Showing papers in "Biotechnology Progress in 2022"

A novel pH‐responsive nanoniosomal emulsion for sustained release of curcumin from a chitosan‐based nanocarrier: Emphasis on the concurrent improvement of loading, sustained release, and apoptosis induction

Abstract: Curcumin application as an anti‐cancer drug is faced with several impediments. This study has developed a platform that facilitates the sustained release of curcumin, improves loading efficiency, and anti‐cancer activity. Montmorillonite (MMT) nanoparticles were added to chitosan (CS)‐agarose (Aga) hydrogel and then loaded with curcumin (Cur) to prepare a curcumin‐loaded nanocomposite hydrogel. The loading capacity increased from 63% to 76% by adding MMT nanoparticles to a chitosan‐agarose hydrogel. Loading the fabricated nanocomposite in the nanoniosomal emulsion resulted in sustained release of curcumin under acidic conditions. Release kinetics analysis showed diffusion and erosion are the dominant release mechanisms, indicating non‐fickian (or anomalous) transport based on the Korsmeyer‐Peppas model. FTIR spectra confirmed that all nanocomposite components were present in the fabricated nanocomposite. Besides, XRD results corroborated the amorphous structure of the prepared nanocomposite. Zeta potential results corroborated the stability of the fabricated nanocarrier. Cytotoxicity of the prepared CS‐Aga‐MMT‐Cur on MCF‐7 cells was comparable with that of curcumin‐treated cells (p < 0.001). Moreover, the percentage of apoptotic cells increased due to the enhanced release profile resulting from the addition of MMT to the hydrogel and the incorporation of the fabricated nanocomposite into the nanoniosomal emulsion. To recapitulate, the current delivery platform improved loading, sustained release, and curcumin anti‐cancer effect. Hence, this platform could be a potential candidate to mitigate cancer therapy restrictions with curcumin.

Proliferation and differentiation of primary bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat

Abstract: Recently, cultured meat obtained from livestock‐derived cells is being considered as a sustainable food source that reduces the use of natural resources. This study aimed to show that nutrients extracted from Chlorella vulgaris were beneficial in the culture of primary bovine myoblasts (PBMs), a major cell source for cultured meat production. Nutrients (glucose, amino acids, and vitamins) present in the animal‐cell culture media were effectively recovered from C. vulgaris using acid hydrolysis treatment. On culture in nutrient‐free inorganic salt solution, cell death was induced in most PBMs after 6 days of cultivation. However, the addition of C. vulgaris extract (CVE) significantly improved PBM viability, which was comparable to the viability in conventional culture medium (Dulbecco's modified Eagle's medium). Furthermore, by adding horse serum to induce differentiation, the formation of myotubes was confirmed when CVE were used. Together, the results showed that CVE could be used as an alternative to the conventional culture medium for PBMs. These findings will not only lower the environmental risks associated with the establishment of this eco‐friendly cell culture system, but also highlight microalgae as a potent nutrient source that can replace conventional grain‐dependent nutrient sources.

Immobilized enzymes as potent antibiofilm agent

Abstract: Biofilm has been a point of concern in hospitals and various industries. They not only cause various chronic infections but are also responsible for the degradation of various medical appliances. Since the last decade, various alternate strategies are being adopted to combat the biofilm formed on various biotic and abiotic surfaces. The use of enzymes as a potent anti‐fouling agent is proved to be of utmost importance as the enzymes can inhibit biofilm formation in an eco‐friendly and cost‐effective way. The physical and chemical immobilization of the enzyme not only leads to the improvement of thermostability and reusability of the enzyme, but also gains better efficiency of biofilm removal. Immobilization of amylase, cellobiohydrolase, pectinase, subtilisin A and β‐N‐acetyl‐glucosaminidase (DspB) are proved to be most effective in inhibition of biofilm formation and removal of matured biofilm than their free forms. Hence, these immobilized enzymes provide greater eradication of biofilm formed on various surfaces and are coming up to be the potent antibiofilm agent.

