Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells.

A modular and multi-functional purification strategy that enables a common framework for manufacturing scale integrated and continuous biomanufacturing.

Biopharmaceutical manufacture is transitioning from batch to integrated and continuous biomanufacturing (ICB). The common framework for most ICB, potentially enables a global biomanufacturing ecosystem utilizing modular and multi-function manufacturing equipment. Integrating unit operation hardware and software from multiple suppliers, complex supply chains enabled by multiple customized single-use flow paths, and large volume buffer production/storage make this ICB vision difficult to achieve with commercially available manufacturing equipment. Thus, we developed SymphonX™, a downstream processing skid with advanced buffer management capabilities, a single disposable generic flow path design that provides plug-and-play flexibility across all downstream unit operations and a single interface to reduce operational risk. Designed for multi-product and multi-process cGMP facilities, SymphonX™ can perform stand-alone batch processing or ICB. This study utilized an Apollo™ X CHO-DG44 mAb-expressing cell line in a steady-state perfusion bioreactor, harvesting product continuously with a cell retention device and connected SymphonX™ purification skids. The downstream process used the same chemistry (resins, buffer composition, membrane composition) as our historical batch processing platform, with SymphonX™ in-line conditioning and buffer concentrates. We used surge vessels between unit operations, single-column chromatography (protein A, cation and anion exchange) and two-tank batch virus inactivation. After the first polishing step (cation exchange), we continuously pooled product for 6 days. These 6 day pools were processed in batch-mode from anion exchange to bulk drug substance. This manufacturing scale proof-of-concept ICB produced 0.54 kg/day of drug substance with consistent product quality attributes and demonstrated successful bioburden control for unit-operations undergoing continuous operation.

Seasonal variations in the sources of natural and anthropogenic lead deposited at the East Rongbuk Glacier in the high-altitude Himalayas.

Lead (Pb) isotopic compositions and concentrations, and barium (Ba) and indium (In) concentrations have been analysed at sub-annual resolution in three sections from a <110 m ice core dated to the 18th and 20th centuries, as well as snow pit samples dated to 2004/2005, recovered from the East Rongbuk Glacier in the high-altitude Himalayas. Ice core sections indicate that atmospheric chemistry prior to ~1,953 was controlled by mineral dust inputs, with no discernible volcanic or anthropogenic contributions. Eighteenth century monsoon ice core chemistry is indicative of dominant contributions from local Himalayan sources; non-monsoon ice core chemistry is linked to contributions from local (Himalayan), regional (Indian/Thar Desert) and long-range (North Africa, Central Asia) sources. Twentieth century monsoon and non-monsoon ice core data demonstrate similar seasonal sources of mineral dust, however with a transition to less-radiogenic isotopic signatures that suggests local and regional climate/environmental change. The snow pit record demonstrates natural and anthropogenic contributions during both seasons, with increased anthropogenic influence during non-monsoon times. Monsoon anthropogenic inputs are most likely sourced to South/South-East Asia and/or India, whereas non-monsoon anthropogenic inputs are most likely sourced to India and Central Asia.

An ultra-clean technique for accurately analysing Pb isotopes and heavy metals at high spatial resolution in ice cores with sub-pg g(-1) Pb concentrations.

Measurements of Pb isotope ratios in ice containing sub-pg g(-1) concentrations are easily compromised by contamination, particularly where limited sample is available. Improved techniques are essential if Antarctic ice cores are to be analysed with sufficient spatial resolution to reveal seasonal variations due to climate. This was achieved here by using stainless steel chisels and saws and strict protocols in an ultra-clean cold room to decontaminate and section ice cores. Artificial ice cores, prepared from high purity water were used to develop and refine the procedures and quantify blanks. Ba and In, two other important elements present at pg g(-1) and fg g(-1) concentrations in Polar ice, were also measured. The final blank amounted to 0.2+/-0.2 pg of Pb with (206)Pb/(207)Pb and (208)Pb/(207)Pb ratios of 1.16+/-0.12 and 2.35+/-0.16, respectively, 1.5+/-0.4 pg of Ba and 0.6+/-2.0 fg of In, most of which probably originates from abrasion of the steel saws by the ice. The procedure was demonstrated on a Holocene Antarctic ice core section and was shown to contribute blanks of only approximately 5%, approximately 14% and approximately 0.8% to monthly resolved samples with respective Pb, Ba and In concentrations of 0.12 pg g(-1), 0.3 pg g(-1) and 2.3 fg g(-1). Uncertainties in the Pb isotopic ratio measurements were degraded by only approximately 0.2%.