Bleed recycling is a novel method to increase the yield of steady-state perfusion processes by concentrating process bleed to selectively remove biomass and recycle the liquid fraction. This results ...in significant product saving which otherwise would go to waste. As long as cells can be concentrated and separated, existing cell separation devices can be used for such an application. However, limited information comparing operation modes and efficiency for bleed recycling applications is available. For the first time, inclined gravity settling has been used as bleed recycling technology and was compared to acoustic separation. Except for lower debris removal, inclined gravity settling showed similar bleed recycling efficiency and no negative impact on cell viabilities, nutrient and metabolite levels and product quality. Additionally considering reduced system complexity and facilitated scale-up, inclined gravity settling was the preferred technology for further evaluation during a 42-day lab-scale perfusion process. Up to a 3.5-fold bleed reduction and an average harvest rate increase of 19% was achieved. Scalability was subsequently tested with a large-scale inclined gravity settler suitable for a 2000 L perfusion process confirming performance of lab-scale experiments. Bleed recycling characterization data from screening experiments combined with scalability demonstration facilitates decision making when considering bleed recycling for novel perfusion process settings to reduce perfusion waste, increase process sustainability and boost overall process yield.
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•Novel application of inclined gravity settling in bleed recycling.•Acoustic separation vs. inclined gravity settling for bleed recycling.•Characterization of bleed recycling for a wide range of process parameters.•Successful bleed recycling automation during long-term perfusion culture.•Scale-up of inclined gravity settling to perfusion manufacturing scale.
Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain make ...it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways.
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For steady operation of perfusion cell cultures, the biomass concentration is controlled by a capacitance signal that triggers a bleed flowrate (non-filtered culture broth). The ...product of interest is also removed by this stream. To diminish the impact on the process yield, an acoustic settler was used in this study to separate the solid from the liquid phase. The concentrated cell fraction was harvested and discarded. The liquid fraction containing the product of interest was recycled to the bioreactor. In the presented study, yield was improved up to 2.25 fold because of the bleed volume reduction that resulted in more harvest collection.
•Use of an acoustic settler to concentrate and recycle perfusion waste stream.•Perfusion process yield was improved 2.25-fold.•Waste reduction in perfusion cell cultures.•Novel application of an existing cell retention device.
Stable operation in mammalian perfusion cell cultures can be achieved using a waste (bleed) stream to remove excess biomass from the reactor. This stream is often considered as a necessary loss sometimes leading to consequent yield drops. In this study, an acoustic settler was used to concentrate the bleed stream in order to harvest concentrated cells and recycle the clarified portion to the bioreactor. The amount of recycled product corresponded to an equivalent increase in harvest flowrate, which led to an increased overall productivity. This equipment was chosen because it can be operated in continuous mode and there are no fouling/clogging risks as it is the case for many other cell retention devices (CRD).
Two similar operating conditions (OC) were tested, one with and another without the acoustic settler working. Both conditions were compared with a strong focus on the bleed and harvest streams to evaluate the yield improvement. The separation efficiency (SE), which is the ability of the system to remove cells from the liquid phase, was calculated and was always superior to 99 %. The bleed fraction in this specific example could be decreased from 59.2 % ± 1.6–3.9 % ± 1.3 without impacting the process or the product quality and leading up to a 2.25-fold yield improvement.
Receptor-tyrosine-kinase-like Orphan Receptor 1 (ROR1) is a tyrosine-protein kinase transmembrane receptor and ROR1 overexpression is associated with a poor prognosis in various cancers, including ...ovarian cancer. Targeting of ROR1 has been evaluated as a novel cancer therapy strategy. This study developed a novel chimeric anti-ROR1 Fab antibody (named ROR1-cFab) and then assessed the antitumor activity of this antibody in ovarian cancer cells, an
model of preclinical cancer therapy. A ROR1-cFab prokaryotic expression vector was constructed from positive fusion cells (splenocytes from mice with high ROR1 immune titers were fused with myeloma cells) after three rounds of sub-clone affinity screening. Then, a variety of assays were employed to assess the binding selectivity and specificity of ROR1-cFab to ROR1 protein. Furthermore, CCK8, flow cytometric apoptosis, wound healing, and Transwell migration assays were used to assess antitumor activity of this newly developed anti-ROR1 antibody in ovarian cancer cells. We demonstrated that ROR1-cFab could specifically bind to ROR1 protein and ROR1-positive ovarian cancer A2780 cells. Functional assays revealed that ROR1-cFab inhibited tumor cell proliferation and migration, as well as inducing apoptosis of ROR1-positive A2780 cells in a dose dependent manner. These effects were not observed in ROR1-negative lose386 cells. In conclusion, ROR1-cFab is a novel anti-ROR1 antibody with a high affinity to ROR1 protein and inhibitory effects on ROR1-positive cells. Future studies will determine whether the ROR1-cFab might be a promising candidate for treatment of ROR1-positive ovarian cancer.