Smart process development: Application of machine‐learning and integrated process modeling for inclusion body purification processes

Abstract: The development of a biopharmaceutical production process usually occurs sequentially, and tedious optimization of each individual unit operation is very time‐consuming. Here, the conditions established as optimal for one‐step serve as input for the following step. Yet, this strategy does not consider potential interactions between a priori distant process steps and therefore cannot guarantee for optimal overall process performance. To overcome these limitations, we established a smart approach to develop and utilize integrated process models using machine learning techniques and genetic algorithms. We evaluated the application of the data‐driven models to explore potential efficiency increases and compared them to a conventional development approach for one of our development products. First, we developed a data‐driven integrated process model using gradient boosting machines and Gaussian processes as machine learning techniques and a genetic algorithm as recommendation engine for two downstream unit operations, namely solubilization and refolding. Through projection of the results into our large‐scale facility, we predicted a twofold increase in productivity. Second, we extended the model to a three‐step model by including the capture chromatography. Here, depending on the selected baseline‐process chosen for comparison, we obtained between 50% and 100% increase in productivity. These data show the successful application of machine learning techniques and optimization algorithms for downstream process development. Finally, our results highlight the importance of considering integrated process models for the whole process chain, including all unit operations.

The function of chitosan/agarose biopolymer on Fe2O3 nanoparticles and evaluation of their effects on MCF‐7 breast cancer cell line and expression of BCL2 and BAX genes

Abstract: In recent decades, magnetic nanoparticles modified with biocompatible polymers have been recognized as a suitable tool for treating breast cancer. The aim of this research was to evaluate the function of chitosan/agarose‐functionalized Fe2O3 nanoparticles on the MCF‐7 breast cancer cell line and the expression of BCL2 and BAX genes. Free Fe2O3 nanoparticles were prepared by hydrothermal method. FTIR, XRD, SEM, DLS, VSM, and zeta potential analyses determined the size and morphological characteristics of the synthesized nanoparticles. The effect of Fe2O3 free nanoparticles and formulated Fe2O3 nanoparticles on induction of apoptosis was studied by double‐dye Annexin V‐FITC and PI. Also, the gene expression results using the PCR method displayed that Fe2O3 formulated nanoparticles induced BAX apoptosis by increasing the anti‐apoptotic gene expression and decreasing the expression of pro‐apoptotic gene BCL2, so the cell progresses to planned cell death. In addition, the results showed that the BAX/BCL2 ratio decreased significantly after treatment of MCF‐7 cells with free Fe2O3 nanoparticles, and the BAX/BCL2 ratio for Fe2O3 formulated nanoparticles increased significantly. Also, to evaluate cell migration, the scratch test was performed, which showed a decrease in motility of MCF‐7 cancer cells treated with Fe2O3 nanoparticles formulated with chitosan/agarose at concentrations of 10, 50, 100, and 200 μg/ml.

Advances in <scp>3D</scp> printing of composite scaffolds for the repairment of bone tissue associated defects

Abstract: The conventional methods of using autografts and allografts for repairing defects in bone, the osteochondral bone, and the cartilage tissue have many disadvantages, like donor site morbidity and shortage of donors. Moreover, only 30% of the implanted grafts are shown to be successful in treating the defects. Hence, exploring alternative techniques such as tissue engineering to treat bone tissue associated defects is promising as it eliminates the above-mentioned limitations. To enhance the mechanical and biological properties of the tissue engineered product, it is essential to fabricate the scaffold used in tissue engineering by the combination of various biomaterials. Three-dimensional (3D) printing, with its ability to print composite materials and with complex geometry seems to have a huge potential in scaffold fabrication technique for engineering bone associated tissues. This review summarizes the recent applications and future perspectives of 3D printing technologies in the fabrication of composite scaffolds used in bone, osteochondral, and cartilage tissue engineering. Key developments in the field of 3D printing technologies involves the incorporation of various biomaterials and cells in printing composite scaffolds mimicking physiologically relevant complex geometry and gradient porosity. Much recently, the emerging trend of printing smart scaffolds which can respond to external stimulus such as temperature, pH and magnetic field, known as 4D printing is gaining immense popularity and can be considered as the future of 3D printing applications in the field of tissue engineering.

Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy

Abstract: Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%–50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.

Bax and Bak knockout apoptosis‐resistant Chinese hamster ovary cell lines significantly improve culture viability and titer in intensified fed‐batch culture process

Abstract: In the field of therapeutic protein production, process intensification strategies entailing higher starting cell seeding densities, can potentially increase culture productivity, lower cost of goods and improve facility utilization. However, increased cell densities often trigger apoptotic cell death at the end of the cell culture process and thus reduce total viable cell count. Apoptosis-resistant Chinese hamster ovary cell lines may offer the possibility to diminish this undesired outcome of the intensified production process. In this study, we have generated and tested Bax/Bak double-knock-out (DKO) apoptosis resistant hosts to express standard and bispecific antibodies, as well as complex molecules in intensified production processes both as pools and single cell clones, and at different scales. In all cases, therapeutic proteins expressed from clones or pools generated from the Bax/Bak DKO hosts showed not only better viability but also enabled extended productivity in the later stages of the 14-day intensified production process. The product qualities of the produced molecules were comparable between Bax/Bak DKO and wild type cells. Overall, we showed that Bax/Bak DKO apoptosis-resistant host cell lines significantly improve viability and volumetric productivity of the intensified production cultures without altering product qualities.

