Achievement of a robust and scalable cell retention device remains a challenge in perfusion systems. Of the two filtration systems commonly used, tangential flow filtration (TFF) systems often have an inferior product sieving profile compared to alternating tangential flow filtration (ATF) systems, which is typically attributed to the ATF's unique alternating flow. Here, we demonstrate that observed performance differences between the two systems are a function of cell lysis and not the alternating flow as previously thought. The peristaltic pump used in typical TFF perfusion systems is shown to be the single major contributor to shear stress and cell lysis. Replacing the peristaltic pump with a low shear centrifugal pump brought cell growth, cell lysis, particle concentration, and product sieving in a TFF perfusion system to levels comparable with that of an ATF. These results provide a correlation where poor product sieving can be partially explained by high shear in cell retention systems and demonstrate that low shear TFF systems are a feasible alternative to ATF systems.

Aim: To assess the efÞ cacy of topical negative pressure moist wound dressing as compared to conventional moist wound dressings in improving the healing process in chronic wounds and to prove that negative pressure dressings can be used as a much better treatment option in the management of chronic wounds. Materials and Methods: This is a prospective comparative study of data from 112 patients with chronic wounds, of which 56 patients underwent topical negative pressure dressings (17 diabetic, 10 pressure sores, nine ischemic, two varicose, 10 post-infective raw areas and eight traumatic -six had bone exposed, two orthopaedic prosthesis exposed). The remaining 56 patients underwent conventional moist dressings (20 diabetic, two ischemic, 15 pressure sores, three varicose, eight post-infective raw areas and eight traumatic -Þ ve had bone exposed, three orthopaedic prosthesis exposed). The results were compared after 10 days. The variables compared were, rate of granulation tissue formation as a percentage of ulcer area covered, skin graft take up as the percentage of ulcer surface area and duration of hospital stay. The variables were compared using Unpaired Student's t test. A "P" value <0.05 was considered signiÞ cant. Results: Out of 56 patients who underwent topical negative pressure dressings, six (10.71%) were failures, due to failure in maintaining topical negative pressure due to defective sealing technique; these were included into the study group. After 10 days, the mean rate of granulation tissue formation was 71.43% of ulcer surface area. All these 56 cases underwent split-thickness skin grafting. The mean graft take-up was 79.29%. The mean hospital stay was 32.64 days. In the remaining 56 patients, the mean rate of granulation tissue formation was 52.85% of ulcer surface area. The mean graft take-up was only 60.45% of the total ulcer surface area. The mean hospital stay was 60.45 days. Conclusion: To conclude, topical negative pressure dressings help in faster healing of chronic wounds and better graft take-up and reduce hospital stay of these patients.

Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

Perfusion processes typically require removal of a continuous or semi-continuous volume of cell culture in order to maintain a desired target cell density. For fast growing cell lines, the product loss from this stream can be upwards of 35%, significantly reducing the overall process yield. As volume removed is directly proportional to cell growth, the ability to modulate growth during perfusion cell culture production thus becomes crucial. Leveraging existing media components to achieve such control without introducing additional supplements is most desirable because it decreases process complexity and eliminates safety and clearance concerns. Here, the impact of extracellular concentrations of sodium (Na) and potassium (K) on cell growth and productivity is explored. High throughput small-scale models of perfusion revealed Na:K ratios below 1 can significantly suppress cell growth by inducing cell cycle arrest in the G0/1 phase. A concomitant increase in cell specific productivity was also observed, reaching as high as 115 pg/cell/day for one cell line studied. Multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrated similar responses to lower Na:K media, indicating the universal applicability of such an approach. Product quality attributes were also assessed and revealed that effects were cell line specific, and can be acceptable or manageable depending on the phase of the drug development. Drastically altering Na and K levels in perfusion media as a lever to impact cell growth and productivity is proposed.

Successes with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered versus unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated natural killer cells have shown promise as a cellular therapy but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound IL-21 and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly-used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in two xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21-expanded NK cells. Collectively, these data support development of mbIL-21-expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.