Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source.

Escherichia coli has been a primary host for the prokaryotic production of antibody fragments (Fabs) and has contributed to several successes in the pharmaceutical industry. Nevertheless, the requirement of disulfide bonds often results in low-yield fermentation and a lack of cost-effectiveness. Despite the improved production of functional Fabs by fermentation below 30 °C, the limited cellular growth needs further work. To address these issues, we investigated the effect of nitrogen supply on the cellular growth and the Fab productivity. We used the anti-human VEGF-A Fab as a model that exhibited poor expression at 37 °C regardless of the amount of nitrogen supplied during fermentation. In stark contrast, the expression yield of soluble Fab with a gross nitrogen supply of 6.91 g/L of broth throughout the fermentation at 25 °C was 332 mg/L. Furthermore, and increased nitrogen supply of 10.9 g/L significantly improved the yield of active form by 59.7% and the cellular growth rate by 39.3%. These results indicate that overdosing of a nitrogen source at low temperature is critical to Fab productivity in E. coli.

Purification of antibody fragments for the reduction of charge variants using cation exchange chromatography.

Recently, antibody fragments have been studied as therapeutic agents because they lack Fc effector function while having affinity similar to their original monoclonal antibody and can be produced using E. coli. Antibody fragments can be purified using affinity chromatography in the capture step, although they need a polishing step because of product-related impurities, mainly charge variants. Unlike monoclonal antibodies, few studies exist regarding the separation of charge variants in antibody variants. In this study, an efficient separation of charge variant method was assessed using a cation exchange chromatography resin with salt and a pH gradient. The SP ImpRes resin and pH gradient exhibited the most effective separation potency using combinations of resin and the separation method. The antibody fragment that did not undergo the charge variant separation process exhibited a difference in the tertiary structure of the protein and in vivo pharmacokinetics. However, the antibody fragment was similar to the reference protein when the charge variant separation process was performed. These results are expected to support efficient charge variant separation of antibody fragments and to be applied to the industrial production of therapeutic antibody fragments.

The prolonged half-lives of new erythropoietin derivatives via peptide addition.

Erythropoietin, or Epo, is a hematopoietic cytokine that promotes erythropoiesis, and recombinant human Epo has been used in the treatment of anemia in various chronic diseases. Here, we have constructed novel Epo derivatives with prolonged half-lives by adding peptides to the carboxy terminus of Epo without using linkers. The fused peptides were selected from the carboxy terminal region of human chorionic gonadotropin (hCG) or human thrombopoietin (hTpo), which promote the proper folding, secretion, and stabilization of bioactive glycoproteins. Addition of these peptides did not interfere with secretion or receptor binding, and significantly increased the in vivo half-life of human Epo, as measured by intravenous administration in rats. The plasma half-life of the Epo constructs was longest when the carboxy terminal 28 aa of the beta subunit of hCG was added (Epo-CGC), a half-life that was slightly longer than NESP (Aranesp), which is the most effective Epo product in current clinical use. The transformation of four Ser glycosylation sites to Ala on the CGC sequence also lengthened the plasma half-life of Epo, indicating that the in vivo stabilizing effect of the hCG peptide was due to both structures within the peptide itself and its O-glycosylations. The application of the carboxy terminal half of hTpo also resulted in remarkably reduced elimination of the Epo chimera (Epo-TpC), possibly due to protection by the TpC sequence. The in vivo hematopoietic activity of Epo derivatives in mice was consistent with their pharmacokinetic profiles. Therefore, these derivatives with prolonged half-lives may provide opportunities for developing new Epo therapeutics with less frequent administration.

Altered oral absorption of alcohol by combined aqueous extracts of four herbal plants in rats.

This study examined the effect of combined aqueous extracts (BHR) of Ginko biloba, Mentha arvensis var. piperascens, Citrus unshiu, and Pueraria lobata var. chinensis on oral absorption of alcohol in rats. The rats were pretreated with BHR, placebo solution identical to BHR without the herbal extract, and isotonic saline. Alcohol was administered orally at 1- and 3-g/kg doses and the absorption profiles were compared. After oral administration of 1-g/kg doses, mean area under the curve (AUC) and C(max) values were significantly reduced in BHR-treated rats (16.1 +/- 10.0 and 0.3 +/- 0.1 mg/ml, respectively) as compared with saline-treated (37.9 +/- 14.4 and 0.7 +/- 0.7 mg/ml, respectively) and placebo solution-treated (63.0 +/- 46.4 and 0.7 +/- 0.4 mg/ml, respectively) rats. Similarly, after administration of 3-g/kg doses, mean AUC and C(max) values in BHR-treated rats (188.1 +/- 119.7 mg(.)min/ml and 1.0 +/- 0.4 mg/ml) were significantly reduced over those in saline-treated rats (571.4 +/- 512.4 mg(.)min/ml and 1.8 +/- 0.9 mg/ml, respectively). The relative oral bioavailability of alcohol calculated as the ratio of AUC(BHR)/AUC(Saline) was 42.5% and 32.9% at 1- and 3-g/kg doses, respectively. The reduced serum alcohol levels as well as the reduced AUC and C(max) after pretreatment with BHR appear to be a result of a reduced systemic absorption not due to an increased metabolic clearance.