What About CE for Vaccine In-Process Methods?
An older publication reported the use of CE as an in-process test for the purification of recombinant hepatitis B vaccine
(6). CE was used to quantify the amount of recombinant hepatitis B to demonstrate removal of impurities and show the presence
and removal of reagents added to the process. A single CE method was able to monitor these parameters in each step of the
10-step process versus the use of a variety of different methods and different technologies.
As expected, the first step (a cell lysate step) showed a plethora of peaks in the electropherogram, including spikes from
particulates in the sample. The next step (filtered lysate) electropherogram looked similar to the cell lysate step, although
some proteins were removed across the microfilter. The following step is a 10-fold concentration step and the electropherogram
reflects the increase in protein concentration. In the fourth step, the main product is absorbed on silica; this peak can
be quantified, along with Triton X-100. The next step is a HIC step followed by treatment with Amberlite. These steps remove
impurities. The seventh step is a diafiltration (100 kD MWCO) and gives a clean electropherogram with no Triton X-100 peak.
The next step, another diafiltration step, looks similar to the previous step electropherogram with a reduction of impurity
peaks. The last two steps are a sterilization and treatment with formaldehyde and thimerosal. No noticeable differences from
the previous steps are noticed, with the exception of the appearance of thimerosal. The CE method was capable of quantifying
thimerosal, Triton X-100 and the recombinant hepatitis B with good linearity (R2 of 0.999 or higher), although no precision or accuracy data was reported.
The quantification of Triton X-100 in an influenza vaccine has been described (7). Triton X-100 is added to limit aggregation
and improve homogeneity during production of the influenza vaccine. A CE method was developed to quantify the Triton X-100
and Triton X-100 ethylene oxide oligomers levels in the process. The Triton X-100 method was inferior to a HPLC method, but
the Triton X-100 ethylene oxide oligomers method performed well. As before, the separation was performed at 25 kV and detected
at 200 nm. The CE method was linear (R2 = 0.99975), precise (4.5%), and showed an acceptable LOD (56 μg/mL).
How About CE for Vaccine Impurity Methods?
Determination of the amount of free carrier protein is important for conjugate vaccines (vaccines which involve attachment
to an immunogenic carrier protein). CE is well suited for this application because of its resolving power; conjugated vaccines
are often well separated from the free protein.
CE has been used to determine the amount of free carrier protein in vaccines for Streptococcus pneumoniae and Neisseria meningiditis
(8). The method was designed to separate free carrier protein from the conjugated polysaccharides. Free protein is a measure
of the effectiveness of the conjugation reaction. The method was able to separate free carrier protein (diphtheria toxoid
[Dt] or tetanus toxoid [Tt]) for a variety of conjugates (Figure 3) including
S. pneumoniae Type 7 conjugate,
S. pneumoniae Type 3 conjugate,
S. pneumoniae Type 18C conjugate as well as S. pneumoniae Type 5 conjugate, S. pneumoniae Type 6B conjugate,
N. meningiditis Type A conjugate,
N. meningiditis Type C conjugate,
N. meningiditis Type W135 conjugate and N. meningiditis Type Y conjugate. The method was able to quantify Dt with linearity (R2 > 0.99) and precision (5.8% RSD). The method was also able to determine the free polysaccharide because there was no significant
overlap between the migration times for the polysaccharides when compared to the migration time of Dt or Tt (Figure 2). No
peaks were observed for S. pneumoniae Type 6B and S. pneumoniae Type 23F.
The same group published an optimized method for the determination of the amount of free protein for Meningococcal Polysaccharide-Diphtheria
toxoid conjugate vaccines at the final bulk stage (9). The meningococcal conjugate migrated was resolved from the free Dt.
The method had a LOD of approximately 15 μg/mL, which would allow for detecting as low as 2% free Diphtheria toxoid protein.