugation method disclosed in Lees et al (Vaccine 26:
190-198, 1996)
19
Table 4: Estimation of 4-PPY in polysaccharide protein
conjugate vaccines
Table 5: Estimation of DMAP in polysaccharide protein
conjugate vaccines
Spi
ke
Rec
overy (DMAP & 4PPY) for all above samples was found to be
between 80-120%. Further MEKC-CE method was found to be
sensitive to capillary temperature variation.Temperature
of about 20oC was found to maintain the viscosity of the
buffer thereby ensuring optimal electrophoretic and EOF
velocities ultimately resulting into optimal migration
time.
Samples 4-PPY (ng/μg of Ps)
Men ATT
Conjugate
Below Detection limit
Men Y-CRM197 Conjugate 0.58
Hib-
CRM197 Conjugate
1.01
PN23FCRM
197 Conjugate
1.61
Samples DMAP (ng/μg of Ps)
Men ATT
Conjugate
Below Detection
limit
Men Y-CRM197 Conjugate 0.71
Hib-
TT Conjugate
0.59
Pneumococcal 9VCRM
197 Conjugate
1.01
20
Further said MEKC method for assaying 4-PPY & DMAP in
samples elucidates substantial improvement in
reproducibility in quantitative analysis, including
migration time and peak height or peak area.MEKC-CE
method provides also a number of advantages over other
analytical techniques: the rapid development of the
analysis method, its analysis speed, reduced consumable
and solvent expenses, low sample amount, simplicity of
operations, and a greater possibility of implementation
of a single set of method conditions for the analysis of
several different samples.
It is to be understood that typical methods of Micellar
electrokinetic capillary chromatography(MEKC)technology
have been applied, and that a person skilled in the art
of MEKC is well aware of minor adjustments that would not
alter the results to be obtained, such as different
measurements at other suitable wavelengths or by the use
of other column material that would not severely alter
the results obtained by the invention.In view of the many
possible embodiments to which the principles of the
disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only
preferred examples of the invention and should not be
taken as limiting the scope of the invention.
21
We Claim:
1. An improved method for assaying a sample suspected
to contain cyanylation conjugation reaction byproducts
of 1-Cyano-4- pyrrolidinopyridinium
tetrafluorborate (CPPT)or 1 -cyano-4-
dimethylaminopyridine tetrafluoroborate
(CDAP),characterized in that the quantification is
performed by a micellar electrokinetic capillary
chromatography method .
2. A method according to claim 1, wherein the
cyanylation conjugation reaction by-products are 4-
pyrrolidinopyridine(4-PPY) for CPPT and 4-
dimethylaminopyridine(DMAP) for CDAP.
3. A method according to claim 1, wherein the method
provides improved peak resolution characterized in
that separate peaks for cyanylation conjugation
reaction by-products and polysaccharide-protein
conjugate can be obtained in a given sample.
4. A method according to claim 1, wherein the MEKC
based method can comprise of following steps:
a) Preparing working standard 100 μg/mL (4PPY or DMAP);
b) Arranging set standards from 31.25 ng/mL to 500
ng/mL;
c) Diluting sample 0.1 to 1mg/mL on basis of
Polysaccharide concentration;
d) Preparing spike of 125ng/mL each for 4PPY and DMAP;
e) Transferring sample and standards in PCR vial;
f) Washing capillary using instrument wash programme ;
g) Running separation method at 17kv voltage for 45 min
using MEKC buffer (280nm);and
22
h) Checking for correlation coefficient and % spike
recovery.
5. A method according to claim 4, wherein the sample
dilution buffer for DMAP is 0.9% NaCl and for 4PPY
is 10mM Tris, pH 7.2.
6. A method according to claim 4, wherein sample
concentration can be between 0.1 to 1 mg/ ml of Ps
of conjugate.
7. A method according to claim 4,wherein sample
injection is carried out under pressure mode at
0.5psi for about 27 sec.
8. A method according to claim 4, wherein the running
solution can comprise of i) a surfactant selected
from a group of sodium dodecyl sulfate
(SDS),cetyltrimethylammonium bromide (CTAB) and
sodium tetradecyl sulfate (STS) and ii)a buffer
selected from a group of Borate ,phosphate ,Good’s
and tris (hydroxymethyl) aminomethane (Tris)buffer.
9. A method according to claim 8, wherein the running
solution comprises of i) SDS having concentration
between 80 mM to about 120 mM and ii) borate having
concentration between 10mM to about 50 mM at pH 9.3.
10. A method according to claim 4, wherein said aqueous
phase can be modified for manipulating selectivity
by using additives selected from a group of
cyclodextrins (CDs), ion-pair reagents, urea,
organic solvents and metal salts.
23
11. A method according to claim 4,wherein said micellar
phase can be modified by adding a second surfactant
to form a mixed micelle or by selecting a different
counter ion.
