Günter Iberer

Analysis of aggregates of human immunoglobulin G using size-exclusion chromatography, static and dynamic light scattering.

Large aggregates (Mr: 10(6)-10(7) g/mol) of human immunoglobulins are present in extremely small concentrations in IgG preparations (<0.1%). Traces of large protein aggregates cannot be determined by conventional size-exclusion chromatography (SEC) using UV detection due to limitations in sensitivity. The conventional analysis of IgG by SEC is limited to dimers and oligomers. Using light scattering it is possible to determine significant differences concerning the aggregate composition and the extent of protein aggregation in samples of different process steps. Two different pilot preparations were analyzed by SEC with UV and static light scattering detection and compared to dynamic light scattering in the batch mode. The change of large aggregates could be monitored and data were corroborated by dynamic light scattering.

Continuous matrix-assisted refolding of proteins.

A refolding reactor was developed for continuous matrix-assisted refolding of proteins. The reactor was composed of an annular chromatography system and an ultrafiltration system to recycle aggregated proteins produced during the refolding reaction. The feed solution containing the denatured protein was continuously fed to the rotating bed perfused with buffer promoting folding of the protein. As the protein passed through the column, it was separated from chaotropic and reducing agents and the refolding process took place. Native proteins and aggregates could be continuously separated due to different molecular size. The exit stream containing aggregates was collected, concentrated by ultrafiltration and recycled to the feed solution. The high concentrations of chaotropic and reducing agents in the feed solution enabled dissociation of the recycled aggregates and consequently were fed again to the refolding reactor. When the initial feed mixture of denatured protein is used up, only buffer-containing chaotropic agents and recycled aggregates are fully converted to native protein. This process resulted in a stoichiometric conversion from the denatured protein to its correctly folded native state. The system was tested with bovine alpha-lactalbumin as model protein. Superdex 75 PrepGrade was used as size-exclusion medium. The yield of 30% active monomer in the batch process was improved to 41% at a recycling rate of 65%. Assuming that the aggregates can be redissolved and recycled into the feed stream in a quantitative manner, a refolding yield close to 100% is possible. The method can be also applied to other chromatographic principles suited for the separation of aggregates.

Continuous Removal of Protein Aggregates by Annular Chromatography

The removal of polymeric proteins from their monomers is a frequently encountered separation task, especially in the polishing step of therapeutic proteins. Continuous separation of protein polymers from monomers by annular chromatography using size exclusion chromatography has been studied regarding the resolution, recovery, fouling, and productivity and has been compared to conventional chromatography. An IgG preparation rich in aggregates was used as a model protein mixture. Under conditions that maximized the throughput, the polymers could be separated from the monomers, but baseline separation could not be achieved. Baseline separation was also not possible in batch mode using equivalent conditions, which was also confirmed by computer simulation. For separation of the aggregates from the product the entire available separation space (360°) was indispensable. Therefore only cyclic, discontinuous regeneration could be carried out. Loading was identified as a critical step, since the concentrated protein solution evaded into the headspace instead of migrating into the gel where viscous fingering often occurs in conventional chromatography. The productivity of annular chromatography was two times higher than that of the conventional batch chromatography, and the buffer consumption was reduced to half the conventional value. These two benefits are especially important for protein separation processes that suffer from low loadability, such as size exclusion chromatography. We have demonstrated that size exclusion can be performed on an industrial scale when it is run continuously with the aid of a pressurized annular chromatograph.

Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities from the components of interest and/or as final step for the separation of purified proteins from their aggregates, in a so-called polishing step. However, the throughput in SEC is low compared to other chromatographic processes as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitation opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation process (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade as sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed flow-rate, eluent flow-rate and rotation rate on the separation efficiency was investigated. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extra column band broadening. The packing quality was proved to be identical for both systems. The productivity of conventional batch SEC was lower compared to continuous SEC, consequently buffer consumption was higher in batch mode.

Continuous purification of a clotting factor IX concentrate and continuous regeneration by preparative annular chromatography.

Preparative continuous annular chromatography, a method to separate proteins in a truly continuous manner, was investigated in an industrial environment. Plasma-derived clotting factor IX concentrate was used as model protein. Separation of vitronectin, a common impurity in commercial available factor IX concentrates, from factor IX was studied and compared to conventional packed bed chromatography in batch mode. As sorbent, Toyopearl DEAE 650M was used. Regeneration was performed simultaneously with the purification of factor IX in continuous mode. All required parameters applied for preparative annular chromatography such as feed flow-rate and elution flow-rate were first estimated from experiments on conventional batch columns. Then preparative annular chromatography and conventional packed beds were compared regarding enrichment, purity and productivity. Three different process scenarios, the optimal batch process,the preparative annular chromatography process and the batch process equivalent to the preparative annular chromatography process were investigated. The productivity of the optimal batch process was higher than that of the preparative annular chromatography and batch process equivalent to the preparative annular chromatography process. Therefore the throughput could not be increased by the use of the continuous chromatographic system.