Long-term thermal stability of group C meningococcal polysaccharide-tetanus toxoid conjugate vaccine.

Abstract

The stability of vaccines during storage and handling is a prerequisite for optimal potency at the time of immunization. Meningococcal group C conjugate vaccines have been successfully incorporated in mass immunization programs, however, thus far no long-term real-time stability studies of these vaccines have been reported. Stability of de-O-acetylated group C meningococcal polysaccharide coupled to tetanus toxoid (GCMP-TT) was evaluated in real time on the basis of immunogenicity and physiochemical properties. The vaccine is formulated as a 0.5 mL suspension containing 10 mug GCMP conjugated to 10-20 mug of TT adsorbed on 0.5 mg aluminum in saline. The single dose syringes were stored under refrigeration (5 +/- 3 degrees C) and at room temperature (25 +/- 2 degrees C) for up to 42 months and at elevated temperature (40 +/- 2 degrees C) for up to 6 months. At both refrigerated and room temperatures, no time-dependent change in animal potency was detectable through 42 months. After the nine months maximum recommended storage period at room temperature, 96% of the baseline serum bactericidal antibody (SBA) titer was maintained. Time-dependent decreases in SBA level and anti-GCMP-TT IgG level were observed at 40 +/- 2 degrees C. No changes in GCMP-TT adsorption and pH occurred in all the studies. Loss of integrity increased over six months at 40 +/- 2 degrees C (p = 0.004). Free sugar content did not change over 36 months under refrigeration. GCMP-TT retained immunogenicity and physicochemical properties under refrigeration and at room temperature (25 +/- 2 degrees C) for up to 42 months.

Full text

Page 1

©2006 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
vaccines (MenC) were developed. Two of these are composed of an O-acetylated capsular
polysaccharide conjugated to the CRM197 mutant of diphtheria toxin. The third consists
of de-O-acetylated capsular polysaccharide conjugated to a tetanus toxoid (GCMP-TT)
The MenC conjugate vaccines have generally proven to be stable over a wide range of
storage temperatures, although repeated freeze-thawing can affect some formulations.9
To date, no long-term real-time stability studies of MenC conjugate vaccines have been
reported. Short-term stability evaluation of MenC conjugate vaccines over a period of
Research Paper
Long-Term Thermal Stability of Group C Meningococcal Polysaccharide-
Tetanus Toxoid Conjugate Vaccine
Shwu-Maan Lee1,*
Robert Petermann2
Quallyna Porte1
Greg Berezuk1
Brian Crowe2
John Shirtz1
1Baxter BioScience; Beltsville, Maryland USA
2Baxter BioScience; Vienna, Austria
*Correspondence to: Shwu-Maan Lee; Baxter BioScience; 12140 Indian Creek
Court; Beltsville, Maryland 20705 USA; Tel.: 301.419.8587; Fax: 301.470.2613;
Email: lees4@baxter.com
Original manuscript submitted: 12/11/06
Manuscript accepted: 12/21/06
Previously published online as a Human Vaccines E-publication:
http://www.landesbioscience.com/journals/vaccines/abstract.php?id=3749
Key woRdS
vaccines, conjugate, meningococcal infections,
immunogenicity, thermal stability, storage
ABBReviAtionS
CI
ELISA