12.A method according to claim 3, wherein said micellar
electrokinetic capillary chromatography can employ
organic modifiers that reduce electro-osmotic flow
and thereby increase peak capacity of the separation
that can be selected from a group of methanol,
ethanol, propan-2-ol,butan-1-ol, butan-2-ol,
acetone, methyl ethyl ketone and acetonitrile.
13. A method according to claim 4, wherein capillary is
a 75 μm I.D.having total length of about 70 cm.
14. A method according to claim 4, wherein applied
voltage is 17kV (current 130μA).
15.A method according to claim 4, wherein capillary
cartridge temperature is between 15°C and about
20°C.
16. A method according to claim 4, wherein detection
methods can be selected from ultraviolet and visible
light absorption, electrochemical detection,
fluorescence and mass spectroscopy, characterized in
that the detection is carried at a wavelength of
280nm.
17.The method of any preceding claim wherein the
capsular polysaccharide is a polysaccharide selected
from a Meningococcal polysaccharide, a Pneumococcal
polysaccharide, a Haemophilus influenzae type B
24
polysaccharide, a Vi polysaccharide of Salmonella
typhi, or a group B Streptococcus polysaccharide.
18.The method of any preceding claim wherein the
polysaccharide can be selected from Meningococcal
group A polysaccharide, a Meningococcal group C
polysaccharide, a Meningococcal group W-135
polysaccharide, a Meningococcal group Y and a
Meningococcal group X.
19. The method of any preceding claim wherein the
carrier protein is a protein selected from the group
consisting of tetanus toxoid (TT), fragment C of TT,
diphtheria toxoid, CRM197, Pneumolysin, protein D,
PhtD, PhtDE,OMPC and N19.
20. The method of any preceding claim,wherein the sample
being assayed for 4-PPY comprises of atleast one
conjugate selected from (a)a conjugate of (i) the
capsular saccharide of serogroup A N meningitidis
and (ii)tetanus toxoid; (b) a conjugate of (i)
capsular saccharide of serogroup C N meningitidis
and (ii)CRM197; (c) a conjugate of (i) capsular
saccharide of serogroup Y N. meningitidis and (ii)
CRM197;(d) a conjugate of (i) capsular saccharide of
serogroup W135 N.meningitidis and (ii)CRM197; and
(d) a conjugate of (i) capsular saccharide of
serogroup X N meningitidis and (ii)tetanus toxoid.
21. The method of any preceding claim wherein the sample
being assayed for DMAP comprises of atleast one
polysaccharide-protein conjugate with polysaccharide
selected from group of Streptococcus pneumoniae type
1, 2, 3, 4, 5, 6A, 6B,7F,8, 9A,9V,9N,10A, 11A
25
,12F,14,15B, 17F, 18C, 19A , 19F, 20, 22F,23F ,33F
and 45.
Dated this 20th day of May 2014
Of Anand and Anand Advocates
Agent for the Applicant
26
ABSTRACT
MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY BASED
ASSAY FOR QUANTIFYING RESIDUAL 4- PYRROLIDINOPYRIDINE (4-
PPY) AND 4- DIMETHYLAMINOPYRIDINE (DMAP)IN POLYSACCHARIDE
- PROTEIN CONJUGATE VACCINES
The present invention relates to a rapid and reproducible
micellar electrokinetic capillary chromatography based
method for quantifying residual 4-pyrrolidinopyridine(4-
PPY)and4-dimethylaminopyridine(DMAP) in polysaccharideprotein
conjugate vaccines prepared by cyanylation
conjugation chemistry using 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate (CPPT) and 1 -
cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP)
respectively.Said method provides separate peaks for
residuals(4PPY/DMAP) and polysaccharide-protein conjugate
in a given sample,does not require any tedious sample
preparation ,is applicable for quantification of 4-PPY
and DMAP in any polysaccharide-protein conjugate bulk or
vaccine formulation samples and is highly
reproducible.Further good precision and linearity of
instant method can be attributed to under-pressure mode
of injection, injection volume,injection duration,
concentration of surfactant and concentration of buffer.

FORM-2
THE PATENT ACT,1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10;rule 13)
" MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY BASED ASSAY FOR QUANTIFYING RESIDUAL 4-
PYRROLIDINOPYRIDINE (4-PPY) AND 4- DIMETHYLAMINOPYRIDINE (DMAP)IN POLYSACCHARIDE - PROTEIN
CONJUGATE VACCINES "
SERUM INSTITUTE OF INDIA LTD., an Indian Institute, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028,
Maharashtra, India.
The following specification particularly describes the invention and the manner in which it is to be performed:
2
Brief Description:
Vaccines that contain protein covalently linked to
carbohydrate have proven remarkably successful in
inducing an immune response to the carbohydrate moiety.
Examples of such vaccines, known as "conjugates" are
available for Haemophilus influenzae type b (e.g.,
ActHib, Hiberix), Neisseria meningitidis types A C W and
Y (e.g., Menactra) and S. pneumoniae (e.g., Prevnar,
Synflorix). In order for the protein to be linked to the
carbohydrate, the latter generally needs to be activated
so that it can be reacted with the protein, either
directly or via a spacer (Dick, W.E. Jr and Beurret, M.