A careful selection of culture mediums and feeds has become necessary to maximize yields of recombinant proteins during bioprocesses of mammalian cells. Supplements contain a variety of concentrate ...nutrients, and their beneficial effects vary according to recombinant cell lines. In this study, the effects of PowerFeed A on growth kinetics, productivity and cellular metabolism were evaluated for two Chinese hamster ovary cell lines producing a monoclonal antibody in a batch culture. Supplemented cultures increased integral viable cell density of CRL-12444 and CRL-12445 cells by 2.4 and 1.6 times through extension of culture time at which viability was above 90% in 72 and 36 h, respectively, and increment of maximal cell concentration in 3.25 × 10
6
cells/ml (69%) for CRL-12445 cells. Product titer augmented 1.9 and 2.5 times for CRL-12444 and CRL-12445 cells, respectively, without changes in growth rate and specific productivity. Feed supplementation also stimulated full consumption of glucose and free glutamine and reduced 10 times lactate accumulation, while ammonium, sodium and potassium remained at similar concentrations at the end of the culture. About 44% of calcium, mainly provided by feed, was consumed by both cell lines. Maximization of cellular growth, viability and protein titer through feeding encourages extending its use to other cell lines and exploring novel combinations with other basal mediums or feeds. A thorough investigation of its impact on protein quality and the molecular mechanisms behind these effects will allow designing effective feeds and strategies to rationally optimize protein production in the biomanufacturing industry.
A 14-month-old male Armenian hamster (Cricetulus migratorius) presented with a spontaneous, subcutaneous, firm mass (4.0 × 2.0 × 1.5 cm) on the ventral neck extending towards the cheek pouch causing ...multifocal small oral ulcerations. This animal was immunized subcutaneously on the dorsal neck for the development of monoclonal antibodies seven months before presentation. The animal was euthanized and necropsy was performed. Histopathology of the mass showed a well demarcated, multilobulated, unencapsulated, highly cellular, neoplastic mass composed of spindle cells arranged in interlacing streams and bundles, with a moderate amount of fibrovascular stroma. The neoplastic cells exhibited indistinct cell borders and a moderate to large amount of eosinophilic, fibrillar cytoplasm, marked anisocytosis and anisokaryosis, binucleated and multinucleated cells, and high mitotic rate. Based on the histomorphologic features of the mass, and the presence of renal tubular hyaline globules and myeloid hyperplasia in the bone marrow, a diagnosis of histiocytic sarcoma was made. The presumptive diagnosis was confirmed by immunohistochemistry, upon which the neoplastic cells showed strong immunoreactivity for the histiocytic cell markers Iba1 and CD11b. Histiocytic sarcomas have been reported in Syrian (Mesocricetus auratus) and Siberian dwarf (Phodopus sungorus) hamsters but, to our knowledge, the current report represents the first case of histiocytic sarcoma described in an Armenian hamster. It is plausible to consider the animal’s experimental immunization history and the development of the histiocytic sarcoma to be related. An association between adjuvanted vaccines and soft-tissue sarcomas has been described in cats and referred to as feline injection-site sarcomas.
A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process ...as well as production of small‐scale drug candidates. The process presented in this article is a proof‐of‐concept of a continuous end‐to‐end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model‐based design and control. The downstream process, consisting of a periodic twin‐column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real‐time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4‐log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.
Antibody cloning from single B cells is an essential tool for characterizing humoral immune responses and obtaining valuable therapeutic and analytical reagents. Antibody cloning from individuals ...with high serologic titers to HIV-1, Influenza, Malaria and ZIKV has led to new insights that inform vaccine design efforts. In contrast to humans and mice, less is known about antibody cloning from single B cells in macaques. Here, we describe a protocol to identify and purify single antigen-specific macaque B cells, and subsequently clone and produce macaque monoclonal antibodies. The sorting strategy requires the use of a combination of fluorochrome labeled antigens and omission of anti-IgG antibodies that can interfere with antigen binding and vice versa. Optimized methods for macaque antibody gene amplification, DNA preparation for antibody production and antibody screening by ELISA are also presented.
•Design of sorting strategies to isolate HIV-1 Envelope-specific B cells.•Envelope baits and anti-isotype antibodies compete for binding to B cell receptor.•Method for next generation sequencing and cloning of macaque antibodies.•Cost-effective protocol to produce and screen monoclonal antibodies.
Due to the increasing economic and social relevance of biotherapeutics, their production processes are continually being reconsidered and reoptimized in an effort to secure higher product ...concentrations and qualities. Monitoring the productivity of cultured cells is therefore a critically important part of the cultivation process. Traditionally, this is achieved by determining the overall product titer by high performance liquid chromatography (HPLC), and then calculating the specific cell productivity based on this titer and an associated viable cell density. Unfortunately, this process is typically time‐consuming and laborious. In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively time‐effective, and potentially cheaper alternative for monitoring the productivity of a cell culture.
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