An innovative strategy to recycle permeate in biologics continuous manufacturing process to improve material efficiency and sustainability.

Intensified perfusion processes are an integral part of continuous manufacturing for biopharmaceuticals which enable agile operations and significant reduction in cost of goods. However, they require large volumes of media to support robust cell growth and maintain high productivity, posing substantial challenges to operations, logistics, and process sustainability. This study explores a novel strategy for reprocessing and reusing permeate from perfusion cultures for mAb production. The concept was initially evaluated by recycling permeate, Protein A flow-through (ProA FT) and CEX processed ProA FT in deep-well plate mock perfusion and ambr® 250 perfusion formats. Further processing of ProA FT through a cation exchange depth filter before recycling reduced process impurities such as host cell proteins (HCPs) and DNA. However, a direct replacement of fresh media with spent media reduces nutrient depth which results in a concomitant reduction in productivity. In ambr® 250 bioreactors, recycling of ProA FT at 25%-50% replacement rates (defined as the fraction of recycled material in media) resulted in a 13%-30% reduction in cumulative productivity while maintaining product quality. To mitigate this, we used media concentrates which allowed independent modulation of media depth by replacing a portion of diluent WFI with recycled material. Results from deep-well mock perfusion studies demonstrated that comparable or higher productivities relative to control can be achieved with this approach. Taken together, our study demonstrates the feasibility of recycling permeate in perfusion cultures. Process mass intensity (PMI) calculations reveal that this approach can meaningfully improve material efficiency by reducing water consumption, thereby enhancing overall bioprocess sustainability.

Targeted Therapies for the Neurofibromatoses.

Over the past several years, management of the tumors associated with the neurofibromatoses has been recognized to often require approaches that are distinct from their spontaneous counterparts. Focus has shifted to therapy aimed at minimizing symptoms given the risks of persistent, multiple tumors and new tumor growth. In this review, we will highlight the translation of preclinical data to therapeutic trials for patients with neurofibromatosis, particularly neurofibromatosis type 1 and neurofibromatosis type 2. Successful inhibition of MEK for patients with neurofibromatosis type 1 and progressive optic pathway gliomas or plexiform neurofibromas has been a significant advancement in patient care. Similar success for the malignant NF1 tumors, such as high-grade gliomas and malignant peripheral nerve sheath tumors, has not yet been achieved; nor has significant progress been made for patients with either neurofibromatosis type 2 or schwannomatosis, although efforts are ongoing.

Insights into the Pathogenesis of NF1-Associated Neoplasms.

Neurofibromatosis type 1 (NF1) is one of the most common neurocutaneous genetic disorders, presenting with different cutaneous features such as café-au-lait macules, intertriginous skin freckling, and neurofibromas. Although most of the disease manifestations are benign, patients are at risk for a variety of malignancies, including malignant transformation of plexiform neurofibromas. Numerous studies have investigated the mechanisms by which these characteristic neurofibromas develop, with progress made toward unraveling the various players involved in their complex pathogenesis. In this review, we summarize the current understanding of the cells that give rise to NF1 neoplasms as well as the molecular mechanisms and cellular changes that confer tumorigenic potential. We also discuss the role of the tumor microenvironment and the key aspects of its various cell types that contribute to NF1-associated tumorigenesis. An increased understanding of these intrinsic and extrinsic components is critical for developing novel therapeutic approaches for affected patients.

Unbiased Microbiome and Metabolomic Profiling of Fecal Samples from Patients with Melanoma.

Gut microbiota influence and modulate host immune responses. In preclinical cancer models, mice lacking gut microbiota have a markedly diminished response to immune checkpoint inhibitor therapy. Further, in melanoma patients, specific commensal gut microbiota have been associated with a positive clinical response to immunotherapy. In order to study the gut microbiome and metabolome, we have developed methods for fecal sample collection and processing, microbiome and metabolome profiling, and bioinformatic analysis. This protocol will be a useful tool for interrogating the taxonomic composition and functional output of a melanoma patient's gut microbiome.

Effective bioreactor pH control using only sparging gases.

pH control is critical in bioreactor operations, typically realized through a two-sided control loop, where CO2 sparging and base addition are used in bicarbonate-buffered media. Though a common approach, base addition could compromise culture performance due to the potential impact from pH excursions and osmolality increase in large-scale bioreactors. In this study, the feasibility of utilizing control of sparge gas composition as part of the pH control loop was assessed in Chinese hamster ovary (CHO) fed-batch cultures. Fine pH control was evaluated in multiple processes at different setpoints in small-scale ambr®250 bioreactors. Desired culture pH setpoints were successfully maintained via air sparge feedback control. As part of the pH control loop, air sparging was increased to improve CO2 removal automatically, hence increase culture pH, and vice versa. The effectiveness of this pH control strategy was seamlessly transferred from ambr®250 to 200 L scale, demonstrating scalability of the proposed methodology. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2743, 2019.

Validation of the Sleep-Wake Scoring of a New Wrist-Worn Sleep Monitoring Device.

STUDY OBJECTIVES: To test the sleep-wake scoring reliability of a new wrist-worn sleep monitoring device. METHODS: Twenty-seven adult good sleepers underwent 1 night of polysomnography (PSG) while wearing both the new device (myCadian [MC]; CurAegis Technologies, Rochester, New York, United States) and commercially available actigraphy (Actiwatch 2 [AW]; Philips Respironics, Murrysville, Pennsylvania, United States) on their nondominant wrist. PSG tests were manually stage scored. After excluding missing data, 20 participants had full-night data on all three devices with 17,734 total 30-second epochs. Using PSG as the gold standard, pooled epoch-by-epoch agreement for sleep and wake was calculated for each device using percent agreement and Cohen kappa statistic. Positive predictive values for both sleep and wake epochs, as well as sleep continuity statistics, were calculated. RESULTS: Percent agreement with PSG-scored wake and sleep was 91.3% for MC (kappa = 0.67) and 87.7% for AW (kappa = 0.50). Positive predictive values for sleep epochs were 94.4% and 90.8% for MC and AW, respectively, and 74.5% and 65.6% for wake. Both devices underestimated wake and overestimated sleep compared to PSG. Descriptively, compared to PSG, sleep latency was higher with MC and wake after sleep onset higher with AW. Total sleep time and sleep efficiency were more similar across devices. CONCLUSIONS: The kappa statistic for MC is consistent with a high level of agreement with PSG. Overall, the reliability of MC compared to PSG scoring was slightly more favorable than that of AW. Findings suggest that MC provides reliable sleep-wake scoring during a nocturnal sleep period for good sleepers.