Glyco-conjugates of bacterial carbohydrate antigens. A
survey and consideration of design and preparation
factors. In: Conjugate Vaccines (Eds Cruse, J.M. and
Lewis, R.E.).
It has been reported previously that the polysaccharideprotein
conjugate purification procedures should remove
residual reagents used for conjugation and the removal of
reagents and reaction by-products such as cyanide, EDAC
and others depending on the conjugation chemistry, should
be confirmed by suitable tests or by validation of the
purification process wherein the acceptable limits should
be approved by the NRA. Refer Section A.3.3.1
WHO/BS/06.2041 2006 “Recommendations to assure the
quality, safety and efficacy of group A meningococcal
conjugate vaccines”
Regulatory authorities do consider potential toxicity of
the identified exogenous process related impurities and
accordingly require that the same should be considerably
lower than the 1.5 μg/dose .Refer EMEA Guideline(EMEA
3
/CHMP /QWP/251344/2006) on ‘The Limits of Genotoxic
Impurities’.
Improved methods for cyanating polysaccharides use 1 -
cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP)
(Lees, A., Producing immunogenic constructs using soluble
carbohydrates activated via organic cyanylating reagents.
U.S. Patent Nos. 5,651,971; 5,693,326; and 5,849,301).
GSK’s Synflorix is an approved 10-valent pneumococcal
polysaccharide-protein conjugate vaccine prepared by
using CDAP based cyanylation conjugation
chemistry.Further GSK’s Nimenrix is a pipeline
multivalent meningococcal polysaccharide-protein
conjugate vaccine prepared by using CDAP based
cyanylation conjugation chemistry. The activation of the
polysaccharide with CDAP introduces a cyanate group in
the polysaccharide and DMAP(4-dimethylamino-pyridine) is
liberated. It is known Synflorix contains residual DMAP
at a level of 65ng per dose.Residual DMAP results in
degradation of polysaccharide .Further DMAP has a rat
LD50 (oral)of 140 mg/kg .
To address above concerns of DMAP and subsequent queries
raised by regulatory agencies , GSK has previously
disclosed in-house assays to quantify residual DMAP
content in CDAP conjugation chemistry based
polysaccharide-protein conjugate vaccines (Synflorix,
Nimenrix)as well as utilized diafiltration for removal of
DMAP.Refer WO00/56359 and WO2007000342.
An isocratic reverse phase HPLC method for the analysis
of 4-Dimethylaminopyridine (DMAP)has been reported
earlier.Refer application notes on DMAP- From MicrSolv.
4
Polysaccharide- protein conjugate vaccines prepared
using 1-Cyano-4- pyrrolidinopyridinium tetrafluorborate
(CPPT)based cyanylation conjugation chemistry are still
in development phase and yet to be approved by regulatory
agencies.During conjugation process 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate gets hydrolysed to
form a reaction by-product“4-Pyrrolidinopyridine (4-
PPY)”.
Pipeline Multivalent conjugate vaccines(5-valent)may
contain residual 4-PPY at a level of 25-30ng per
dose.Further 4-PPY has a rat LD50 (oral) of 176 mg/kg.
To address above concerns of 4-PPY and queries that could
be raised by regulatory agencies , there is an urgent
need to develop an assay to quantify residual 4-PPY
content in CPPT conjugation chemistry based
polysaccharide-protein conjugate vaccines.Particularly,
quantification of 4-PPY would be necessary for pipeline
multivalent meningococcal polysaccharide protein
conjugate vaccine compositions comprising capsular
saccharide from serogroups X and capsular saccharide from
A, C, W135 and Y prepared by using 1-Cyano-4-
pyrrolidinopyridinium tetrafluorborate based cyanylation
conjugation chemistry.Refer WO2013/114268.
Capillary electrophoresis (CE) combines the
quantification and handling benefits of HPLC with the
separating power of conventional electrophoretic
techniques. As compared with HPLC, CE offers an important
advantage, namely a wide variation of analytical
conditions.
Free solution capillary electrophoresis, also called
capillary zone electrophoresis (CZE), is the most common
5
mode of capillary electroseparation, because it is
performed in a capillary filled only with an electrolyte
solution at a selected pH value and ionic composition.
CZE is applicable to a wide range of charged substances,
including pharmaceuticals, amino acids, peptides, and
proteins. The separation in CZE is based on the
differences in the electrophoretic mobilities, resulting
in different velocities of migration of ionic species in
the electrophoretic buffer contained in the capillary.
Neutral analytes can also be separated by a variation of
CZE termed micellar electrokinetic capillary
chromatography(MEKC). In this technique a surfactant,
usually sodium dodecyl sulfate, is added to the solution
of the neutral analytes. As the sodium dodecyl sulfate is
negatively charged, it moves in the opposite direction to
the electro-osmotic flow. Neutral analytes are swept
along with the electro-osmotic flow and are separated
based on their partitioning between the surfactant and
the surrounding aqueous phase. This technique was
introduced by Terabe (Anal. Chem. vol.57, pp. 834-841
(1985) and vol. 56, pp. 111-113, (1984))and have been
extended by Sepaniak, Fujiwara, Honda, and others to
pharmaceutical preparations Refer William E. Werner et al
“Size-Dependent Separation of Proteins Denatured in SDS
by Capillary Electrophoresis Using a Replaceable Sieving
Matrix” Analytical Biochemistry 212 (1993) 253-258.
