Continuous Removal of Protein Aggregates by Annular Chromatography

Abstract

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 degrees ) 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.

Continuous Matrix‐assisted Refolding of Inclusion‐body Proteins: Effect of Recycling

“…A higher flow rate of regeneration buffer resulted in damage of the upper gel layer and additional dispersion effects at the outlet of the nozzle in the surrounding glass beads. Similar effects have been observed by Iberer et al [20] and Buchacher et al [21].…”

“…Dispersion effects caused by the density and viscosity differences between the feed stream, regeneration buffer and eluent buffer resulted in an increase of outlet fractions containing the regenerate. Broadening of the regenerate peak during extended operation might lead to reduction of resolution of the various protein containing fractions and therefore limitation of such a continuous process [21].…”

A multipurpose capacitive biosensor for assay and quality control of human immunoglobulin G

“…The standard method for the determination of aggregate content combines high-performance size-exclusion chromatography (SEC) with UV detection (Buchacher et al, 2001). However, traces of large protein aggregates cannot be detected by UV absorbance due to its limited sensitivity since these aggregates usually exist in low abundance, often smaller than 0.3% of the total hIgG content (Ahrer et al, 2003).…”

Preparative continuous annular chromatography (P-CAC), a review

“…The recovery ratio or yield is influenced by operating conditions, the stability of the protein, the source of the protein, and the presence of degrading enzymes. Buchacher et al [26] showed that this equation is also valid for annular chromatography. The recovery ratio is defined as throughput of protein at a certain purity where m p is the amount of pure protein:…”

“…The nonlinear multicomponent Langmuir isotherm correctly predicts the decrease in resolution as the concentration is increased. Buchacher et al [26] observed progressive fouling at high feed concentrations (50 mg IgG/ml) and, consequently, the separation resolution deterioriated.…”