Kv3.1-containing K(+) channels are reduced in untreated schizophrenia and normalized with antipsychotic drugs.

Neuronal firing is a fundamental element of cerebral function; and, voltage-gated potassium (K(+)) channels regulate that firing through the repolarization of action potentials. Kv3-type channels (Kv3.1-Kv3.4) represent a family of voltage-gated K(+) channels that have fast-spiking properties. Kv3.1 channel subunits are predominantly localized to cortical parvalbumin (PV)-positive, inhibitory interneurons. The firing properties of these interneurons participate in establishing the normal gamma oscillations and synchrony of cortical neuronal populations, thought to be the signature of higher information processing in human brain. Schizophrenia (SZ) is associated with abnormalities in cortical gamma synchrony and in information processing, particularly with dysfunction in working memory and executive function. Here, we report the distribution of Kv3.1b and Kv3.2 protein in normal human brain, showing that Kv3.1b is limited to neocortical areas, whereas Kv3.2 is abundantly represented in neo- and subcortical regions. In SZ cases, levels of Kv3.1b protein are decreased in the neocortex, but only in cases without antipsychotic drug (APD) treatment; Kv3.1 levels are normal in antipsychotic-treated cases. Kv3.2 is not different in distribution or in level between normal and SZ cases, nor influenced by APD, in any region tested. The apparent increase in Kv3.1b protein levels by APDs in SZ neocortex was confirmed in laboratory rodents treated with chronic APDs. These findings show a decrease in Kv3.1b channel protein in SZ neocortex, a deficit that is restored by APDs. This alteration could be fundamentally involved in the cortical manifestations of SZ and in the therapeutic response to APDs.

Process characterization for metal-affinity chromatography of an Fc fusion protein: a design-of-experiments approach.

The utility of a design-of-experiments approach was investigated for process characterization of a metal-affinity chromatographic purification process for an Fc fusion protein. This approach gave a better understanding of some of the key process variables as well as robustness for this step in the purification process. Single-variable experiments were employed to screen some of the potentially important variables in this step. Ranges for these variables were set based on prior experience in clinical manufacturing with similar processes. Following these experiments, a fractional factorial study was employed to further investigate the most important variables and their interactions. Key operational variables that had an impact on step yield and eluate purity were identified. In addition, the study helped identify a worst-case scenario for the step purity and helped assure that the rest of the process would successfully purify the product. This paper demonstrates the utility of a design-of-experiments approach for the characterization and validation of process chromatography steps in downstream processing. In addition, this study emphasizes the utility of robustness studies early in process development and establishes a strategy for future robustness studies.

Purification of oligonucleotides by high affinity, low molecular weight displacers.

High affinity, low molecular weight anionic displacers were successfully employed for the purification of antisense oligonucleotides. Several important structural characteristics were identified that contribute to the affinity of low molecular weight displacers to a hydrophilized polystyrene divinyl benzene anion exchanger. Sulfonic acid groups were found to possess higher affinity than carboxylic acid and phosphate functionalities, and nonspecific interactions (particularly hydrophobic interactions) were shown to play a major role in the retention process on this stationary phase material. Using this information, two high affinity, low molecular weight displacers were identified. These molecules are relatively inexpensive organic dyes that possess multiple sulfonic acid moieties, as well as aromatic functionalities, which increase nonspecific interactions with the stationary phase. These high affinity displacers, which can be readily detected, were then employed to displace several strongly retained antisense oligonucleotides that could not be displaced by previously established low molecular weight displacers. The displacement process resulted in very high purities of the antisense oligonucleotides. The results presented in this paper are significant in that they demonstrate that low molecular weight displacers for ion-exchange chromatography can possess equal to or greater affinities than their higher molecular weight counterparts, when nonspecific interactions with the stationary phase are exploited. In addition, the results illustrate the high resolutions possible with displacement chromatography and demonstrate an attractive technology for the process scale purification of oligonucleotides.

Hydrophobic displacement chromatography of proteins.

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.

Structural characteristics of low-molecular-mass displacers for cation-exchange chromatography.

The relative efficacy of a variety of low-molecular-mass displacers was examined using a displacer ranking plot. This method enables an evaluation of the dynamic affinity of a variety of displacers over a range of operating conditions. Several homologous series of molecules were evaluated to provide insight into the effects of various structural features on displacer efficacy. The results indicate that linear flexible geometries may have advantages over branched or cyclic structures. Data also indicate that the spreading out of charges may increase affinity. The incorporation of aromatic moieties in these displacers, particularly near the surface of the molecules, appears to result in a dramatic increase in displacer affinity. The ability of several high-affinity low-molecular-mass displacers a very strongly bound cationic protein is also examined. The results confirm the predictions of the theory and indicate that it is indeed possible to displace highly bound macromolecules with low-molecular-mass dispatchers. The work presented in this paper indicates that non-specific interactions can be exploited for producing high-affinity low-molecular-mass displacers.

Purification of an antigenic vaccine protein by selective displacement chromatography.

A recent advance in the state of the art of displacement chromatography has been the development of selective displacement chromatography. In this process, the bioproduct of interest is selectively displaced while impurities with lower retention are eluted in the induced salt gradient and higher retained impurities are desorbed after the breakthrough of the displacer front. In this manuscript, selective displacement chromatography is employed to purify an antigenic vaccine protein (AVP) from an industrial process stream. Displacers were screened and an operating regime plot was employed to establish appropriate conditions for selective displacement. The selective displacement process was successful and resulted in AVP that was equivalent in purity to product obtained at commercial production scale after conventional step gradient chromatography. Methods used to characterize the purified protein include size-exclusion chromatography, SDS-PAGE, isoelectric focusing, N-terminal amino acid sequence analysis, and amino acid composition analysis. This is the first report of the purification of a commercially and pharmaceutically significant protein using selective displacement chromatography and thereby sets the stage for the implementation of selective displacement chromatography for the downstream processing of biologicals.

Structural characteristics of low-molecular-mass displacers for cation-exchange chromatography. II. Role of the stationary phase.

The relative efficacy of a variety of low-molecular-mass displacers was examined on three different stationary phase materials. Several homologous series of displacer molecules were evaluated on these ion-exchange resins using a displacer ranking plot based on the steric mass action model. The results demonstrate that while aromaticity and hydrophobicity can play a significant role in the affinity of displacer molecules on polymethacrylate based and hydrophilized polystyrene-divinylbenzene based materials, this effect is much less pronounced on an agarose based resin. The work presented in this paper demonstrates that different structural features of low-molecular-mass displacers can dominate their affinity on various stationary phase materials employed and provides rules of thumb for the design of high affinity, low-molecular-mass displacers for a variety of commercial cation-exchange materials.