Development of a miniature bioreactor model to study the impact of pH and DOT fluctuations on CHO cell culture performance as a tool to understanding heterogeneity effects at large‐scale

Abstract: Understanding the impact of spatial heterogeneities that are known to occur in large‐scale cell culture bioreactors remains a significant challenge. This work presents a novel methodology for mimicking the effects of pH and dissolved oxygen heterogeneities on Chinese hamster ovary (CHO) cell culture performance and antibody quality characteristics, using an automated miniature bioreactor system. Cultures of 4 different cell lines, expressing 3 IgG molecules and one fusion protein, were exposed to repeated pH and dissolved oxygen tension (DOT) fluctuations between pH 7.0–7.5 and DOT 10%–30%, respectively, for durations of 15, 30, and 60 min. Fluctuations in pH had a minimal impact on growth, productivity, and product quality although some changes in lactate metabolism were observed. DOT fluctuations were found to have a more significant impact; a 35% decrease in cell growth and product titre was observed in the fastest growing cell line tested, while all cell lines exhibited a significant increase in lactate accumulation. Product quality analysis yielded varied results; two cell lines showed an increase in the G0F glycan and decrease in G1F, G2F, and Man5; however, another line showed the opposite trend. The study suggests that the response of CHO cells to the effects of fluctuating culture conditions is cell line specific and that higher growing cell lines are most impacted. The miniature bioreactor system described in this work therefore provides a platform for use during early stage cell culture process development to identify cell lines that may be adversely impacted by the pH and DOT heterogeneities encountered on scale‐up. This experimental data can be combined with computational modeling approaches to predict overall cell culture performance in large‐scale bioreactors.

Biosynthesis of 1,3‐propanodiol and 2,3‐butanodiol from residual glycerol in continuous cell‐immobilized Klebsiella pneumoniae bioreactors

Abstract: In recent years, residual glycerol from biodiesel synthesis made this chemical a cheap, readily available carbon source to bioprocess, which is also a form to reduce costs in the fuel industry. We propose and describe a bioprocess using fluidized and packed‐bed continuous bioreactors to convert this residual glycerol into value‐added products such as 1,3‐propanediol (1,3‐PD) and 2,3‐butanediol (2,3‐BD), largely used in the chemical industry. The bacterium Klebsiella pneumoniae BLh‐1, strain isolated by us, was immobilized in the permeable support of polyvinyl alcohol (LentiKats®). After testing different dilution rates (D) for all bioreactor configurations, the best obtained productivities of 1,3‐PD was 8.69 g L−1 h−1 at a D = 0.45 h−1, and 2.99 g L−1 h−1 at a D = 0.30 h−1 for 2,3‐BD, both in the packed‐bed configuration. In the fluidized‐bed reactor, the highest productivity values achieved were 4.48 and 1.16 g L−1 h−1 for 1,3‐PD and 2,3‐BD, respectively, both at D = 0.33 h−1. These results show the potential of setting up a bioprocess based on continuous cultures using immobilized K. pneumoniae BLh‐1 in PVA matrices in order to efficiently convert the abundant surplus of glycerol into commercially important chemicals such as 1,3‐PD and 2,3‐BD.

Impact of different trace elements on metabolic routes during heterotrophic growth of C. ljungdahlii investigated through online measurement of the carbon dioxide transfer rate.

