Fouling Behavior during Sterile Filtration of Different Glycoconjugate Serotypes Used in Conjugate Vaccines.

PURPOSE: Sterile filtration can be a particular challenge when processing very large glycoconjugate vaccines. The objective of this study was to examine the sterile filtration performance of a series of glycoconjugate vaccines produced by coupling different polysaccharide serotypes to an immunogenic protein. METHODS: Sterile filtration was performed at constant filtrate flux using 0.22 µm pore size Durapore® polyvinylidene fluoride membranes. Glycoconjugates were characterized by dynamic light scattering, rheological measurements, and nanoparticle tracking analysis (NTA). Confocal microscopy was used to examine glycoconjugate capture profiles within the membrane. Transmembrane pressure data were analyzed using a recently developed fouling model. RESULTS: All glycoconjugates deposited in a narrow band near the entrance of the Durapore® membranes. The rate of fouling varied significantly for the different serotypes, with the fouling parameter correlated with the fraction of glycoconjugates larger than 200 nm in size. CONCLUSIONS: The fouling behavior and sterile filter capacity of the different glycoconjugate serotypes are determined primarily by the presence of large species (>200 nm in size) as determined by nanoparticle tracking analysis. The modified intermediate pore blockage model provides a framework for predicting the sterile filtration performance for these glycoconjugate vaccines.

Purification of a conjugated polysaccharide vaccine using tangential flow diafiltration.

Conjugated vaccines prepared from the capsular polysaccharide of Streptococcus pneumoniae can provide immunization against invasive pneumococcal disease, meningitis, and otitis media. One of the critical steps in the production of these vaccines is the removal of free (unreacted) polysaccharides from the protein-polysaccharide conjugate. Experimental studies were performed to evaluate the effects of membrane pore size, filtrate flux, and solution conditions on the transmission of both the conjugate and free polysaccharide through different ultrafiltration membranes. Conjugate purification was done using diafiltration performed in a linearly-scalable tangential flow filtration cassette. More than 98% of the free polysaccharide was removed within a 5-diavolume diafiltration process, which is a significant improvement over previously reported results for purification of similar conjugated vaccines. These results clearly demonstrate the opportunities for using ultrafiltration/diafiltration for the final purification of conjugated vaccine products.

Stereospecific interactions between histidine and monoclonal antibodies.

Histidine is a frequently used buffer in the final formulation of many commercialized monoclonal antibodies (mAbs), with histidine helping to stabilize the antibody during storage in addition to its buffering function. The objective of this study was to examine the stereospecificity of any histidine-antibody interactions using a combination of experimental studies and molecular dynamics simulations. Isothermal titration calorimetry provided evidence of weak stereospecific interactions, with the antibody showing approximately two to four additional interaction sites for d- versus l-histidine. The greater interactions with d-histidine were confirmed by measurements of the net protein charge using electrophoretic light scattering. The reduction in the net negative charge of the antibody in d-histidine led to significantly different behavior during diafiltration due to Donnan exclusion effects. Molecular dynamics simulations corroborated the presence of additional d-histidine interaction sites. These results provide the first demonstration of weak stereospecific interactions between l- and d-histidine and a mAb and the implications of these interactions for antibody formulation.