GCMP-TT de-O-acetylated group C
meningococcal polysaccharide
coupled to tetanus toxoid
kDa kilodalton
MenC meningococcal group C vaccine
SBA serum bactericidal antibody
TT tetanus toxoid
95% confidence interval;
enzyme-linked immunosorbent
assay
ABStRACt
The stability of vaccines during storage and handling is a prerequisite for optimal
potency at the time of immunization. Meningococcal group C conjugate vaccines have
been successfully incorporated in mass immunization programs, however, thus far no
long‑term real‑time stability studies of these vaccines have been reported. Stability of
de‑O‑acetylated group C meningococcal polysaccharide coupled to tetanus toxoid
(GCMP‑TT) was evaluated in real time on the basis of immunogenicity and physio‑
chemical properties. The vaccine is formulated as a 0.5 mL suspension containing 10 mg
GCMP conjugated to 10–20 mg of TT adsorbed on 0.5 mg aluminum in saline.
The single dose syringes were stored under refrigeration (5 ± 3˚C) and at room
temperature (25 ± 2˚C) for up to 42 months and at elevated temperature (40 ± 2˚C) for
up to 6 months. At both refrigerated and room temperatures, no time‑dependent change
in animal potency was detectable through 42 months. After the nine months maximum
recommended storage period at room temperature, 96% of the baseline serum bacteri‑
cidal antibody (SBA) titer was maintained. Time‑dependent decreases in SBA level and
anti‑GCMP‑TT IgG level were observed at 40 ± 2˚C. No changes in GCMP‑TT adsorption
and pH occurred in all the studies. Loss of integrity increased over six months at 40 ±
2˚C (p = 0.004). Free sugar content did not change over 36 months under refrigeration.
GCMP‑TT retained immunogenicity and physicochemical properties under refrigeration
and at room temperature (25 ± 2˚C) for up to 42 months.
intRoduCtion
A major concern in the formulation of vaccines is the ability to preserve biological
activity during storage and handling until use. Careful attention to storage and handling
is recognized as vital to ensure optimal potency of vaccines at the time of immunization.
With many vaccines, loss of potency over time occurs because of thermal instability.1,2 The
standard approach has been “cold chain management” that maintains vaccines between
2–8˚C from the time of manufacture to the time of administration. In the developing
world where the availability of electrical power and refrigeration are often inadequate, heat
storage, handling and heat stability of vaccines have been major areas of concern. However,
even though refrigeration is readily available in the developed world, breaks in the cold
chain are common. Inadequate storage and handling have been reported in 16–20% of
physician offices and health care facilities in the United States and Canada.3,4
Conjugate vaccines, in which a bacterial capsular polysaccharide is covalently conju-
gated to a carrier protein such as tetanus or diphtheria toxoid, have proven superior to
polysaccharide vaccines in several respects, especially the reliable induction of immunity
in children <2 years of age, and the induction of immunologic memory in vaccine
recipients.5-7 Conjugate vaccine technology has been successfully employed in childhood
vaccines for the prevention of bacterial meningitis pathogens, including vaccines against
Neisseria meningitidis Group C and Haemophilus influenzae serotype b.
Polysaccharide-protein conjugates are relatively stable compared to other vaccines such
as live attenuated bacterial vaccines, but such conjugates tend to degrade by hydrolysis
of the polysaccharide chain.8 During the 1990s, three meningococcal group C conjugate
[Human Vaccines 3:1, 27-32, January/February 2007]; ©2007 Landes Bioscience
www.landesbioscience.com Human Vaccines 27

Page 2

five weeks at a variety of temperatures has
demonstrated that the structural stability of
the polysaccharide chains and of the protein
carrier vary between conjugate vaccines.10-12
GCMP-TT displayed higher relative thermal
stability as compared with CRM197 conjugate
vaccines studied previously using similar tech-
niques.12
The rate of decline in vaccine potency at
a given temperature over real time provides a
reliable measure of thermal stability. Vaccine
shelf life at refrigerated temperatures has gener-
ally been two years. The approved shelf life of
GCMP-TT was recently extended from 36 to 42
months at 5 ± 3˚C. In addition, GCMP-TT is
also approved for nine months at room temper-
ature (25 ± 2˚C) for a single period within
the 42 months shelf-life period. The present
investigation addresses the thermal stability
of GCMP-TT up to 42 months with respect
both to immunogenicity and physicochemical
properties at refrigerated and room temper-
atures. Thermal stability of GCMP-TT at
elevated temperature is also examined.
MAteRiALS And MethodS
Vaccine. GCMP-TT (NeisVac-C, bulk
conjugate manufactured at Baxter Bioscience,
Beltsville, Maryland) is comprised of the
de-O-acetylated form of the group C menin-
gococcal polysaccharide coupled to tetanus
toxoid by reductive amination.13 Designed
for intramuscular injection, GCMP-TT
is formulated as a 0.5 mL suspension in a
prefilled single-dose syringe (type I glass) of
10 mg de-O-acetylated group C meningococcal
polysaccharide (from strain C11) conjugated to
10–20 mg of tetanus toxoid adsorbed on 0.5 mg aluminum in saline.
The adjuvant, aluminum hydroxide gel, was obtained from Brenntag
Biosector, Denmark.
Study design. A total of ten lots formulated and filled in full-scale
production batches at sites in Bloomington, Indiana, USA and
Vienna, Austria were tested (Table 1). All stability determinations
were performed on final filled syringes stored at either 5 ± 3˚C,
Table 1? Batches?included?in?the?report
Study numbers
1
2*
3
4
5
6
7
Lot numbers
801036, 801039, 801277
440103DF, 440203GB, 440303HC
801036, 801039, 801277
801125
801449, 801450, 801451
801036, 801039, 801277
440303HC
Storage Condition
5 ± 3˚C
5 ± 3˚C
25 ± 2˚C
25 ± 2˚C
25 ± 2˚C
40 ± 2˚C
40 ± 2˚C
time Points
0, 1, 2, 3, 6, 9, 12, 15, 18, 24, 36, and 42 months
0, 3, 6, 9, 12, 18, 24, 36, and 42 months
1, 3, and 6 months
1, 2, 3, and 4 weeks
0, 3, 6, 9, 12, 18, 24, 36, and 42 months
1, 3, and 6 months
1, 2, 3, and 6 months
*Study number 2 is on-going, currently at 36 months
Figure 1. GCMP‑TT stability of six lots for up to 42 months at 5 ± 3˚C based on (A) SBA and (B)
IgG titers. Data points show empirical measured values for each GCMP‑TT lot. Data are shown for
studies 1 and 2 with three lots each; study 1 has been concluded, study 2 is ongoing with 12–18
months data shown. Solid line indicates best‑fit linear regression function, and dashed lines indicate
the CI of the regression. Indicated p values are for hypothesis of zero slope.
Meningococcal Vaccine Stability
25 ± 2˚C, or 40 ±2˚C. In conformance with the International
Conference on Harmonization Guideline of Stability Testing of
Biotechnological/Biological Products, the following parameters were
tested: potency, sterility, pH, % adsorption, conjugate integrity, and
Appearance. Free saccharide was also determined in selected lots. All
parameters were tested with the same methods used for release of the
final product.
28 Human Vaccines 2007; Vol. 3 Issue 1

Page 3

Meningococcal Vaccine Stability
Potency. The immunogenicity of samples
was evaluated using Swiss Webster mice
(Harlan, Indianapolis, Indiana, USA). Mice,
10 in each group, were injected with 0.2 mL
of the diluted vaccine sample at 10 mg/mL, a
negative control, or a positive control, on days
0, 14 and 28. The animals were exsanguinated
on day 38, and sera were collected and stored
frozen until they were analyzed. All proce-
dures were completed in a manner that was
compliant with animal care guidelines.
The immune response of the murine serum
was determined by both serum bactericidal
antibody (SBA) titer14 and serogroup C-specific
murine IgG measured by enzyme-linked
immunosorbent assay (ELISA).15 The SBA
titer reflects functional antibodies, whereas the
ELISA measures total or isotype-specific serum
antibody response. The SBA assay utilized baby
rabbit complement (Pel-Freeze Biologicals,
Rogers, Arkansas, USA) and Group C, strain
11 N. meningitides (O-acetyl positive strain).
The IgG ELISA utilized de-O-acetylated
GCMP-human serum albumin conjugate as
the coating antigen. Test samples consisted
of serum pools prepared from equal aliquots
of individual mouse sera in each group, and
all sera were tested in duplicate. Bactericidal
activity of ≥5,000 was required to meet speci-
fications.
Physicochemical methods. MenC polysac-
charide is a sialic acid homopolymer. Sialic
acid content, an indication of % adsorption
of the conjugated vaccine to the aluminum
hydroxide adjuvant, was determined using
a resorcinol-based method similar to that
described by Svennerholm.16 To 1 mL of
sample and standards containing 5–50 mg of
N-acetylneuramic acid, 1 mL of resorcinol
reagent (0.2% resorcinol and 0.25 mM copper sulfate in 9.6 N
hydrochloric acid) was added. The mixture was heated at 100˚C for
30 min, cooled in a water bath for 10 min, and 2 mL of extraction
solution (85:15 of 1-butyl acetate: 1-butanol) was added. The absor-
bance of the upper organic phase was measured at 580 nm.
For % adsorption, the vaccine sample was spun for 10 min at
15,000 g and the gel layer was recovered upon removal of the super-
natant. Sialic acid content was determined for the vaccine sample
and its gel sample. The % adsorption is the ratio of sialic acid in
the adjuvant gel to the total sialic acid in the vaccine. Adsorption of
≥87% was required to meet specifications.
The conjugate integrity of the vaccine was determined by capillary
electrophoresis using a P/ACE 5510 or MDQ CE system (Beckman
Coulter, Fullerton, California, USA). Capillary electrophoresis can
detect both conformational and covalent changes. GCMP-TT was
desorbed from the aluminum hydroxide adjuvant by incubating
the gel phase of the vaccine in 0.05M sodium phosphate, 0.06 M
sodium citrate, pH 7.2 at 95˚C for 20 min. The migration profile of
the desorbed conjugate was compared with its precursor GCMP-TT
bulk material. In order to meet the specification, differences in the
migration profile could not be greater than 20%.
Free sugar, another measure of conjugate integrity, reduces the
immunogenicity of the vaccine. The conjugate vaccine was desorbed
from the aluminum hydroxide adjuvant and free sugar was separated
from the bulk conjugate by 100 kDa ultrafiltration. The free sugar
was hydrolyzed with 190 mM TFA at 85˚C for 30 min and the sialic
acid content was determined by high performance anion exchange
chromatography coupled with pulsed amperometric detection
(HPAE-PAD), using a Dionex DX-600 chromatography system.
Samples were also tested for sterility and pH was determined by
the standard potentiometric method. Appearance was assessed by
visual inspection under a fluorescent lamp, and the samples were
required to remain as a homogenous semi-opaque white to off-white
color suspension.
Statistical analysis. Stability over time regarding immunogenicity
and physicochemical properties was evaluated by linear regression.
The statistical significance of time-dependent change was assessed
under the null hypothesis of a zero regression slope. When significant
time-dependent change was evident, the percent of baseline values
retained at various time points and corresponding 95% confidence
intervals (CI) were estimated from the regression analysis. All anal-
yses were carried out using Stata 9.1 statistical software (Stata Corp.,
College Station, Texas).
Figure 2. Stability of 7 GCMP‑TT lots for up to 42 months at 25 ± 2˚C based on (A) SBA and (B) IgG
titers. Data are shown for studies 3–5. Graphic conventions as in Figure 1. Retained activity and CI
at nine months estimated from the regression.
www.landesbioscience.com Human Vaccines 29

