Biotechnology Progress最新文献

Recent advances in gene editing technologies have transformed the genetic engineering of Chinese hamster ovary (CHO) hosts, enabling the development of cell lines with improved stability and productivity. In this study, we employed the programmable nuclease (PN) Cas-CLOVER to precisely target the Glutamine synthetase (GS) locus in CHO cells. A total of 30 unique serum-free, suspension-adapted CHO-K1 candidate host cell lines were subjected to Cas-CLOVER-mediated gene editing, generating over one hundred potential GS knockout (GSKO) clones. A subset of the GSKO clones was subsequently validated using three orthogonal methods to confirm complete knockout of the GS gene in 98 clones. Randomly selected GSKO clones were utilized to produce standard monoclonal antibodies. The resulting pools demonstrated enhanced productivity, with up to a 14.5-fold increase in titer compared to their wild-type parental hosts. These findings highlight the potential of gene editing approaches to significantly improve recombinant protein production in CHO expression systems, offering valuable insights for biopharmaceutical manufacturing applications.

In vitro transcription (IVT) is a powerful method to generate RNA which not only facilitates RNA research but also plays a key role in the development and manufacture of RNA-based vaccines. mRNA is produced via the IVT process with a DNA template that contains information for the target antigen. However, as many disease-causing viruses mutate quickly and the cost of raw materials is high for the IVT reaction, there is a need for a system to develop a cost-effective and efficient IVT process platform. In this paper, we showed how total nucleoside-5'-triphosphates (NTPs) input, Mg²⁺ concentration and NTP preparation methods can influence IVT reaction yield and purity level of the final RNA constructs of different lengths and sequences. We propose an IVT design that will result in high RNA yield, high RNA integrity and low double-stranded RNA (dsRNA) concentrations for multiple RNA sequences. The approach presented here could significantly contribute to the development of a cost-effective, easy-to-adopt IVT process platform for RNA manufacturing.

Lentiviral vectors (LVVs) offer distinct advantages including large payload capacity and stable transduction of non-dividing cells, making them well-suited for ex vivo modification of stem or immune cells. However, chromatographic purification of LVVs is hindered by low recoveries, co-elution of product related impurities, and vector instability. In this study, we evaluated arginine hydrochloride (ArgHCl) as an alternative eluent to sodium chloride using CIMmultus™ QA (CIM QA) monoliths. Screening of solution conditions identified that phosphate buffer concentrations between 100-200 mM enhanced infective particle stability. During anion exchange screening experiments using a CIM QA 96-well plate, ArgHCl improved both infectious particle and p24 recoveries, which was corroborated in linear gradient elution (LGE) experiments using the monolith in a disk format. Furthermore, fractions eluted with ArgHCl exhibited improved colloidal stability compared to those eluted with NaCl. Increasing the ArgHCl gradient endpoint concentration to 1.5 M ArgHCl yielded infectious particle recoveries of 71%. Analysis of gradient fractions with nanoflow cytometry revealed a two-peak elution profile, with the more strongly retained peak enriched in vesicular stomatitis virus glycoprotein (VSV-G) positive particles, corresponding to greater infectious particle recoveries. ArgHCl also improved the chromatographic resolution between the initial impurity peak and the secondary peak, enabling improved separation. These findings support the use of ArgHCl and phosphate buffers to enhance recovery during the purification of LVVs using AEX chromatography and highlight the utility of nanoflow cytometry for rapid detection of product-related impurities.

Biopharmaceuticals are becoming one of the most successful clinical therapeutic products for treating various disorders and are gradually being utilized across nearly all areas of medicine. They have revolutionized the treatment of numerous diseases and continue to represent a significant area of research and development. Presently, host cell systems like bacteria, yeast, insects, and mammalian cells dominate the production of both therapeutic and diagnostic proteins. This review explores the strengths and limitations of these existing host systems for recombinant protein production, emphasizing the promising potential of microalgal systems for expressing therapeutic and diagnostic proteins. It accentuates the advantages of microalgae, such as their rapid growth rates, scalability, and sustainability. We delve into the intricacies of glycosylation patterns in microalgae, comparing them with those in other expression systems. This review highlights recent advancements in algal-based protein expression systems for diagnostic and therapeutic applications. It also outlines a strategic roadmap for future developments in biopharmaceutical production, emphasizing how each expression system's unique characteristics can help meet modern medicine's growing demands.