Abstract: Synthesis gas fermentation using acetogenic clostridia is a rapidly increasing research area. It offers the possibility to produce platform chemicals from sustainable C1 carbon sources. The Wood-Ljungdahl pathway (WLP), which allows acetogens to grow autotrophically, is also active during heterotrophic growth. It acts as an electron sink and allows for the utilization of a wide variety of soluble substrates and increases ATP yields during heterotrophic growth. While glycolysis leads to CO2 evolution, WLP activity results in CO2 fixation. Thus, a reduction of net CO2 emissions during growth with sugars is an indicator of WLP activity. To study the effect of trace elements and ventilation rates on the interaction between glycolysis and the WLP, the model acetogen Clostridium ljungdahlii was cultivated in YTF medium, a complex medium generally employed for heterotrophic growth, with fructose as growth substrate. The recently reported anaRAMOS device was used for online measurement of metabolic activity, in form of CO2 evolution. The addition of multiple trace elements (iron, cobalt, manganese, zinc, nickel, copper, selenium, and tungsten) was tested, to study the interaction between glycolysis and the Wood ljungdahl pathway. While the addition of iron(II) increased growth rates and ethanol production, added nickel(II) increased WLP activity and acetate formation, reducing net CO2 production by 28%. Also, higher CO2 availability through reduced volumetric gas flow resulted in 25% reduction of CO2 evolution. These online metabolic data demonstrate that the anaRAMOS is a valuable tool in the investigation of metabolic responses i.e. to determine nutrient requirements that results in reduced CO2 production. Thereby the media composition can be optimized depending on the specific goal. This article is protected by copyright. All rights reserved.

Examining organic acid production potential and growth‐coupled strategies in Issatchenkia orientalis using constraint‐based modeling

Abstract: Growth‐coupling product formation can facilitate strain stability by aligning industrial objectives with biological fitness. Organic acids make up many building block chemicals that can be produced from sugars obtainable from renewable biomass. Issatchenkia orientalis is a yeast strain tolerant to acidic conditions and is thus a promising host for industrial production of organic acids. Here, we use constraint‐based methods to assess the potential of computationally designing growth‐coupled production strains for I. orientalis that produce 22 different organic acids under aerobic or microaerobic conditions. We explore native and engineered pathways using glucose or xylose as the carbon substrates as proxy constituents of hydrolyzed biomass. We identified growth‐coupled production strategies for 37 of the substrate‐product pairs, with 15 pairs achieving production for any growth rate. We systematically assess the strain design solutions and categorize the underlying principles involved.

Exploring the potential of machine learning for more efficient development and production of biopharmaceuticals

Abstract: Principles of Industry 4.0 direct us to predict how pharmaceutical operations and regulations may exist with automation, digitization, artificial intelligence (AI), and real time data acquisition. Machine learning (ML), a sub‐discipline of AI, involves the use of statistical tools to extract the desired information either through understanding the underlying patterns in the information or by development of mathematical relationships among the critical process parameters (CPPs) and critical quality attributes (CQAs) of biopharmaceuticals. ML is still in its infancy for directly supporting the quality‐by‐design based development and manufacturing of biopharmaceuticals. However, adoption of ML‐based models in place of conventional multi‐variate‐data‐analysis (MVDA) is increasing with the accumulation of large‐scale data. This has been majorly contributed by the real‐time monitoring of process variables and quality attributes of products through the implementation of process analytical technology in biopharmaceutical manufacturing. All aspects of healthcare, from drug design to product distribution, are complex and multidimensional. Thus, ML‐based approaches are being applied to achieve sophistication, accuracy, flexibility and agility in all these areas. This review discusses the potential of ML for addressing the complex issues in diverse areas of biopharmaceutical development, such as biopharmaceuticals design and assessment of early stage development, upstream and downstream process development, analysis, characterization and prediction of post‐translational modifications (PTMs), formulation, and stability studies. Moreover, the challenges in acquisition, cleaning and structuring the bioprocess data, which is one of the major hurdles in implementation of ML in biopharma industry, have also been discussed. Regulatory perspectives on implementation of AI/ML in the biopharma sector have also been briefly discussed. This article is a bird's eye view on the recent developments and applications of ML in overcoming the challenges for adopting “Industry – 4.0” in the biopharma industry.

Dentin regeneration based on tooth tissue engineering: A review

Abstract: Missing or damaged teeth due to caries, genetic disorders, oral cancer, or infection may contribute to physical and mental impairment that reduces the quality of life. Despite major progress in dental tissue repair and those replacing missing teeth with prostheses, clinical treatments are not yet entirely satisfactory, as they do not regenerate tissues with natural teeth features. Therefore, much of the focus has centered on tissue engineering (TE) based on dental stem/progenitor cells to create bioengineered dental tissues. Many in vitro and in vivo studies have shown the use of cells in regenerating sections of a tooth or a whole tooth. Tooth tissue engineering (TTE), as a promising method for dental tissue regeneration, can form durable biological substitutes for soft and mineralized dental tissues. The cell‐based TE approach, which directly seeds cells and bioactive components onto the biodegradable scaffolds, is currently the most potential method. Three essential components of this strategy are cells, scaffolds, and growth factors (GFs). This study investigates dentin regeneration after an injury such as caries using TE and stem/progenitor cell‐based strategies. We begin by discussing about the biological structure of a dentin and dentinogenesis. The engineering of teeth requires knowledge of the processes that underlie the growth of an organ or tissue. Then, the three fundamental requirements for dentin regeneration, namely cell sources, GFs, and scaffolds are covered in the current study, which may ultimately lead to new insights in this field.

