Evaluation of a single-use disk stack centrifuge for improved efficiency and sustainability at 1000 L GMP manufacturing scale.

Direct depth filtration is an established technology for single-use harvest operation. Advantages of direct depth filtration include familiarity with depth filtration in downstream processes and simplicity of the operation. Drawbacks include low capacity, large footprint, labor-intensive set-up, high water use, and high waste in the form of discarded filters. Single-use centrifugation is emerging as an alternative to depth filtration for the single-use harvest step. Within the single-use centrifugation space, disc stack centrifugation represents the newest entrant. In this study, we evaluated the performance of the GEA kytero single-use disc stack centrifuge to clarify two monoclonal antibody-producing cell culture fluids. The separation performance of the GEA kytero centrifuge varied between the two cell culture fluids, with differences in centrate turbidity and centrate filterability measured. A comparison was then performed to determine resource savings, compared to direct two-stage depth filtration, when using a GEA kytero centrifuge to harvest a 1000 L bioreactor. The analysis concluded that replacement of the first stage of depth filters with a GEA kytero centrifuge has the potential to decrease the required second stage depth filtration area by up to 80%. The decrease in depth filter area resulting from the use of the GEA kytero would result in a decrease in the harvest step footprint, a decrease in buffer volume required to prime and rinse depth filters, and a decrease in the volume of plastic waste. An economic comparison of the GEA kytero single-use centrifuge against a direct depth filtration step found that for a 1000 L harvest step, the GEA kytero centrifuge may reduce costs by up to 20% compared with two-stage direct depth filtration.

Use of ultrafiltration/diafiltration for the processing of antisense oligonucleotides.

Ultrafiltration/diafiltration (UF/DF) has been the hallmark for concentrating and buffer exchange of protein and peptide-based therapeutics for years. Here we examine the capabilities and limitations of UF/DF membranes to process oligonucleotides using antisense oligonucleotides (ASOs) as a model. Using a 3 kDa UF/DF membrane, oligonucleotides as small as 6 kDa are shown to have low sieving coefficients (<0.008) and thus can be concentrated to high concentrations (≤200 mg/mL) with high yield (≥95%) and low viscosity (<15 centipoise), provided the oligonucleotide is designed not to undergo self-hybridization. In general, the oligonucleotide should be at least twice the reported membrane molecular weight cutoff for robust retention. Regarding diafiltration, results show that a small amount of salt is necessary to maintain adequate flux at concentrations exceeding about 40 mg/mL. Removal of salts along with residual solvents and small molecule process-related impurities can be robust provided they are not positively charged as the interaction with the oligonucleotide can prevent passage through the membrane, even for common divalent cations such as calcium or magnesium. Overall, UF/DF is a valuable tool to utilize in oligonucleotide processing, especially as a final drug substance formulation step that enables a liquid active pharmaceutical ingredient.

Considerations for operational space definition and optimization of a no-salt flowthrough hydrophobic interaction chromatography purification step.

No-salt flowthrough hydrophobic interaction chromatography (HIC) has been shown to effectively remove process and product-related impurities from bioprocess streams. In this publication, a panel of six antibodies has been used to demonstrate operating principles for the application of no-salt flowthrough HIC in antibody purification processes. The results indicate that no-salt flowthrough HIC provides robust aggregate clearance across operating conditions including flow rate, and variations in resin ligand density. Additionally, HMW reduction has an optimal pH range relative to the isoelectric point of each molecule and high molecular weight (HMW) reduction can be improved by altering the total protein load and/or HMW concentration to drive binding of high molecular weight species to the resin.

Occupational exposure to mixtures of organic solvents increases the risk of neurological symptoms among printing workers in Hong Kong.

LEARNING OBJECTIVES: Summarize the nature and extent of exposure to organic solvents to which printing workers in this study were subjected. Compare the numbers of neurological symptoms in exposed and non-exposed workers and how they related to level of exposure, as defined by specific job title or by air sampling. Clarify the relationship between exposure to organic solvents and individual psychoneurological and other symptoms. ABSTRACT: The health effects of low-dose occupational exposure to organic solvents remains unclear. A cross-sectional survey was conducted among 762 male printing workers to assess the impacts of exposure to mixtures of n-hexane, toluene, isopropyl alcohol, and benzene on neurological and other symptoms. After controlling for age, smoking, alcohol drinking, past exposure history, working hours and shift work, current exposure to solvent mixtures was significantly associated with the total number of neurological symptoms and with the prevalence of specific symptoms of the nervous system and mucous membrane irritation. The adjusted odds ratio of neurovegetative lability (1.7-5.9), abnormal or reduced smell (1.6-4.1), memory loss (1.8), and mucous membrane irritation symptoms (1.5-4.6) significantly increased in the exposed group, especially when the summation index of exposure exceeded one.