Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System.

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

Risk factors for death in 224 cases of hepatocellular carcinoma after transcatheter arterial chemoembolization.

BACKGROUND/AIMS: Transcatheter arterial chemoembolization (TACE) has been reported as effective therapy for unresectable hepatocellular carcinoma (HCC), however, few have described methods for predicting prognosis, especially in patients treated by repeated TACE. To determine risk factors for death and try to predict the prognosis, we evaluated clinical data. METHODOLOGY: We retrospectively analyzed the clinical parameters of 224 patients with unresectable HCC treated with repeated TACE from January 1997 to December 2007. TACE was repeated when recurrence was diagnosed by tumor marker elevation and/or dynamic computed tomography findings. Factors affecting survival were evaluated using multivariate analysis after univariate analysis. Next, we combined the score for each significant factor into a single prognostic score and added up the positive factors in each case, then analyzed the significance of prognosis, after which the results were compared with other prognostic scoring systems. RESULTS: Multivariate analysis revealed that bilobular HCC, alpha-fetoprotein (> or = 400 ng/ml), tumor invasion of the portal vein, tumor size (> or = 10 cm), and albumin (< 2.8 g/dl) were related to poor prognosis, and developed a prognostic scoring system from those. According to that score, patients were classified into 5 groups. CONCLUSION: Our scoring system was easily performed and the results showed that repeated TACE should not be administered to patients with scores of 3 or more.

Efficacy of radiofrequency ablation therapy compared to surgical resection in 164 patients in Japan with single hepatocellular carcinoma smaller than 3 cm, along with report of complications.

BACKGROUND/AIMS: Radiofrequency ablation therapy (RFA) has become widely used against hepatocellular carcinoma (HCC), mainly because of its ease of use, lower level of invasiveness, and high level of effectiveness. To compare the efficacy and safety of RFA with surgery, we retrospectively investigated relevant patient clinical data. METHODOLOGY: The patients with a single HCC (3 cm > or =, Child-Pugh A or B) who were treated with RFA (RFA-group: n=105) or surgery (Surgery-group: n=59) from January 2000 to June 2007 were enrolled. RESULTS: The ratio of patients classified as Child-Pugh B was greater in RFA-group (24.8% vs. 8.5%, P=0.011), though the sizes of the tumors were not significantly different. There were no significant differences for survival rates or disease free survival rates after 3- and 5-years (RFA-group vs. Surgery-group; survival: 87.8 and 59.3% vs. 91.4 and 59.4%, disease free survival: 58.7 and 24.6% vs. 64.3 and 22.4%, respectively). As for complications, in RFA-group there was no severe complication while there was 1 case of postoperative hepatic failure death and 2 with a postoperative abscess in Surgery-group. CONCLUSIONS: In the present study, patients with a single HCC smaller than 3cm who underwent RFA had a lower frequency of severe complications, thus the efficacy of RFA was thought to be equal to a surgical procedure.