Lamb DH et al discloses determination of free carrier
protein in protein-polysaccharide conjugate vaccines by
micellar electrokinetic chromatography.Refer J Chromatogr
A. 2000 Oct 13; 894(1-2):311-8.However it does not
disclose methods to quantify residuals(4PPY,DMAP)in
polysaccharide-protein conjugate vaccines prepared by
6
cyanylation conjugation chemistry.
Existing methods for quantification of residuals(4PPY and
DMAP) are not suitable for in-process analysis and suffer
from following setbacks i)do not provide separate peaks
for residuals(4PPY/DMAP) and polysaccharide-protein
conjugate in a given sample ii)require complex sample
preparation iii)long analysis time iv) additional time
for column stabilization and v) are imprecise due to
possibility of interference of sample matrix and
appearance of ghost peaks.
Present invention eliminates the foregoing shortcomings
of the prior art methods and thereby provides a rapid and
reproducible micellar electrokinetic capillary
chromatography based method for quantifying residual 4-
pyrrolidinopyridine(4-PPY) and 4-dimethylaminopyridine(
DMAP)in polysaccharide- protein conjugate vaccines
prepared by cyanylation conjugation chemistry using CPPT
and CDAP respectively.
Summary of Invention:
The present invention arises from the surprising finding
that it is possible to quantify 4-PPY and DMAP in
polysaccharide- protein conjugate vaccine formulations
and polysaccharide-protein conjugate bulk by utilizing a
novel micellar electrokinetic capillary chromatography
based assay.
Instant micellar electrokinetic capillary chromatography
based assay for 4PPY or DMAP quantification in
polysaccharide-protein conjugate samples has following
characteristics:
7
1.Said method is applicable for quantification of 4-PPY
or DMAP in any polysaccharide- protein conjugates(having
capsular polysaccharides from Streptococcus Pneumoniae,
Neisseria meningitidis , Haemophilus influenzae)prepared
by using CPPT or CDAP conjugation chemistry respectively.
2.It can be utilized for 4PPY or DMAP quantification for
in-process(bulk polysaccharide-protein conjugate) as well
as final polysaccharide-protein conjugate vaccine
formulation.
3.Provides separate peaks for residuals(4PPY/DMAP) and
polysaccharide-protein conjugate in a given sample
4.It can quantify 4-PPY and DMAP in any given sample
wherein 0.03125 μg/ml is the lowest detectable limit
observed.
5.It utilizes an under-vacuum/pressure mode of injection
at pressure of about 0.3psi to 0.5psi for a duration
between 20 and 25 seconds ,capillary temperature between
20 and 25oC, SDS as surfactant at a concentration between
80mM and 120mM,Borate buffer with pH between 9 to 9.5
having a concentration between 10mM and 50 mM.
6. Using 0.9% NaCl as sample dilution buffer for DMAP and
10mM Tris having pH 7.2 as sample dilution buffer for
4PPY instead of WFI to maintain stability of conjugate in
sample.
7.It is a rapid method with a total duration of 45
minutes.
8
8.It does not require any tedious sample preparation.
9.None of the sample matrix interferes with the 4-PPY
peak.
10.It shows good precision,linearity and better
reproducibility in migration times and peak areas.
Description of the figures
Figure 1:Spectral scan of 4PPY (10μg/ml) showed max.
Absorbance at 280nm.
Figure 2:4PPY standard 500ng/mL observed at migration
time 37.896 using CE-MEKC method.
Figure 3:MenA -TT Conjugate was found to be more
purified with Respect to 4PPY
Figure 4:The chromatogram showed different peaks for MenY
-CRM Conjugate and 4PPY (Migration Time 38.112)
Figure 5The chromatogram showed different peaks for
PNU23F-CRM Conjugate and 4PPY (Migration Time 37.496)
Figure 6:The chromatogram showed different peaks for HIB
-CRM Conjugate and 4PPY (Migration Time 37.104)
Figure 7:The chromatogram showed different peaks for
Multivalent Conjugates and 4PPY (Migration Time 38.938)
.
9
Figure 8:Spectral scan of DMAP (10μg/ml) showed max
Absorbance at 280nm
Figure 9:DMAP standard 500ng/mL observed at migration
time 38.133 using CE-MEKC method
Figure 10:The chromatogram showed different Migration
Time PNU 9V -CRM Conjugate and DMAP (Migration Time
37.833)
Figure 11:The chromatogram showed different Migration
Time for HIB -TT Conjugate and DMAP (Migration Time
39.030)
Figure 12:The chromatogram showed different Migration
Time for MenY Conjugate and DMAP (Migration Time
38.867)
Figure 13: In MenA -TT Conjugate, DMAP was found to be
Less than detection limit
Detailed Description
An important aspect of the instant invention is that said
4-PPY and DMAP quantification assay comprises of
following steps:
1. Preparation of working standard 100 μg/mL
(4PPY/DMAP).
