Development of an N-1 perfusion process and optimized scale-down models for implementation in a platform CHO cell culture manufacturing process

The use of N-1 perfusion, coupled with high-inoculum fed batch in CHO cell culture manufacturing processes, has been shown to increase volumetric productivity and shorten the duration of the fed-batch production phase. Implementation of N-1 perfusion as part of a platform process requires the ability to screen multiple clones and to optimize media and process parameters in a high-throughput manner. We have developed an N-1 perfusion process, along with a series of scale-down models for N-1 perfusion using shake flasks, cell culture tubes, and deep-well plates. Process parameters for scale-down models were optimized to maximize comparability of growth profiles and cell culture performance relative to 5L N-1 perfusion bioreactors. Scale-down models were used to inoculate fed-batch experiments in Ambr15 micro-bioreactors at high seeding density, in order to compare growth and productivity profiles to those observed in 5L bench scale bioreactors. Multiple cell lines derived from different CHO hosts were evaluated in order to verify the robustness of the scale-down models. Results demonstrated that cell growth and viability in the optimized scale-down models were comparable to those observed in 5L N-1 perfusion bioreactors. Furthermore, growth, productivity, and product quality profiles from high-inoculum fed-batch experiments were comparable regardless of inoculum source. Optimized scale down models of N-1 perfusion, coupled with Ambr15 fed-batch production micro-bioreactors, have now been integrated into a high-throughput and robust workflow to enable DOE and screening experiments for clone selection, media development and parameter optimization in a platform N-1 perfusion process for monoclonal antibody manufacturing

Integration of upstream and downstream for a hybrid continuous process development and manufacturing for a stable monoclonal antibody produced in CHO cell culture

Process intensification by continuous operation has been successfully applied in the chemical industry, when batch processes matured several decades ago. Fully integrated upstream and downstream continuous processing has also shown great potential for increased productivity and reduced cost in biomanufacturing using mammalian cell culture. After a few decades of development, continuous or perfusion cell culture has demonstrated for manufacturing of labile proteins or low-titer processes. Due to significant challenges implementing fully integrated continuous biomanufacturing and the fact that fed-batch cell culture has not yet matured, fed-batch cell culture and batch chromatography steps are still predominant for stable protein manufacturing in the industry. In comparison to perfusion cell culture, continuous or semi-continuous downstream processing for stable monoclonal antibodies (mAbs) has developed within less than a decade. Due to a high titer, e.g., 8-10 g/L, already achieved via fed-batch cell culture, which challenges the processing capacity for batch downstream commercial manufacturing, the demand of continuous chromatography operation dramatically increases. Here, we present a case study developing a hybrid continuous upstream and downstream as our next generation process for production of a stable mAb. For upstream, we implemented N-1 perfusion seed, which significantly increased the seeding density for fed-batch production. After media and process parameter optimization, the product titer for the intensified fed-batch process with high-seed increased more than 100% over the original fed-batch process. It should be noted that the original fed-batch process was optimized and used for clinical manufacturing at 1000-L scale. For next generation downstream, we developed multi-column chromatography for Protein A step, automated VI step and integrated pool-less polishing chromatography steps with increased productivity and reduction in resin requirement, buffer consumption and processing time. The next generation process with perfusion N-1 seed and continuous chromatography steps has been scaled up in 500-L bioreactor, and now has been demonstrated for full implementation in a GMP manufacturing facility at the 2000-L scale. We will present full set of data to compare the original optimized batch process at 1000-L scale and the next generation process at 2000-L scale for the stable mAb production using CHO cell culture. We believe that the hybrid continuous process is relatively easy to develop and implement in GMP manufacturing with significantly higher productivity than conventional fed-batch process for now, while the hybrid continuous process lays a good foundation for us to further develop and implement fully continuous upstream and downstream process in manufacturing with even higher productivity in future

Next generation manufacturing for biologics: Integration of a hybrid model for continuous manufacturing concepts into a clinical facility

The “one size fits all” concept is rarely applicable in life, this is also true for the concept of continuous manufacturing where specific applications will differ based upon the requirements of the end user. This is the scenario we describe here in which aspects of continuous manufacturing for both upstream and downstream biologics manufacturing are being incorporated to address the current pipeline needs within Bristol-Myers Squibb. The application is for stable, easily expressed monoclonal antibody processes that require moderate volumes and throughputs, such as for most oncology or immuno-oncology therapies. However, this is countered by challenges of an expanding pipeline that necessitates looking beyond the current platform philosophy for how to modify the process with the goal to increase overall productivity in a flexible manner. BMS recently constructed a clinical biologics manufacturing facility on the Devens, Massachusetts campus with operations being initiated in two phases. The first phase start-up aligned with a traditional, but flexible (i.e., based upon disposable technologies) upstream and downstream processes and was rapidly brought on line. The second phase is the design and construction within that same manufacturing building purposely left unfinished to allow for the process development group to design and demonstrate a next generation concept for manufacturing. With respect to the upstream process, the decision was made to maintain a fed-batch production bioreactor philosophy, but to employ much higher inoculation densities through use of perfusion culture at the seed bioreactor stage generating the inoculum. This results in cultures with shorter durations and opportunities for increased titer. Selection of the overall cycle time is an optimization between cadence and bioreactor throughput. With respect to the downstream processes, numerous continuous manufacturing technologies were evaluated to handle the increased titers being generated in the bioreactors. These downstream technologies include continuous harvest technologies, multicolumn continuous chromatography for capture, integrated pool-less polishing steps, automated viral inactivation, single pass TFF and in-line diafiltration. The advantages for manufacturing cadence and overall throughput, as well as other outcomes including efforts to decrease perfusion media usage, and a significant reduction in downstream resin costs will be presented. Once the second phase is implemented, the facility will accommodate both traditional as well as this hybrid model for continuous manufacturing interchangeably. The overall benefit to support multiple clinical products and the higher titer/throughputs are expected to reduce the number of batches as well as eliminate resupply batches for clinical supply