A rapid assessment of manufacturability for drug candidates is crucial for advancing a prospective biotherapeutic from a candidate to a bulk drug substance. A lot-to-lot approach to manufacturability is adopted where each biologic batch is assessed for manufacturability as a bulk, unfractionated pool. Manufacturers may explore a more granular approach, independently enriching and evaluating the filterability of antibody variants within each lot, especially within the confines of relative hydrophobicity and surface charge. This study examined the use of bind-and-elute chromatography to alter the proportions of monoclonal antibody (mAb) proteoforms in eluate sub-pools from a mixed-mode chromatography resin-packed column. Filterability of each sub-pool through a virus-retaining filter was subsequently examined. Circular dichroism and Fourier transform infrared spectroscopy were performed for each sub-pool to probe for higher-order structure differences between mAb variants enriched therein. Bioanalytical techniques were also used to assess colloidal stability, surface hydrophobicity, surface charge, and size differences. Results showed that basic charge variants, high-mannose glycovariants, high relative hydrophobicity proteoforms, and high-molecular-weight species were enriched in the last-eluting (terminal) sub-pools. The first sub-pool and the final sub-pool showed the most fouling propensity on VPro virus filters. Circular dichroism showed that enriched proteoforms in the last sub-pool possessed a higher percentage of bends. Most secondary structures did not vary significantly between sub-pools. Diffusion interaction parameter was highly negative across all sub-pools and the bulk unfractionated pool. These results provide a design space for identifying and depleting problematic mAb variants before the crucial virus filtration step.

For biofuels production, hemicellulose pre-hydrolysate is considered an attractive feedstock rich in fermentable sugars. The lignocellulosic biomass comprises, along with sugars, several inhibitors that can hamper its efficient conversion. In this work, mixed cultures of Ureibacillus thermosphaericus and Cupriavidus taiwanensis were used for the first time to detoxify the pre-hydrolysate. The nutrient source was first optimized in synthetic media with mono-cultures to detoxify phenolic compounds, and a medium containing inorganic salts was selected. Afterwards, the efficiency of phenolic degradation was compared in a single-compound solution and in a mixture. The simultaneous co-culture showed the highest degradation efficiency (90% at 2.8 g/L of phenolic compounds). Finally, the detoxification of a raw pre-hydrolysate was conducted, and a maximum degradation of 14% of the phenolics was obtained using sequential inoculation of Ureibacillus thermosphaericus followed by Cupriavidus taiwanensis addition.

In this work we present a workflow for developing two-step, flow-through polishing processes for monoclonal antibodies (mAbs). The approach is demonstrated using redissolved precipitates from three CHO-derived mAbs generated by a continuous, PEG/ZnCl₂-mediated precipitation capture process. Size Exclusion Chromatography (SEC) fingerprinting and a percent SEC clearance (PSC) metric are developed to enable simultaneous quantification of monomer yield and impurity removal during high-throughput screening and scale-down column studies. Batch slurry plate screens are used to evaluate multimodal anion exchange (MMA) resins and an activated carbon composite adsorber under varying pH and ionic strengths, assessing partition coefficients and PSC values against both low-molecular-weight (LMW) and high-molecular-weight (HMW) impurities. Top candidates were then assessed in single-column, higher-loading flow-through experiments using the redissolved precipitates as feeds. Activated carbon emerged as a highly effective first polishing step for LMW impurity removal under acidic, low-conductivity conditions, while MMA resins provided complementary LMW and HMW clearances in a subsequent flow-through step. The two-step processes achieved overall mAb recoveries of 80%-87%, reduced HMW species from >1.7% down to 1.1%, and decreased host-cell protein levels from >10,000 to <40 ppm for all three mAbs. SEC fingerprints showed the ability to identify orthogonal impurity removal opportunities between the two polishing materials, validating the screening methodology for a process devoid of bind-elute processing steps. This work demonstrates that SEC-based impurity profiling and PSC metrics can guide the development of flow-through polishing processes and offer a useful intensification strategy to alleviate DSP bottlenecks and reduce reliance on affinity capture.