Role of membrane structure on the filtrate flux during monoclonal antibody filtration through virus retentive membranes

Abstract: Virus removal filtration is a critical step in the manufacture of monoclonal antibody products, providing a robust size‐based removal of both enveloped and non‐enveloped viruses. Many monoclonal antibodies show very large reductions in filtrate flux during virus filtration, with the mechanisms governing this behavior and its dependence on the properties of the virus filter and antibody remaining largely unknown. Experiments were performed using the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters using a highly purified monoclonal antibody. The filtrate flux for a 4 g/L antibody solution through the Viresolve® Pro decreased by about 10‐fold when the filter was oriented with the skin side down but by more than 1000‐fold when the asymmetric filter orientation was reversed and used with the skin side up. The very large flux decline observed with the skin side up could be eliminated by placing a large pore size prefilter directly on top of the virus filter; this improvement in filtrate flux was not seen when the prefilter was used inline or as a batch prefiltration step. The increase in flux due to the prefilter was not related to the removal of large protein aggregates or to an alteration in the extent of concentration polarization. Instead, the prefilter appears to transiently disrupt reversible associations of the antibodies caused by strong intermolecular attractions. These results provide important insights into the role of membrane morphology and antibody properties on the filtrate flux during virus filtration.

Optimization of an artificial neural network topology using response surface methodology for microbial fuel cell power prediction

Abstract: Microbial fuel cells (MFCs) are among the newest bioelectrical devices that have attracted significant attention because they convert biodegradable organic matter to electricity. MFC design can be improved by understanding and predicting the performance of MFC under different conditions and substrate concentrations. However, few mathematical models have been investigated due to problems caused by the high sensitivity of MFC systems. In this research, a multilayer neural network (NN) was used to predict the generated power of a cell with three inputs (concentration, time, and resistance). Response surface methodology with factors including the number of first layer neurons, number of second layer neurons, training epochs, validation check, and training percentage was used to obtain the optimum structure of the network, and mean squared error (MSE). NN had the minimum MSE when the Number of neurons in the first and second hidden layers, the training epochs, validation check, training percentage were 28, 20, 1000, 100, and 70, respectively. This built network had an excellent ability to predict, and R2 was 98%. According to the results, increasing COD concentration increases generated power and system utilization time. In addition, reducing the external resistance up to 100 Ω can lead to more power obtained.

Development of targeted gene delivery system based on liposome and PAMAM dendrimer functionalized with hyaluronic acid and TAT peptide: In vitro and in vivo studies

Abstract: The development of gene delivery systems is essential to improve their transfection efficiency and cytotoxicity. Combination of lipid and polymeric nanoparticles with the characteristics of both systems have been considered as a next‐generation gene delivery platform. In the current study, we designed a novel and efficient targeted gene delivery system based on liposome and PAMAM dendrimer in cancer cells. Two polymeric formulations containing polyamidoamine‐TAT (PAMAM‐TAT) and PAMAM‐TAT‐Hyaluronic acid (HA) and two lipopolymeric carriers including PAMAM‐TAT‐Liposome and PAMAM‐TAT‐HA‐Liposome were complexed with the enhanced green fluorescent protein (EGFP) plasmid and then evaluated in terms of physicochemical characteristics. The cytotoxicity and transfection efficiency of these synthetized carriers were accomplished against murine colon carcinoma cell line (C26). The biodistribution of polyplexes and lipoployplexes was also evaluated in the C26 tumor bearing mice. The results showed no significant toxicity for all designed nanoparticles (NPs) in C/P4. The highest gene expression was observed using lipopolyplex PAMAM‐TAT‐HA‐Liposome in C/P4 (ratio polymer/DNA; wt/wt). Biodistribution study demonstrated more aggregation of targeted lipopolyplex in tumor cells than other nanoparticles (NPs). It could be concluded that the developed targeted lipopolymeric complex could serve as promising nanotherapeutic system for gene therapy.

Protein adsorption on core‐shell resins for flow‐through purifications: Effect of protein molecular size, shape, and salt concentration

Abstract: This work addresses the functional properties of the core‐shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose‐based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.

