Hindered diffusion of proteins in mixture adsorption on porous anion exchangers and impact on flow-through purification of large proteins.

Complex adsorption kinetics behaviors of proteins in mixtures hampers chromatographic process development and complicates model-based prediction of separation. We investigated the adsorption characteristics of mixtures comprised of a larger protein (secretory immunoglobulins or thyroglobulin) and a smaller protein (serum albumin or green fluorescence protein) on the small-pore anion exchanger Q Sepharose FF. Confocal laser scanning microscopy measurements revealed that binding of the large protein was extremely slow and eventually stopped completely after the adsorption front penetrated just a few µm into the particle. Binding capacities after 24 h of incubation were nevertheless around 35 mg/mL of particle which is relatively high when considering that only a fraction of the particle was saturated, suggesting that locally-high bound protein concentrations are attained in a layer close to the particle surface. During mixture adsorption, the bound protein layer also significantly hindered diffusion of the smaller proteins into the particles resulting in about three times slower adsorption kinetics compared to single component adsorption. The combined effects of restricted diffusion and protein binding explain why flow-through purification of these mixtures with the small-pore resin Q Sepharose FF is effective under practical conditions. In this resin, diffusion of secretory immunoglobulins (or thyroglobulin) is restricted in the small pores so that despite their intrinsically greater affinity for the resin, much less binds compared to small proteins. Using the large-pore resin POROS 50 HQ results in faster transport, but also in more binding of secretory immunoglobulins (or thyroglobulin) compared to smaller protein impurities, preventing effective flow-through purification.

Secretory immunoglobulin purification from whey by chromatographic techniques.

Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process. Several chromatographic stationary phases were tested. A combination of two anion-exchange steps resulted in the highest purity. The key step was the use of a small-porous anion exchanger operated in flow-through mode. Diffusion of SIg into the resin particles was significantly hindered, while the main impurities, IgG and serum albumin, were bound. In this step, initial purity was increased from 66% to 89% with a step yield of 88%. In a second anion-exchange step using giga-porous material, SIg was captured and purified by step or linear gradient elution to obtain fractions with purities >95%. For the step gradient elution step yield of highly pure SIg was 54%. Elution of SIgA and SIgM with a linear gradient resulted in a step yield of 56% and 35%, respectively. Overall yields for both anion exchange steps were 43% for the combination of flow-through and step elution mode. Combination of flow-through and linear gradient elution mode resulted in a yield of 44% for SIgA and 39% for SIgM. The proposed process allows the purification of biologically active SIg from animal whey in preparative scale. For future applications, the process can easily be adopted for purification of recombinant secretory immunoglobulin species.

A nonchromatographic process for purification of secretory immunoglobulins from caprine whey.

Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non-chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30-40-fold on a 500 kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re-dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans-membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by-product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra-/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:642-653, 2017.