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The extent of afucosylation, which refers to the absence of core fucose on Fc glycans, can correlate positively with the antibody-dependent cellular cytotoxicity (ADCC) activity of a monoclonal antibody (mAb). Therefore, it is important to maintain consistent afucosylation during cell culture process scale-up in bioreactors for a mAb with ADCC activity. However, there is currently a lack of understanding about the impact of partial pressure of carbon dioxide (pCO2 )-a parameter that can vary with bioreactor scale-on afucosylation. Using a small-scale (3 L) bioreactor model that can modulate pCO 2 levels through modified configurations and gassing strategies, we identified three cell culture process parameters that influence afucosylation of a mAb produced by a recombinant Chinese Hamster Ovary (CHO) cell line: pCO 2 , media hold duration (at 37°C), and manganese. These three-independent parameters demonstrated a synergistic effect on mAb afucosylation; increase in pCO 2 , media hold duration, and manganese consistently increased afucosylation. Our investigations into the underlying mechanisms through proteomic analysis indicated that the synergistic interactions downregulated pathways related to guanosine diphosphate-fucose synthesis and fucosylation, and upregulated manganese transport into the CHO cells. These new findings highlight the importance of considering potential differences in culture environment and operations across bioreactor scales, and understanding the impact of their interactions on product quality.

The current study documented the implementation of three-dimensional conformal radiotherapy and assessed the tumor control and toxicity of such treatment in a large, multisite community practice.The authors retrospectively reviewed their first 222 consecutive patients with clinically localized (N0) prostate carcinoma treated with a 6-field conformal technique from October 1993 through March 2000. Standardized target definitions, dose planning constraints, and gantry angles were utilized to develop the treatment plan. Patients were categorized by low, intermediate, and high risk. Low risk was defined as T1a-T2a disease, a Gleason score7, and prostate-specific antigen (PSA) level/= 10.0 ng/mL (n = 47 [21%]). Intermediate risk was defined as T2b disease, a Gleason score6, or PSA level10.01 ng/mL (n = 60 [27%]). High risk was defined as 2 of the above risk factors or as T3 disease, a Gleason score7, or a PSA level20 (n = 115 [52%]). Biochemical disease recurrence was defined in accordance with the American Society for Therapeutic Radiology and Oncology definition. Urinary and bowel toxicity were graded using the Radiation Therapy Oncology Group morbidity scoring system.The median follow-up after radiotherapy for surviving patients was 47 months (range, 0-99 months). The 2 and 5-year actuarial biochemical control rates for all patients were 84% and 78%, respectively. Using logistic regression analysis, lower dose (75.6 gray [Gy] vs. 75.6 Gy; P = 0.006), higher risk group (P = 0.033), higher stage (P = 0.045), and higher PSA level (P = 0.001) were significantly associated with biochemical disease recurrence. Toxicity was not significantly correlated with a higher radiotherapy dose.Dose escalation to 75.6 Gy using a 6-field conformal technique was feasible in the authors' community practice and resulted in acceptable toxicity and early biochemical outcomes.

The addition of Fluosol-DA carbogen breathing to melphalan treatment of the FSa-IIC fibrosarcoma was assessed by tumor growth delay and cell survival assays. Melphalan (10 mg/kg) administered intraperitoneally (i.p.) was preceded by Fluosol-DA (0.3 ml) administered intravenously (i.v.) and followed by 1 hr of carbogen breathing; this resulted in a tumor growth delay of 9.5 +/- 1.4 days or an approximately 3-fold increase compared to melphalan alone. Melphalan produced about 1.7 logs of cell killing; neither carbogen breathing nor Fluosol-DA pretreatment altered the cell killing observed. There was a 10-fold increase in tumor-cell killing when Fluosol-DA was administered immediately prior to melphalan administration followed by carbogen breathing for 1 hr. Density gradient separation identified a population of denser FSa-IIC cells which showed increased sensitivity to melphalan after Fluosol-DA administration. There was no additional toxicity to bone marrow as measured by CFU-GM with the combination of melphalan/Fluosol-DA/O2 compared to melphalan alone.