Scale‐up issues during sterile filtration of glycoconjugate vaccines

Abstract: Several recent studies have provided important insights into the factors controlling the sterile filtration of glycoconjugate vaccines; however, this work has been limited to small‐scale disk filters with very uniform flow distribution. The objective of this study was to examine the scale‐up of the sterile filtration step using a glycoconjugate drug substance made from a single polysaccharide serotype. Experimental data were obtained during constant flux filtration through 0.22 μm Durapore® polyvinylidene difluoride (PVDF) membranes, both with small discs and with the Opticap® XL2 pleated cartridge. The transmembrane pressure increased rapidly during the glycoconjugate filtration due to membrane fouling, with the rate of pressure increase being more pronounced in the pleated cartridge. Additional insights into the fouling behavior were obtained using confocal microscopy by in situ labeling of the glycoconjugate captured within the filter media using an Alexa Fluor fluorescent dye. Glycoconjugate deposition occurred only within the first 5–15 μm of the 0.22 μm Durapore® membrane at both scales, with more variability in the deposition pattern observed for the pleated filter due to the non‐uniform flow distribution in the Opticap® XL2 cartridge. These results provide important insights into the underlying fouling behavior during sterile filtration of glycoconjugate vaccines as well as a framework for the scale‐up of the sterile filter step in glycoconjugate biomanufacturing.

Development of virus‐like particles‐based vaccines against coronaviruses

Abstract: Severe acute respiratory syndrome coronavirus (SARS‐CoV), Middle East respiratory syndrome coronavirus (MERS‐CoV), and the current severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are the most impactful coronaviruses in human history, especially the latter, which brings revolutionary changes to human vaccinology. Due to its high infectivity, the virus spreads rapidly throughout the world and was declared a pandemic in March 2020. A vaccine would normally take more than 10 years to be developed. As such, there is no vaccine available for SARS‐CoV and MERS‐CoV. Currently, 10 vaccines have been approved for emergency use by World Health Organization (WHO) against SARS‐CoV‐2. Virus‐like particle (VLP)s are nanoparticles resembling the native virus but devoid of the viral genome. Due to their self‐adjuvanting properties, VLPs have been explored extensively for vaccine development. However, none of the approved vaccines against SARS‐CoV‐2 was based on VLP and only 4% of the vaccine candidates in clinical trials were based on VLPs. In the current review, we focused on discussing the major advances in the development of VLP‐based vaccine candidates against the SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2, including those in clinical and pre‐clinical studies, to give a comprehensive overview of the VLP‐based vaccines against the coronaviruses.

New technical concept for alternating tangential flow filtration in biotechnological cell separation processes

Abstract: Robust cell retention devices are key to successful cell culture perfusion. Currently, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) are most commonly used for this purpose. TFF, however, suffers from poor fouling mitigation, which leads to high filtration resistance and product retention, and ATF suffers from long residence times and cell accumulation. In this work, we propose a filtration system for alternating tangential flow filtration, which takes full advantage of the fouling mitigation effects of alternating flow and reduces cell accumulation. We have tested this novel setup in direct comparison with the XCell ATF® as well as TFF with a model feed comprising yeast cells and bovine serum albumin as protein at harsh permeate to feed flow conditions. We found that by avoiding the dead‐end design of a diaphragm pump, the proposed filtration system exhibited a reduced filtration resistance by approximately 20% to 30% (depending on feed rate and permeate flow rate). A further improvement of the novel setup was reached by optimization of phase durations and flow control, which resulted in a fourfold extension of process duration until hollow fiber flow channel blockage occurred. Thus, the proposed concept appears to be superior to current cell retention devices in perfusion technology.

Toward optimal clearance: A universal affinity‐based mass spectrometry approach for comprehensive ELISA reagent coverage evaluation and HCP hitchhiker analysis

Abstract: In the control strategy for process related impurities in biopharmaceuticals, the enzyme linked immunosorbent assay (ELISA) is the method of choice for the quantification of host cell proteins (HCPs). Besides two dimensional‐western blots (2D‐WB), the coverage of ELISA antibodies is increasingly evaluated by affinity purification‐based liquid chromatography–tandem mass spectrometry (AP‐MS) methods. However, all these methods face the problem of unspecific binding issues between antibodies and the matrix, involving the application of arbitrarily defined thresholds during data evaluation. To solve this, a new approach (optimized AP‐MS) was developed in this study, for which a cleavable linker was conjugated to the ELISA antibodies enabling the subsequent isolation of specifically interacting HCPs. By comparing both approaches in terms of method variability and the number of false positive or negative hits, we could demonstrate that the optimized AP‐MS method is very reproducible and superior in the identification of antibody detection gaps, while previously described strategies suffered from over‐ or underestimating the coverage. As only antibody associated HCPs were identified, we demonstrated that the method is beneficial for hitchhiker analysis. Overall, the method described herein has proven as a powerful tool for reliable coverage determination of ELISA antibodies, without the need to arbitrarily exclude HCPs during the coverage evaluation.