2. Arranging set standards from 31.25 ng/mL to 500
ng/mL.
3. Diluting sample 0.1 to 1mg/mL on basis of
Polysaccharide concentration.
4. Preparing spike of 125ng/mL each 4PPY & DMAP.
10
5. Transferring sample and standards in PCR vial.
6. Washing capillary using instrument wash programme.
7. Running separation method at 17kv voltage for 45 min
using MEKC buffer (280nm).
8. Checking for correlation coefficient and % spike
recovery
A second aspect of the instant invention is that said
running solution can comprise of a surfactant selected
from but not limited to sodium dodecyl sulfate
(SDS),cetyltrimethylammonium bromide (CTAB) and sodium
tetradecyl sulfate (STS).
A third aspect of the instant invention is that said
micellar phase can be modified by adding a second
surfactant to form a mixed micelle or by selecting a
different counter ion.
A fourth aspect of the instant invention is that said
running solution can comprise of a buffer selected from
but not limited to Borate , Phosphate,Good’s and Tris
buffer.
Preferably the running solution comprises of 80mM to
about 120 mM of SDS, and 10mM to about 50 mM borate at pH
9.3.
Further the separation voltage can be between 15kV and
20kV.
Yet another aspect of the instant invention is that
aqueous phase can be modified for manipulating
selectivity by using additives selected from a group of
but not limited to cyclodextrins (CDs), ion-pair
11
reagents, urea, organic solvents and metal salts.
Further as per the instant invention said micellar
electrokinetic capillary chromatography can employ
organic modifiers that reduce electro-osmotic flow and
thereby increase peak capacity of the separation that can
be selected from but not limited to methanol, ethanol,
propan-2-ol,butan-1-ol, butan-2-ol, acetone, methyl ethyl
ketone and acetonitrile.
The cyanylation conjugation reaction by-products or
residuals (4-PPY, DMAP) in a sample present at a
concentration from about 0.03125 μg/ml to about 1μg/ml
can be detected by instant micellar electrokinetic
capillary chromatography based assay .
According to a preferred embodiment of instant invention
said micellar electrokinetic capillary chromatography
based assay for 4PPY or DMAP quantification in
polysaccharide-protein conjugate samples can employ
following operating conditions:
1. Sample dilution buffer: For DMAP - 0.9% NaCl and for
4PPY -10mM Tris, pH 7.2
2. Running solution:SDS - 100mM in Borate buffer -
25mM, pH 9.2
3. Capillary: 75 μm I.D. Total length 70 cm
4. Applied voltage:17kV (current 130μA)
5. Capillary Cartridge Temperature : 20°C
6. Sample solvent: water
7. Sample concentration: 0.1 to 1 mg/ ml of Ps of
conjugate .
8. Injection end:Positive end
9. Injection volume/duration: 0.5psi for 27
sec(Injection under vacuum mode/pressure mode)
12
10. Detection: 280 nm (PDA)
Detection methods which may be readily apparent include
ultraviolet and visible light absorption, electrochemical
detection, fluorescence and mass spectroscopy.
Another aspect of the invention is that above method for
4PPY and DMAP quantification can be applicable to all
polysaccharide-protein conjugate vaccines wherein said
polysaccharide is a bacterial capsular polysaccharide
selected from the group consisting of Neisseria
meningitidis groups A ,C , W135 Y and X, Haemophilus
influenzae,Group A Streptococcus, Group B Streptococcus,
Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6A, 6B,
7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,
19F, 20, 22F, 23F, 33F,Escherichia coli, Francisella
tularensis, Klebsiella, Moraxella catarrhalis,
Porphyromonas gingivalis, Pseudomonas aeruginosa,
Burkholderia cepacia, Salmonella typhi, Salmonella
typhimurium, Salmonella paratyphi, Shigella dysenteriae,
Shigella flexneri, Shigella sonnei, Vibrio
cholera,Enterococcus faecalis, Enterococcus
faecium,Mycobacterium tuberculosis, Staphylococcus aureus
and Staphylococcus epidermidis.
The carrier protein of said polysaccharide-protein
conjugates can be selected from a group of but not
limited to CRM 197,diphtheria toxoid,tetanus toxoid,
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 adhesin protein (PsaA),pneumococcal PhtD,
13
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),particularly CRM197.
Preferably the above method for 4PPY quantification can
be applicable to a multivalent meningococcal
polysaccharide-protein conjugate composition that can
comprise of (a)a conjugate of (i) the capsular
saccharide of serogroup A N meningitidis and (ii)tetanus
toxoid; (b) a conjugate of (i) capsular saccharide of
serogroup C N meningitidis and (ii)CRM197; (c) a
conjugate of (i) capsular saccharide of serogroup Y N.
meningitidis and (ii) CRM197;(d) a conjugate of (i)
capsular saccharide of serogroup W135 N.meningitidis and
(ii)CRM197; and (d) a conjugate of (i) capsular
saccharide of serogroup X N meningitidis and (ii)tetanus
toxoid.
