The availability of clinical-scale downstream processing strategies for cell-based products presents a critical juncture between basic research and clinical development. Aqueous two-phase systems (ATPS) facilitate the label-free, scalable, and cost-effective separation of cells, and are a versatile tool for downstream processing of cell-based therapeutics. Here, we report the application of a previously developed robotic screening platform, here extended to enable a multiplexed high-throughput cell partitioning analysis in ATPS. We investigated the influence of polymer molecular weight and tie-line length on the resolution of five model cell lines in "charge-sensitive" polyethylene-glycol (PEG)-dextran ATPS. We show, how these factors influence cell partitioning, and that the combination of low molecular weight PEGs and high molecular weight dextrans enable the highest resolution of the five cell lines. Furthermore, we demonstrate that the separability of each cell line from the mixture is highly dependent on the polymer molecular weight composition and tie-line length. Using a countercurrent distribution model we demonstrate that our screenings yielded conditions that theoretically enable the isolation of four of the five cell lines with high purity (>99.9%) and yield.

The availability of preparative-scale downstream processing strategies for cell-based products presents a critical juncture between fundamental research and clinical development. Aqueous two-phase systems (ATPS) present a gentle, scalable, label-free, and cost-effective method for cell purification, and are thus a promising tool for downstream processing of cell-based therapeutics. Here, the application of a previously developed robotic screening platform that enables high-throughput cell partitioning analysis in ATPS is reported. In the present case study a purification strategy for two model cell lines based on high-throughput screening (HTS)-data and countercurrent distribution (CCD)-modeling, and validated the CCD-model experimentally is designed. The obtained data are shown an excellent congruence between CCD-model and experimental data, indicating that CCD-models in combination with HTS-data are a powerful tool in downstream process development. Finally, the authors are shown that while cell cycle phase significantly influences cell partitioning, cell type specific differences in surface properties are the main driving force in charge-dependent separation of HL-60 and L929 cells. In order to design a highly robust purification process it is, however, advisable to maintain constant growth conditions.

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Cross-flow membrane microfiltration was used under optimal conditions to recover met-growth hormone inclusion bodies (IBs) from Escherichia coli cell lysate by removal of the host-cell (bacterial) proteins (HCP) under minimal fouling conditions. This is the first step of a two-step process in which the goal was to isolate IBs at high yield from the HCP. These undesired soluble HCP were removed by passing them through the membrane while retaining the insolubles, including the aggregated IBs. Experiments were conducted at constant permeate flux with flat-sheet membranes of different pore sizes and chemistry, with feeds of varying pH and ionic strengths to determine the optimum combination for HCP removal. Diafiltration, the washing away of impurities with protein-free buffer, was then employed to ensure removal of the host cell proteins at the optimum conditions. About 90% removal of the HCP was obtained in about 5 diavolumes, maintaining high protein transmission and low membrane fouling.