Comprehensive studies on building a scalable downstream process for mRNAs to enable mRNA therapeutics

Abstract: In recent years, mRNA‐based therapeutics have been a fast‐growing new class of biologics that can, in principle, encode any protein(s) directly in patients to treat various diseases. mRNA vaccines have been proven to work efficiently, have high potency, and can be rapidly developed and deployed, which is critical for a quick responses in the case of a pandemic. Such agile development is enabled by rapid synthesis of RNA in vitro using recombinant enzymes rather than relying on lengthy and complex cell culture processes. mRNA exhibits physical and chemical properties differing from protein‐based therapeutics. It is highly negatively charged and the hydroxyl group makes mRNA less stable and more susceptible to hydrolysis and nucleophilic cleavage. This novel work shares comprehensive studies carried out to compare the performance of various mRNA purification strategies by considering its scalability and critical quality attributes. In addition, the paper provides insights on how to establish a scalable mRNA purification process that consists of ultrafiltration/diafiltration and chromatography steps with good recoveries. Alternative Oligo(dT) based columns were further explored aiming to improve total process recovery. With Oligo(dT) as a capture step, overall recoveries of 70% can be achieved for mRNAs studied here that encode anti‐influenza immunoglobulin G monoclonal antibodies.

Application of Ambr15 system for simulation of entire SARS‐CoV‐2 vaccine production process involving macrocarriers

Abstract: The Ambr15 system is an automated, high‐throughput bioreactor platform which comprises 24 individually controlled, single‐use stirred‐tank reactors. This system plays a critical role in process development by reducing reagent requirements and facilitating high‐throughput screening of process parameters. However, until now, the system was used to simulate processes involving cells in suspension or growing on microcarriers and has never been tested for simulating cells growing on macrocarriers. Moreover, to our knowledge, a complete production process including cell growth and virus production has never been simulated. Here, we demonstrate, for the first time, the amenability of the automated Ambr15 cell culture reactor system to simulate the entire SARS‐CoV‐2 vaccine production process using macrocarriers. To simulate the production process, accessories were first developed to enable insertion of tens of Fibra‐Cel macrocarries into the reactors. Vero cell adsorption to Fibra‐Cels was then monitored and its adsorption curve was studied. After incorporating of all optimized factors, Vero cells were adsorbed to and grown on Fibra‐Cels for several days. During the process, culture medium was exchanged, and the quantity and viability of the cells were followed, resulting in a typical growth curve. After successfully growing cells for 6 days, they were infected with the rVSV‐ΔG‐Spike vaccine virus. The present results indicate that the Ambr15 system is not only suitable for simulating a process using macrocarriers, but also to simulate an entire vaccine production process, from cell adsorption, cell growth, infection and vaccine virus production.

Copper impurity of iron raw material contributes to improved cell culture performance

Abstract: Cell culture medium (CCM) formulations are chemically defined to reduce lot‐to‐lot variability and complexity of the medium while still providing all essential nutrients supporting cell growth and productivity of various cell lines. However, raw material impurities may still introduce variations and inconsistencies to final CCM formulations. In one of our previous studies (Weiss et al. Biotechnol Prog. 2021;37(4):e3148), we have demonstrated the impact of iron raw material impurity on Chinese hamster ovary (CHO) cell performance and critical quality attributes (CQAs) of recombinant proteins within the Cellvento® 4CHO CCM platform by identifying manganese impurity as the main root cause for improved cell performance and altered glycosylation profiles. This study sought to investigate the impact of iron raw material impurities within another medium platform, namely EX‐CELL® Advanced CHO Fed‐Batch‐Medium. As opposed to previously published results, in this platform, copper instead of manganese impurity present within the used ferric ammonium citrate (FAC) iron source was responsible for an improved cell performance of a CHOZN® cell line and a slight difference in CQAs of the produced recombinant protein. The use of tightly controlled raw material specifications or the use of low impurity iron sources is therefore crucial to minimize the impact of impurities on cell performance in any CCM platform and thereby guarantee consistent and reproducible cell culture processes.