One important aspect of the instant invention is that
above method for DMAP quantification can be applicable to
an immunogenic composition comprising a multivalent
polysaccharide-protein conjugate mixture consisting of
capsular polysaccharides from 12 F and 15B and atleast
one additional polysaccharide selected from 1, 2, 3, 4,
5, 6A, 6B,7F,9A,9V,9N,10A, 11A, 14,17F, 18C, 19A , 19F,
20, 22F,23F ,33F and 45.
Further the above method for DMAP quantification can be
applicable to a multivalent pneumococcal polysaccharideprotein
conjugate composition that can comprise of
14
capsular polysaccharides from serogroups 1 , 4, 5, 6A,
6B, 9V, 14,19A , 19F & 23F conjugated individually to
CRM197.
Examples
I) Preparation of MenA-TT Conjugate (CPPT Conjugation
chemistry)
Polysaccharide preparation:
MenA-TT conjugate was prepared by cyanylation and
carbodiimide chemistry. The native polysaccharide was
sized reduced by mechanical means. The sized reduced PS
(100-120kD on SEC-HPLC) was diafiltered & concentrated on
10kD cuttoff membrane. To 6mg/ml PS, freshly prepared
solution of CPPT(dissolved 114 mg/ml in acetonitrile)
added the pH was shifted to 9.5 with NaOH solution and
hold for upto 5 min. Adipic acid dihydrazide (ADH)
dissolved 100mg/ml in water was added to the PS solution.
The reaction was stirred for 1 hrs at 250C. The reaction
was quenched by addition of excess glycine solution. The
reaction mixture was diafiltered on 10kD TFF in MES
buffer to remove residuals and unreacted components. The
sample was stored at 2-80C until use. The PS content was
measured by phosphorous assay and extent of
derivatization was measured by TNBS assay.
Carrier Protein (TT) Preparation:
Tetanus toxoid was chromatographically purified to remove
aggregates that might cause adverse effect in the
conjugation process. Total TT load was 5-10% v/v of
packed column (toyopearl HW-65F, Tosoh biosciences) with
30cm/hr linear flow rate. The fractions were collected on
the basis of chromatogram obtained on UV detector of the
15
chromatography system. The fractions were pooled on the
basis of aggregate removal seen on SEC-HPLC. The
collected fractions were subjected to concentrationdiafiltration
on 30kD TFF in MES buffer. The protein
content was measured by Lowry assay.
Conjugation of Men A to TT:
The ADH-derivatized PS, purified TT and EDAC (1-ethyl-3-
(3-dimethyl-aminopropyl) carbodiimide) were mixed the
ratio of 1:0.8:1.5 by weight under stirring condition.
The reaction was monitored on HPLC for protein
conversion. The reaction was quenched after 3 hours by
raising pH with 10mM PB+EDTA pH 8.0 buffer.
Conjugate Purification:
The conjugate was purified on 300kD cut off membrane by
passing 30 volumes of 100mM PBS and followed by 30
volume10mM Tris pH 7.2. The sucrose-citrate stabilizer
was added to bulk conjugate and stored below -200C until
use. The purified bulk conjugate was analyzed for total
and unbound (free) PS, Total and unbound(free) protein
and residual 4-PPY in the conjugate bulk.
Table 1
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Men A-TT
Purified
Conjugate
1.1 2.23 4.10 2.27
II) Preparation of MenY-CRM197 Conjugate (CPPT
Conjugation chemistry)
16
Polysaccharide preparation:
Men Y PS sizing was carried out by chemical means . The
chemically sized PS was targeted to achieve size of 75-
100kDa on SEC-HPLC and then the polysaccharide was
diafiltered on 10kD cuttoff membrane.
Carrier Protein (CRM197) Preparation:
The carrier Protein derivatization process was same as
described above for MenC-CRM conjugate.
Conjugation of Men Y to CRM197:
MenY PS was mixed with CPPT (113 mg/ml in acetonitrile) ,
pH adjusted to 9.5 immediately after addition and then
with ADH-derivatized CRM197 in the ratio of 1:1.5:1.25 by
weight under stirring condition. The reaction was
monitored on HPLC. The reaction was quenched by addition
of excess glycine solution.
Conjugate Purification:
The conjugate was purified on 300kD cut off membrane by
passing 30 volumes of 100mM PBS and followed by 10
volume10mM Tris pH 7.2 and then 20 volumes of 10mM Tris
on 100kD cut off membrane. The sucrose-citrate stabilizer
mixture was added to the bulk conjugate and stored below
-200C until use.
The purified bulk conjugate was analyzed for total and
unbound (free) PS, Total and unbound(free) protein and
residual 4-PPY in the conjugate bulk.