Radiofrequency Ablation of Benign Thyroid Nodules and Recurrent Thyroid Cancers: Consensus Statement and Recommendations

Thermal ablation using radiofrequency is a new, minimally invasive modality employed as an alternative to surgery in patients with benign thyroid nodules and recurrent thyroid cancers. The Task Force Committee of the Korean Society of Thyroid Radiology has developed recommendations for the optimal use of radiofrequency ablation for thyroid nodules. These recommendations are based on a comprehensive analysis of the current literature, the results of multicenter studies, and expert consensus

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Identification and ultrastructural characterization of Acanthamoeba bacterial endocytobionts belonging to the Alphaproteobacteria class.

The detection and identification of two endocytobiotic bacterial strains, one affiliated to the "Candidatus Caedibacter acanthamoebae"/"Ca. Paracaedimonas acanthamoeba", and another to the endosymbiont of Acanthamoeba UWC8 and "Ca. Jidaibacter acanthamoeba" are described. For endocytobiont screening, we developed a PCR method with a set of broad-range bacterial 16S rRNA primers to substitute the commonly used but technically demanding fluorescent in situ hybridization technique. Our PCR test alone without sequencing failed to discriminate the endocytobiont-containing and endocytobiont-free Acanthamoeba sp. due to the presence of mismatched primers to host mitochondrial DNA. We highlighted the need to perform bacterial primer checking against the Acanthamoeba genome to avoid false positive detection in PCR. Although the genetic aspect of "Ca. Caedibacter acanthamoebae"/"Ca. Paracaedimonas acanthamoeba" and the endosymbiont of Acanthamoeba UWC8/"Ca. Jidaibacter acanthamoeba" are well studied, knowledge pertaining to their morphologies are quite vague. Hence, we used transmission electron microscopy to examine our endocytobionts which are affiliated to previously described intracellular bacteria of Acanthamoeba sp. We used good-quality TEM images for the localization and the fate of the current endocytobionts inside different life stages of the hosts. Furthermore, to the best of our knowledge, our TEM findings are the first to provide morphological evidence for the clearance of defective Acanthamoeba endocytobionts via an autophagic-like process

Creative Music Therapy in an Acute Care Setting for Older Patients with Delirium and Dementia

Background/Aims: The acute hospital ward can be unfamiliar and stressful for older patients with impaired cognition, rendering them prone to agitation and resistive to care. Extant literature shows that music therapy can enhance engagement and mood, thereby ameliorating agitated behaviours. This pilot study evaluates the impact of a creative music therapy (CMT) programme on mood and engagement in older patients with delirium and/or dementia (PtDD) in an acute care setting. We hypothesize that CMT improves engagement and pleasure in these patients. Methods: Twenty-five PtDD (age 86.5 ± 5.7 years, MMSE 6/30 ± 5.4) were observed for 90 min (30 min before, 30 min during, and 30 min after music therapy) on 3 consecutive days: day 1 (control condition without music) and days 2 and 3 (with CMT). Music interventions included music improvisation such as spontaneous music making and playing familiar songs of patient's choice. The main outcome measures were mood and engagement assessed with the Menorah Park Engagement Scale (MPES) and Observed Emotion Rating Scale (OERS). Results: Wilcoxon signed-rank test showed a statistically significant positive change in constructive and passive engagement (Z = 3.383, p = 0.01) in MPES and pleasure and general alertness (Z = 3.188,p = 0.01) in OERS during CMT. The average pleasure ratings of days 2 and 3 were higher than those of day 1 (Z = 2.466, p = 0.014). Negative engagement (Z = 2.582, p = 0.01) and affect (Z = 2.004, p = 0.045) were both lower during CMT compared to no music. Conclusion: These results suggest that CMT holds much promise to improve mood and engagement of PtDD in an acute hospital setting. CMT can also be scheduled into the patients' daily routines or incorporated into other areas of care to increase patient compliance and cooperation

Multimodal prerehabilitation for elderly patients with sarcopenia in colorectal surgery

Sarcopenia, which is characterized by progressive and generalized loss of skeletal muscle mass and strength, has been well described to be associated with numerous poor postoperative outcomes, such as increased perioperative mortality, postoperative sepsis, prolonged length of stay, increased cost of care, decreased functional outcome, and poorer oncological outcomes in cancer surgery. Multimodal prehabilitation, as a concept that involves boosting and optimizing the preoperative condition of a patient prior to the upcoming stressors of a surgical procedure, has the purported benefits of reversing the effects of sarcopenia, shortening hospitalization, improving the rate of return to bowel activity, reducing the costs of hospitalization, and improving quality of life. This review aims to present the current literature surrounding the concept of sarcopenia, its implications pertaining to colorectal cancer and surgery, a summary of studied multimodal prehabilitation interventions, and potential future advances in the management of sarcopenia