In persons with primary and acquired antibody deficiencies, replacement therapy with IGIV is effective in preventing severe acute bacterial infections (24
). Using ELISA and OPAs, we compared anti-Hib and antipneumococcal antibody levels in current U.S.-licensed IGIV products. IGIV purification methods are manufacturer specific and unique with respect to specific conditions during fractionation and the combination of methods used to decrease aggregate formation, remove proteins associated with adverse events, and provide viral clearance (21
). Another difference among these products may be titers of antibodies in the starting plasma pool. Some products are derived from recovered plasma, harvested from whole-blood donations of volunteer donors. Other products are manufactured from source plasma, obtained by plasmapheresis of paid donors. The age and vaccination status of these donors may differ, thereby influencing the antibody profile. Additionally, plasma and blood collections from different regions may have different antibody profiles because of disease prevalence or vaccination coverage.
In order to examine the differences in antibody levels to pathogens that are important for PIDD patients, we tested seven IGIV products, five lots each for anti-Hib and antipneumococcal antibody concentrations. We also separated three IGIV products into IgG subclasses and measured the pathogen-specific subclass concentration. Finally, we assessed the functional activity of the total IgG and its subclasses against pneumococci using the OPA.
Differences in Hib antibody concentrations, as measured by ELISA, were found among products. Among all products, there was less than a twofold difference. Products B and G had somewhat lower anti-Hib levels than the mean of all products. Product C had the highest specific IgG1 and IgG2 anti-Hib titers, consistent with the overall finding for total IgG. The manufacturing of product B includes a heat treatment step to inactivate potential virus (36
). According to one study, heat treatment at a low pH and low sodium concentration was found not to have any detrimental effects on IgG structure (5
). However, a study on the thermal stability of Ig indicated that IgG may aggregate following heat treatment, and the Fab portion of IgG is most sensitive to heat (40
). Product G is manufactured by using the Kistler-Nitschmann method and includes a low pH and trace pepsin treatment step for virus inactivation (36
). It would be difficult to implicate any of these methods in the absence of direct experimentation, since differences in the starting plasma pool titers could be responsible for the observations. In any case, given a typical infusion of 300 to 400 mg/kg, anti-Hib levels should achieve the putative protective level of 0.15 μg/ml for all products (16
). It may be anticipated that anti-Hib titers in IGIV will increase over time as a result of childhood vaccination recommendations.
Differences were found in antipneumococcal antibody levels among pneumococcal serotypes and IGIV products. The relative antibody concentration against the five pneumococcal types in the seven different IGIV products reflects differences in normal antibody concentrations in adults (9
). Antibody levels to serotypes 14 and 19F are normally severalfold higher than those to types 4, 6B, and 9V, which is consistent with our study. In general, variation among products was at most two- to threefold, and lot-to-lot variation within products was much less. No product had consistently highest or lowest specific antibody levels when all serotypes were considered. Antibodies to serotype 4 were 0.049 ± 0.008 μg/mg of IgG. Most of the IGIV products were in that range, except product A, which had the highest anti-type 4 concentration (0.065 ± 0.010 μg/mg of IgG). Most of the products had a specific antibody concentration to serotypes 6B and 9V within the mean of 0.092 ± 0.022 and 0.088 ± 0.023 μg/mg of IgG, respectively. Antibody profiles to serotypes 14 and 19F were similar, averaging 0.41 ± 0.10 and 0.32 ± 0.08 μg/mg of IgG, respectively. Interestingly, product E, which had specific antibody concentrations similar to those of products C and D for the other serotypes, had lower anti-14 and anti-19F levels. Product E is known to have slightly low IgG3 concentrations, due to the use of small amounts of trypsin during manufacturing. Both IgG1 and IgG3 are susceptible to digestion by trypsin (33
), potentially explaining the lower levels of antibody to serotypes 14 and 19F observed for this product.
In order to address the distribution of specific antibodies within IgG subclasses, we selected three IGIV products, B, C, and G. According to the literature, the predominant subclass to Hib, pneumococcal, and other bacterial PS antigens in adults is IgG2 (35
). The ability to respond to the Hib vaccine when given in combination with the pertussis vaccine has been shown to coincide with maturation of the antibody response and secretion of IgG2 (32
). Also, a direct correlation between IgG2 levels in adults and antibody responses to PS antigens has been observed (34
). In accordance with these reports, we found that IgG2 contained the highest proportion of anti-Hib and antipneumococcal antibodies relative to the other IgG subclasses, although both IgG1 and IgG3 antibodies were also represented (Fig. and Table ). In addition, IgG2 had the best overall antipneumococcal opsonophagocytic activity of the three subclasses (Table ). On a per-milligram-of-IgG-subclass basis, IgG1 contains nearly as much antipneumococcal antibody as IgG2 (Table ). Product C had the highest specific IgG1 and IgG2 concentration against all serotypes of pneumococcus. Interestingly, product G tended to have more antipneumococcal and anti-Hib antibody content in IgG3 when this was compared with IgG3 from other products.
Our results contrast with those of Hamill et al. (13
). These authors performed a similar study with similar methods on four IGIV products, using ELISA. They found the predecessors of product G to have low titers to serotype 4 and of product F to have much higher titers against serotype 19F compared to the other products tested, whereas our studies demonstrate a near equivalence. As in our study, there was little lot-to-lot variation within manufacturers. The most likely explanation is that changes have occurred both in manufacturing methods and possibly in donor epidemiology in the intervening 12 years since the prior study. Consistent with our results, similar lot-to-lot antipneumococcal antibody levels were observed among a limited number of lots of three U.S. products in 2002 (20
There was not a strong correlation in our study between the pneumococcal ELISA and OPA results, which is consistent with another study (4
). The lack of correlation may be attributable to differences in antibody avidity or complement fixation activity, which were not examined in this study. In addition, the OPA can have high intra- and/or interassay variability. While a functional test such as the OPA is desirable, more research needs to be performed for assay optimization.
The minimum concentration of pneumococcus-specific antibody associated with long-term protection against invasive disease in vaccinated infants is typically between 0.15 and 0.5 μg/ml (4
). The average trough levels that would be achieved for a 20-kg child administered 400 mg of IGIV/kg as a one-time first dose, estimating a volume of distribution of 100 ml/kg, would be 0.10, 0.18, 0.17, 0.77, and 0.58 μg/ml for serotypes 4, 6B, 9V, 14, and 19F, respectively. While the average trough level for serotype 4 is less than 0.15 μg/ml, the IGIV preparations may still be effective, as it is not known whether the estimated protective level is the true lowest possible protective antipneumococcal concentration. In addition, trough levels of antibodies rise if regular infusions (every 3 to 4 weeks) are given, so that actual trough levels in a regularly infused patient are likely to be greater than the calculations above reflect (22
). A comparison of the protective antibacterial IgG concentrations measured by others and the levels of specific IgG measured in our ELISA suggests there is sufficient antibody, even in the IGIV with the lowest specific antibody concentrations, to provide protection against invasive pneumococcal disease.
Although we found differences in specific antibody levels among preparations, all of these products have been licensed based upon their ability to prevent bacterial infections in PIDD patients. As donor epidemiology and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients.