Biotechnology Progress最新文献

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.

In the biopharmaceutical industry, effective process control strategies are essential for enhancing drug substance quality and yield. This study presents dielectric spectroscopy as a novel approach for in-line monitoring of cell apoptosis, enabling earlier detection of apoptotic events and related cellular changes. Utilizing permittivity measurements, we assessed Cole-Cole parameters, specifically critical frequency (f_c) and delta epsilon (Δε), as key performance indicators (KPIs) for real-time monitoring of cell health in CHO cell cultures during batch and perfusion processes. Our findings demonstrate that variations in these parameters correlate with cellular stress responses, such as nutrient limitation and high shear conditions, providing timely signals for process monitoring and control. By integrating these in-line measurements, we can enhance feeding strategies, ultimately improving cell viability and productivity. This approach not only streamlines the monitoring process but also offers a robust framework for proactive adjustments in bioprocessing, thereby optimizing overall performance and resource utilization.

Hydrophobic interaction chromatography (HIC) provides a powerful alternative impurity control method for antisense oligonucleotide purification relative to traditionally used anion exchange (AEX) and/or reverse phase methods. HIC is particularly effective in clearing process-related solvents and small molecules by ≥3 log₁₀ as well as failure sequences (sometimes called early eluting impurities (EEIs) by ≥90%). Additionally, HIC reduces harder to remove product-related impurities. These include branchmers (late eluting impurities (LEIs), oligonucleotides missing a single nucleotide (N-1 impurities), oligonucleotides lacking appropriate phosphorothioate sulfurization (P = O₁ impurity), and other synthesis-related impurities. To optimize the purification process, variables such as resin ligand, salt types, processing conditions, types of gradients, and loading ratios were systematically evaluated to achieve 90% yield and maximal impurity resolution. Loading the column at 32%-78% of its dynamic binding capacity (DBC), combined with stepwise wash and elution gradients, provided effective resolution of impurities in crude oligonucleotide mixtures. The desorption of the purified product was achieved in low lyotropic salt concentrations (typically ≤50 mM) using a stepwise gradient. This approach retained non-polar impurities such as LEIs within the column. When properly designed, HIC is an all-aqueous, scalable, cost effective and predictable purification process. It can be implemented as a stand-alone method or integrated into a dual-column process alongside orthogonal techniques, such as AEX, to achieve even higher levels of product purity.

Continued advancements in recombinant CHO expression of therapeutic mAbs have led to improved productivity but have also increased the HCP burden on the downstream purification process. In this work, we developed an in silico mediated workflow to facilitate the rapid development of non-protein A three-step processes for the effective removal of HCPs from a CHO-derived mAb therapeutic. Null CCF and pure mAb retention patterns were generated using linear gradient screens on a set of strategically selected resins, membrane adsorbers, and novel adsorbents. HCP characterization of key fractions was then carried out using RPLC "HCP fingerprinting" and the resulting retention database was processed using an in silico tool to generate a list of all possible three-step sequences subject to design constraints. Top-ranked processes generated by the tool were then evaluated and refined at the bench scale to produce several successful processes consisting of bind-elute capture followed by either a bind-elute and flowthrough step (91.4 ppm HCP with a cumulative product yield of 78.7%) or two flowthrough steps with no salt (96.1 ppm HCP with a cumulative yield of 81.4%).