Page 4

Meningococcal Vaccine Stability
ReSuLtS
Immunogenicity. In serum samples obtained
from mice immunized with vaccine that was
incubated for up to 42 months at 5 ± 3˚C,
no time-dependent change in SBA titer was
detectable (Fig. 1A). All SBA titers exceeded the
5,000 lower limit of specification by 1.8 fold or
more. No time-dependent change was observed in
murine anti-GCMP-TT IgG for up to 42 months
at 5 ± 3˚C (Fig. 1B).
When vaccine was stored for up to 42 months
at 25 ± 2˚C, the SBA titers produced in mice did
not change significantly (Fig. 2A). All SBA titers
were above the 5,000 lower limit of specification by
at least 1.6 fold. Over the nine months maximum
recommended period of exposure for GCMP-TT
to 25 ± 2˚C, SBA equivalent to 96% of the
baseline level was maintained. A non-significant
trend of diminishing murine anti-GCMP-TT IgG
ELISA values in samples incubated at 25 ± 2˚C
was observed (Fig. 2B). Nevertheless, through
nine months at 25 ± 2˚C, ELISA values equaling
95% of the baseline level persisted.
Although all SBA titers produced by vaccine
incubated at 40 ± 2˚C for up to six months
surpassed the minimum 5,000 specification by
1.2 fold or more, titers exhibited a pattern of
significant decrease (Fig. 3A). By six months,
the percent of baseline SBA retained was only
30%. IgG also declined significantly over time at
40 ± 2˚C (Fig. 3B), retaining only 39% of baseline
activity at six months.
Physicochemical properties. Both GCMP-TT
adsorption and pH remained unchanged over
42 months at both 5 ± 3˚C and 25 ± 2˚C and over
6 months at 40 ± 2˚C (Fig. 4A–C and G–I). All
pH measurements for all the studies fell within
the range of 5.4–6.5. Significantly increased loss
of integrity was observed over 6 months at 40 ±
2˚C but not over 42 months at 5 ± 3˚C and 25 ±
2˚C (Fig. 4D–F).
In study number 2, free sugar content was sequentially deter-
mined at periodic intervals up to 36 months when stored at 5 ± 3˚C
(Table 2). There was no evidence of change in percent free sugar over
time (p = 0.51).
Sterility was maintained in all samples at all times tested.
Appearance criteria were fulfilled in all lots except lot 801036 in
study numbers 3 and 6 which failed Appearance test at the six
months time point following incubation at both 25 ± 2˚C and
40 ± 2˚C. This lot passed Appearance test at 1 and 3 months. Small
white particles were seen in the six-month samples, which seemed to
be an aggregation of the aluminum hydroxide adjuvant. This is an
isolated incidence and these out of specification results do not impact
the overall stability profile. In study number 5, three lots passed the
entire battery of tests when stored at 25 ± 2˚C for 42 months.
diSCuSSion
According to World Health Organization recommendations for
the production and control of MenC vaccines, adequate stability
studies are an essential part of vaccine development.9,17 Vaccines
against the same disease from different manufacturers should be
considered individually because they may display different stability
and reactogenicity profiles.
Data on retained vaccine potency at temperatures other than
the recommended refrigerated storage temperature are clinically
useful because cold chain conditions cannot always be maintained.
Furthermore, studies under stress conditions may be useful in
determining whether short-term accidental exposure to undesired
conditions, such as during transport, can compromise product
quality.
The meningococcal Group C conjugate vaccine used in the
current study contains 10 mg of GCMP conjugated to 10–20 mg of
TT and adsorbed on 0.5 mg of aluminum in 0.5 mL of saline. In
Figure 3. Stability of 4 GCMP‑TT lots for up to six months at 40 ± 2˚C based on (A) SBA and (B)
IgG titers. Graphic conventions as in (Fig. 1). Tabulated numerical values and CI are regression
estimates at the specified time points. Measurements at 0 months not performed; starting values
at 0 months for calculation of retained activity extrapolated based on regression intercept. Data
were obtained from study 6 at 1‑, 3‑, and 6 months for three lots, and study 7 at 1‑, 2‑, 3‑ and
6 months for one lot. Overlapping numbers occur at several time points and fewer than 4 circles
are seen at 1, 3 and 6 months time points. For example, SBA titers at the three months time point
for two lots overlap (15.600 and 15.500 appear as a single circle).
30 Human Vaccines 2006; Vol. 3 Issue 1

