Virus clearance by activated carbon for therapeutic monoclonal antibody purification.

In our previous study, activated carbon (AC) was employed in the purification process of therapeutic monoclonal antibody (mAb) as a replacement for Protein A affinity chromatography. In addition, we established an innovative column-free flow-through purification process using AC filter. In these investigations, the effective clearance of impurities (high-molecular-weight species, low-molecular-weight species, host cell proteins, and DNA) was observed compared to the conventional Protein A platform purification process. In this study, virus removal capability of our established AC process (AC filter) was investigated using two model viruses, Murine Leukemia Virus (MuLV) and Minute Virus of Mice (MVM) with the combination of two filtration methods (single-pass filtration and re-circulation filtration) using three kinds of mAbs. We found effective clearance of both MuLV and MVM (>3 log reduction factor, LRF) in all mAbs. Not only filtration method but also re-circulation duration didn't affect LRF, and >3 LRF of virus removal could be achieved by only single-pass filtration. From these results, it is expected that AC will be a promising candidate for the virus removal unit operation for mAb purification processes.

Improving impurities clearance by amino acids addition to buffer solutions for chromatographic purifications of monoclonal antibodies.

The performance of amino acids in Protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography for monoclonal antibody purification was investigated. Glycine, threonine, arginine, glutamate, and histidine were used as buffer components in the equilibration, washing, and elution steps of these chromatographies. Improved clearance of impurity, high molecular weight species (HMW) and host cell proteins (HCP) was observed in the purification processes when using the amino acids as base-buffer constituents, additives or eluents compared with that of buffers without these amino acids. In addition, we designed a buffer system in which the mobile phases were composed of only a single amino acid, histidine, and applied it to the above three chromatographies. Effective HMW and HCP clearance was also obtained in this manner. These results suggest that amino acids may enhance impurity clearance during the purification of monoclonal antibodies.

Measurement of sialic acid content is insufficient to assess bioactivity of recombinant human erythropoietin.

Assessment of biological potency and its comparison with clinical effects are important in the quality control of therapeutic glycoproteins. Animal models are usually used for evaluating bioactivity of these compounds. However, alternative methods are required to simplify the bioassay and avoid ethical issues associated with animal studies. Negatively charged sialic acid residues are known to be critical for in vivo bioactivity of recombinant human erythropoietin (rhEPO). In this study, we used capillary zone electrophoresis, a charge-based separation method, to estimate the sialic acid content for predicting in vivo bioactivity of rhEPO. In vivo bioactivities of rhEPO subfractions were measured and compared with sialylation levels. The results obtained indicated that in vivo bioactivity of rhEPO is not simply correlated with the sialylation level, which suggests that it is difficult to predict biological potency from the sialic acid content alone. N-Glycan moieties as well as sialic acid residues may have a significant impact on in vivo bioactivity of rhEPO.

Rational method for designing efficient separations by chromatography on polystyrene-divinylbenzene resins eluted with aqueous ethanol.

A rational method for designing separation processes by chromatography with polystyrene-divinylbenzene (PS-DVB) resins of different particle diameters (10-400microm) was developed. As model samples, catechin and epigallocatehin gallate (EGCG) were chosen and the mobile phase was an ethanol-water mixture. Linear gradient elution experiments were carried out with different gradient slopes, and the peak ethanol concentration was plotted against the normalized gradient slope. The plots were similar regardless of particle diameter. From these plots the two parameters describing the distribution coefficient K as a function of ethanol concentration I were determined. The K-I curves obtained were verified by isocratic elution experiments, and the conditions at which a baseline separation of the two polyphenols is possible was sought, and confirmed experimentally. Stepwise elution experiments were also designed and performed successfully on the basis of the K-I curve.

Treatment with anti-TGF-beta antibody ameliorates chronic progressive nephritis by inhibiting Smad/TGF-beta signaling.

BACKGROUND: Although short-term treatment with anti-transforming growth factor-beta (TGF-beta) antibody (alphaT) has been shown to prevent early glomerular lesions, its long-term effects and molecular mechanisms, including intracellular signaling, remain poorly understood. We examined whether alphaT treatment induces prevention of renal insufficiency and fibrosis, and affects the TGF-beta/Smad signaling pathway in rats with chronic progressive anti-thymocyte serum (ATS) nephritis induced by repeated ATS injections on days 0 and 7. METHODS: Nephritic and non-nephritic rats were treated with either alphaT or control immunoglobulin (Ig)G twice weekly for 4 weeks from days 7 to 35 (each group, N= 21). Renal lesions and cortical expression of TGF-beta1, TGF-beta2, TGF-beta3, type II TGF-beta receptor (TbetaRII), Smads, type I collagen, and plasminogen activator inhibitor-1 were examined by immunohistochemistry, Western blot, and/or real-time reverse transcription polymerase chain reaction (RT-PCR). The binding of Smad3 in renal cortical cell nuclei to the Smad-binding element (SBE) was investigated by the electrophoretic mobility shift assay. RESULTS: Nephritic rats developed heavy proteinuria, renal insufficiency, and increased extracellular matrix deposition resulting in renal fibrosis. Cortical expression levels of TGF-beta1, TGF-beta2, TbetaRII, and Smad2, but not TGF-beta3, Smad3, and Smad4 were increased. Expression and preferential localization of phosphorylated Smad2/3 in the glomerular and tubular cell nuclei, and Smad3-SBE complex-forming activity were also increased. Four-week alphaT treatment resulted in marked amelioration of chronic progressive ATS nephritis at 8 weeks. CONCLUSION: In chronic progressive ATS nephritis, the TGF-beta/Smad signaling was up-regulated. TGF-beta blockade by alphaT suppressed the progression of renal scarring, at least in part, via inhibition of activated TGF-beta/Smad signaling.