Table 2
17
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Men Y-CRM197
Purified
Conjugate
1.46 3.59 <4.43 <4.09
III) Preparation of Pneumococcal conjugate 23F-CRM197-ADH
Conjugate (CPPT Conjugation chemistry)
Polysaccharide preparation:
In case of Pneumococcal 23F Ps, sizing was carried out by
mechanical means and sizing was continued until the PS
size was reached to 150-170 kD on SEC-HPLC and then the
polysaccharide was diafiltered-concentrated on 10kD
cuttoff membrane.
Carrier Protein (CRM197) Preparation:
In order to conjugate Pneumococcal 23F Ps to CRM197, the
carrier protein was derivatized by ADH linker prior to
conjugation.
The native CRM197 was allowed to react with ADH linker in
presence of EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)
carbodiimide) in MES buffer. The reaction was then
quenched by increasing pH by sodium carbonate solution
under stirring for 15min. The ADH-derivatized CRM197 was
then diafiltered on 10kD cuttoff membrane using 10mM
carbonate buffer pH 9.5 by passing about 35 volumes of
buffer. The sample was submitted to content analysis and
extent of derivatization.
18
Conjugation of Pneumococcal 23F-CRM197:
Pneumococcal 23F PS was mixed with CPPT( in acetonitrile)
and then ADH-derivatized CRM197 in the ratio of
1:1.25:1.25 by weight under stirring condition. The
reaction was monitored on HPLC. The reaction was quenched
after 3 hours by addition of excess glycine solution.
Conjugate Purification:
The crude conjugate was purified on 300kD cut off
membrane by passing 30 volumes of 10mM PBS and followed
by 20 volume10mM Tris pH 7.2. The purified bulk conjugate
was analyzed for total and unbound (free) PS, total and
unbound(free) protein and residual 4-PPY in the conjugate
bulk.
Table 3
Test Sample
Total PS
mg/ml
Total
Protein
mg/ml
% Free
PS
% Free
Protein
Pneumococcal
23F-CRM197
Conjugate
0.87 1.58 1.67 2.11
IV) Preparation of Pneumococcal (9V- CRM
197),Meningococcal(Men A-TT Conjugate ,Men Y-CRM197
Conjugate) & Hib-TT conjugates by CDAP conjugation
chemistry
Polysaccharides were conjugated to carrier by using CDAP
conjugation method disclosed in Lees et al (Vaccine 26:
190-198, 1996)
19
Table 4: Estimation of 4-PPY in polysaccharide protein
conjugate vaccines
Table 5: Estimation of DMAP in polysaccharide protein
conjugate vaccines
Spi
ke
Rec
overy (DMAP & 4PPY) for all above samples was found to be
between 80-120%. Further MEKC-CE method was found to be
sensitive to capillary temperature variation.Temperature
of about 20oC was found to maintain the viscosity of the
buffer thereby ensuring optimal electrophoretic and EOF
velocities ultimately resulting into optimal migration
time.
Samples 4-PPY (ng/μg of Ps)
Men ATT
Conjugate
Below Detection limit
Men Y-CRM197 Conjugate 0.58
Hib-
CRM197 Conjugate
1.01
PN23FCRM
197 Conjugate
1.61
Samples DMAP (ng/μg of Ps)
Men ATT
Conjugate
Below Detection
limit
Men Y-CRM197 Conjugate 0.71
Hib-
TT Conjugate
0.59
Pneumococcal 9VCRM
197 Conjugate
1.01
20
Further said MEKC method for assaying 4-PPY & DMAP in
samples elucidates substantial improvement in
reproducibility in quantitative analysis, including
migration time and peak height or peak area.MEKC-CE
method provides also a number of advantages over other
analytical techniques: the rapid development of the
analysis method, its analysis speed, reduced consumable
and solvent expenses, low sample amount, simplicity of
operations, and a greater possibility of implementation
of a single set of method conditions for the analysis of
several different samples.
It is to be understood that typical methods of Micellar
electrokinetic capillary chromatography(MEKC)technology
have been applied, and that a person skilled in the art
of MEKC is well aware of minor adjustments that would not
alter the results to be obtained, such as different
measurements at other suitable wavelengths or by the use
of other column material that would not severely alter
the results obtained by the invention.In view of the many
possible embodiments to which the principles of the
disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only
preferred examples of the invention and should not be
taken as limiting the scope of the invention.
21
We Claim:
1. An improved method for assaying a sample suspected
to contain cyanylation conjugation reaction byproducts
of 1-Cyano-4- pyrrolidinopyridinium
tetrafluorborate (CPPT)or 1 -cyano-4-
dimethylaminopyridine tetrafluoroborate
(CDAP),characterized in that the quantification is
performed by a micellar electrokinetic capillary
chromatography method .
2. A method according to claim 1, wherein the
cyanylation conjugation reaction by-products are 4-
pyrrolidinopyridine(4-PPY) for CPPT and 4-
dimethylaminopyridine(DMAP) for CDAP.