Page 5

Meningococcal Vaccine Stability
this long-term thermal stability report, the methods used to measure
the stability of the vaccine include animal potency, pH, sterility,
Appearance, % adsorption, conjugate integrity and % free sugar.
To validate several of these methods as stability indicating, several
preliminary short-term stress studies were performed. For example, at
60˚C for 4 weeks, free sugar increase was observed together with SBA
decrease. At pH 3, a change in the conjugate peak mobility and an
increase of free sugar peak in the capillary electrophoresis profile were
observed. No attempt was made to establish a quantitative correla-
tion between integrity and free sugar assays.
Animal testing was conducted at 1/5 human dose (2 mg per
animal), which fell on the plateau region of the dose response curve.
The 2 mg dose was chosen to avoid animal variations (Peter Fusco,
unpublished results) and consequently the assay was insensitive to
small changes. The stability indicating method validation clearly
demonstrated that under thermal stress conditions, degradation of
the vaccine by potency change could be readily detected.
The relation between SBA and GCMP IgG ELISA was discussed
by Michon et al.14 SBA for C11, an O-acetylated positive strain, is
highly correlated with de-O-acetylated GCMP specific IgG ELISA. It
was believed that the GCMP-based protective epitope on the bacte-
rium exists in a de-O-acetylated form and this form, de-O-acetylated
GCMP-human serum albumin conjugate, was used as the coating
antigen for IgG ELISA.
Figure 4. Sequential measurements of (A–C) adsorption, (D–F) loss of integrity and (G–I) pH at 5 ± 3˚C, 25 ± 2˚C and 40 ± 2˚C, respectively, from the
10 GCMP‑TT lots as summarized in Table 1. Graphic conventions as in Figure 1.
Table 2
?
Percent?free?sugar?results?for?the?on‑going?study? ?
number?2;?syringes?were?stored?at?5?±?3˚C








Lot numbers
3 months
6 months
9 months
12 months
18 months
24 months
36 months
440103dF
2.9
5.0
7.3
2.5
4.5
4.7
8.3
440203GB
5.1
5.1
2.3
8.1
3.8
6.7
<2.8
440303hC
6.3
2.6
1.7
10
1.3
5.7
6.0
www.landesbioscience.com Human Vaccines 31