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The extent to which the glomerular basement membrane (GBM) contributes to the charge selectivity of the glomerular capillary wall has been controversial. To reexamine this issue, the size and charge selectivity of filters made from isolated rat GBM were assessed, using polydisperse Ficoll and Ficoll sulfate as test macromolecules. Ficoll sulfate, a novel tracer with spherical shape synthesized for this purpose, exhibited little or no binding to serum albumin, thereby avoiding a major difficulty that has been reported with dextran sulfate. The sieving coefficients of Ficoll sulfate were not different from those of Ficoll at physiological ionic strength, although the values for Ficoll sulfate were depressed at low ionic strength. These results confirm that the GBM possesses fixed negative charges but suggest that its charge density is insufficient to confer significant charge selectivity under physiological conditions, where electrostatic interactions are relatively well screened. The sieving coefficients of Ficoll sulfate and Ficoll were elevated significantly and by similar amounts when bovine serum albumin (BSA) was present in the retentate at 4 g/dl. This could be explained as the combined effect of two nonspecific physical factors, namely, the reduction in filtration velocity due to the osmotic pressure of BSA and the effect on macromolecular partitioning of repulsive solute-solute interactions. The view that BSA does not affect the intrinsic properties of the GBM is supported also by the absence of an effect on the hydraulic permeability of isolated GBM. The sieving coefficient of BSA was roughly half that of Ficoll or Ficoll sulfate of similar Stokes-Einstein radius. Given the finding of negligible charge selectivity, this difference may be attributed to the nonspherical shape of albumin. The results suggest that, to the extent that isolated GBM is similar to GBM in vivo, the charge selectivity of the glomerular capillary wall must be due to the endothelial and/or epithelial cell layers.

Formulation of protein biopharmaceuticals as highly concentrated liquids can improve the drug substance storage and supply chain, improve the target product profile, and allow greater flexibility in dosing methods. The Donnan effect can cause a large offset in pH from the target value established with the diafiltration buffer during the concentration and diafiltration of charged proteins with ultrafiltration membranes. For neutral formulations, the pH will typically increase above the diafiltration buffer pH for basic monoclonal antibodies and decline below the diafiltration buffer pH for acidic Fc-fusion proteins. In this study, new equations for the Donnan effect during the diafiltration and concentration of proteins in solutions containing monovalent and divalent ions were derived. The new Donnan models obey mass conservation laws, account for the buffering capacity of proteins, and account for protein-ion binding. Data for the pH offsets of an Fc-fusion protein and a monoclonal antibody were predicted in both monovalent and divalent buffers using these equations. To compensate for the pH offset caused by the Donnan effect, diafiltration buffers with pH and excipient values offset from the ultrafiltrate pool specifications can be used. The Donnan offset observed during the concentration of an acidic Fc-fusion protein was mitigated by operating at low temperature. It is important to account for the Donnan effect during preformulation studies. The excipients levels in an ultrafiltration pool may differ from the levels in a protein solution obtained by adding buffers into concentrated protein solutions due to the Donnan effect.