As adeno-associated viral vectors (AAV) continue to advance through the clinical pipeline, effective downstream purification strategies must be developed to ensure bulk drug purity and safety. AAV are produced within mammalian cells, bringing forth risks associated with viral contamination. Although existing downstream operations provide some degree of viral inactivation and removal, regulatory agencies have recommended the incorporation of a dedicated virus removal filtration step to ensure robust viral clearance. Recently published studies have demonstrated that membrane filters with nominal pore sizes between 35 and 50 nm can provide effective AAV transmission while removing larger viruses, although these results were obtained over a limited range of conditions. This study represents the first investigation into the effects of filtrate flux and process disruptions on virus reduction filtration for AAV. Experiments were performed using purified AAV capsids and carboxylate-modified polymeric nanoparticles with a nominal diameter of 20 nm. Initial results confirmed that both systems exhibited nearly identical transient transmission profiles during virus filtration. Virus filtration performed at various filtrate fluxes (between 20 and 185 L/m²/h) revealed that moderately higher AAV yield may be obtained at lower fluxes. The data were analyzed using a modified internal polarization model, which was extended to account for the effects of process disruptions on transient particle transmission and recovery. Process disruptions were employed to increase AAV yield beyond 99% without compromising overall clearance of large viruses. At least a 4-log reduction in xenotropic murine leukemia virus (XMuLV) was observed under all conditions tested, even following multiple process pauses.

Natural killer (NK) cells have shown potential for allogeneic cell-based cancer immunotherapies. For development of economical off-the-shelf allogeneic therapies, maximal expansion of the NK cells from each donor must be achieved while maintaining efficacy and uniformity of the cell product. The standard method for robust expansion utilizes weekly stimulation with engineered feeder cells derived from the K562 cell line. However, the effects of repeated stimulation on NK cell growth, metabolism, and function are not well understood. In this study, we demonstrated a distinct shift in growth kinetics and metabolism around week 3-4 of repeated K562 feeder cell stimulation, followed by a change in cytokine secretion and killing ability. Seahorse metabolic flux assays and transcriptomics suggested a transition from glycolytic metabolism to oxidative metabolism after the first week of stimulation, but the shift in growth kinetics generally correlated to reduced metabolic activity. Collectively, these results indicate that serial stimulation sustains large-fold NK cell expansion that can be exploited for NK cell therapy; however, this expansion has important impacts on NK cell growth, metabolism, and function. Careful characterization is critical when developing large-scale biomanufacturing processes to ensure efficacy of the final cellular product.

Despite significant advances in continuous manufacturing of monoclonal antibodies, the implementation of continuous virus inactivation (CVI) remains challenging due to standardization gaps that could compromise product quality and safety. This study identified limitations in minimum residence time (mRT) prediction for packed bed reactors (PBR) utilized for CVI. This work focused on characterizing the residence time distribution (RTD) behavior of tracers with varying molecular properties in four PBR configurations. The results demonstrated that tracer molecular size impacted mRT prediction, with larger molecules showing shorter residence times than smaller molecule tracers under identical conditions. During scale-up from 16 to 26 mm diameter columns, mRT was not maintained, suggesting that traditional chromatography scale-up principles may not be directly applicable to CVI using PBRs. Overall, this work established a helpful foundational understanding of how process material properties impact mRT prediction-a critical process parameter that would directly impact virus inactivation efficacy in integrated CVI systems.