3. A method according to claim 1, wherein the method
provides improved peak resolution characterized in
that separate peaks for cyanylation conjugation
reaction by-products and polysaccharide-protein
conjugate can be obtained in a given sample.
4. A method according to claim 1, wherein the MEKC
based method can comprise of following steps:
a) Preparing working standard 100 μg/mL (4PPY or DMAP);
b) Arranging set standards from 31.25 ng/mL to 500
ng/mL;
c) Diluting sample 0.1 to 1mg/mL on basis of
Polysaccharide concentration;
d) Preparing spike of 125ng/mL each for 4PPY and DMAP;
e) Transferring sample and standards in PCR vial;
f) Washing capillary using instrument wash programme ;
g) Running separation method at 17kv voltage for 45 min
using MEKC buffer (280nm);and
22
h) Checking for correlation coefficient and % spike
recovery.
5. A method according to claim 4, wherein the sample
dilution buffer for DMAP is 0.9% NaCl and for 4PPY
is 10mM Tris, pH 7.2.
6. A method according to claim 4, wherein sample
concentration can be between 0.1 to 1 mg/ ml of Ps
of conjugate.
7. A method according to claim 4,wherein sample
injection is carried out under pressure mode at
0.5psi for about 27 sec.
8. A method according to claim 4, wherein the running
solution can comprise of i) a surfactant selected
from a group of sodium dodecyl sulfate
(SDS),cetyltrimethylammonium bromide (CTAB) and
sodium tetradecyl sulfate (STS) and ii)a buffer
selected from a group of Borate ,phosphate ,Good’s
and tris (hydroxymethyl) aminomethane (Tris)buffer.
9. A method according to claim 8, wherein the running
solution comprises of i) SDS having concentration
between 80 mM to about 120 mM and ii) borate having
concentration between 10mM to about 50 mM at pH 9.3.
10. A method according to claim 4, wherein said aqueous
phase can be modified for manipulating selectivity
by using additives selected from a group of
cyclodextrins (CDs), ion-pair reagents, urea,
organic solvents and metal salts.
23
11. A method according to claim 4,wherein said micellar
phase can be modified by adding a second surfactant
to form a mixed micelle or by selecting a different
counter ion.
12.A method according to claim 3, wherein said micellar
electrokinetic capillary chromatography can employ
organic modifiers that reduce electro-osmotic flow
and thereby increase peak capacity of the separation
that can be selected from a group of methanol,
ethanol, propan-2-ol,butan-1-ol, butan-2-ol,
acetone, methyl ethyl ketone and acetonitrile.
13. A method according to claim 4, wherein capillary is
a 75 μm I.D.having total length of about 70 cm.
14. A method according to claim 4, wherein applied
voltage is 17kV (current 130μA).
15.A method according to claim 4, wherein capillary
cartridge temperature is between 15°C and about
20°C.
16. A method according to claim 4, wherein detection
methods can be selected from ultraviolet and visible
light absorption, electrochemical detection,
fluorescence and mass spectroscopy, characterized in
that the detection is carried at a wavelength of
280nm.
17.The method of any preceding claim wherein the
capsular polysaccharide is a polysaccharide selected
from a Meningococcal polysaccharide, a Pneumococcal
polysaccharide, a Haemophilus influenzae type B
24
polysaccharide, a Vi polysaccharide of Salmonella
typhi, or a group B Streptococcus polysaccharide.
18.The method of any preceding claim wherein the
polysaccharide can be selected from Meningococcal
group A polysaccharide, a Meningococcal group C
polysaccharide, a Meningococcal group W-135
polysaccharide, a Meningococcal group Y and a
Meningococcal group X.
19. The method of any preceding claim wherein the
carrier protein is a protein selected from the group
consisting of tetanus toxoid (TT), fragment C of TT,
diphtheria toxoid, CRM197, Pneumolysin, protein D,
PhtD, PhtDE,OMPC and N19.
20. The method of any preceding claim,wherein the sample
being assayed for 4-PPY comprises of atleast one
conjugate selected from (a)a conjugate of (i) the
capsular saccharide of serogroup A N meningitidis
and (ii)tetanus toxoid; (b) a conjugate of (i)
capsular saccharide of serogroup C N meningitidis
and (ii)CRM197; (c) a conjugate of (i) capsular
saccharide of serogroup Y N. meningitidis and (ii)
CRM197;(d) a conjugate of (i) capsular saccharide of
serogroup W135 N.meningitidis and (ii)CRM197; and
(d) a conjugate of (i) capsular saccharide of
serogroup X N meningitidis and (ii)tetanus toxoid.
21. The method of any preceding claim wherein the sample
being assayed for DMAP comprises of atleast one
polysaccharide-protein conjugate with polysaccharide
selected from group of Streptococcus pneumoniae type
1, 2, 3, 4, 5, 6A, 6B,7F,8, 9A,9V,9N,10A, 11A
25
,12F,14,15B, 17F, 18C, 19A , 19F, 20, 22F,23F ,33F
and 45.