Transfer of continuous manufacturing process principles for mAb production in a GMP environment: A step in the transition from batch to continuous

Abstract: Implementation of continuous in lieu of batch upstream processing (USP) and downstream process (DSP) for the production of recombinant therapeutic protein is a significant paradigm change. The present report describes how the first kilograms of monoclonal antibody were produced with equipment originally designed for batch operations while using continuous manufacturing processes and principles. Project timelines for the delivery of clinical material have driven this ambition and helped the transition. Nevertheless, because of equipment availability, a tradeoff between the envisaged continuous downstream process (cDSP) operations and the ones described in this article had to be taken. A total of 2.1 kg of monoclonal antibody were produced in two GMP runs for clinical trials. For USP, a 200‐L single‐use pilot scale bioreactor was upgraded to enable perfusion operation. DSP steps were designed to be easily transferable to cDSP for later clinical or commercial productions. An in‐line conditioning buffer preparation strategy was tested in a discontinuous way to prove its efficiency and the purification cascade was structured in parallel to the continuous collection of antibody‐containing cell culture supernatant. This strategy will avoid any process change when later moving to the continuous equipment that is currently under qualification. Alignment between small‐scale references runs and the GMP runs in terms of productivity and quality confirmed that the presented approach was valid. Thus, we demonstrate that existing fed‐batch infrastructure can be adapted to continuous manufacturing without significant additional investments. Such approach is useful to evaluate next‐generation manufacturing processes before making large investments.

Understanding the interaction of proteins to ion exchange chromatographic supports: A surface energetics approach

Abstract: Ion exchange chromatography is one of the most widely used chromatographic technique for the separation and purification of important biological molecules. Due to its wide applicability in separation processes, a targeted approach is required to suggest the effective binding conditions during ion exchange chromatography. A surface energetics approach was used to study the interaction of proteins to different types of ion exchange chromatographic beads. The basic parameters used in this approach are derived from the contact angle, streaming potential, and zeta potential values. The interaction of few model proteins to different anionic and cationic exchanger, with different backbone chemistry, that is, agarose and methacrylate, was performed. Generally, under binding conditions, it was observed that proteins having negative surface charges showed strong to lose interaction (20 kT for Hannilase to 0.5 kT for IgG) with different anionic exchangers (having different positive surface charges). On the contrary, anionic exchangers showed almost no interaction (0–0.1 kT) with the positively charged proteins. An inverse behavior was observed for the interaction of proteins to cationic exchangers. The outcome from these theoretical calculations can predict the binding behavior of different proteins under real ion exchange chromatographic conditions. This will ultimately propose a better bioprocess design for protein separation.

Design and optimization of membrane chromatography for monoclonal antibody charge variant separation

Abstract: The manufacturing scale implementation of membrane chromatography to purify monoclonal antibodies has gradually increased with the shift in industry focus toward flexible manufacturing and disposable technologies. Membrane chromatography are used to remove process‐related impurities such as host cell proteins (HCPs) and DNA, leachates, and endotoxins, with improved productivity and process flexibility. However, application of membrane chromatography to separate product‐related variants such as charge variants has not gained major traction due to low‐binding capacity. The work reported here demonstrates that a holistic process development strategy to optimize static binding (pH and salt concentration) and dynamic process (membrane loading, flowrate, and gradient length) parameters can alleviate the capacity limitations. The study employed high throughput screening tools and scale‐down membranes for intermediate and polishing purification of the model monoclonal antibody. An optimized process consisting of anion exchange and cation exchange membrane chromatography reduced the acidic variants present in Protein A eluate from 89.5% to 19.2% with 71% recovery of the target protein. The membrane chromatography process also cleared HCP to below limit of detection with 6‐ to 30‐fold higher membrane loading, compared to earlier reported values. The results confirm that membrane chromatography is effective in separating closely related product variants when supported by a well‐defined process development strategy.

Generation of a host cell line containing a MAR‐rich landing pad for site‐specific integration and expression of transgenes

Abstract: In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix‐Attachment Region (MAR)‐rich landing pads (LPs), which allow for the simultaneous and site‐specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR‐based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP‐containing host cell lines can be used for the site‐specific integration of several GOIs and thus, generation of transgene‐expressing stable recombinant clones. Transgene expression was shown by site‐specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi‐specific antibody (bsAb). The genetic stability of the herein described LP‐based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR‐rich landing pads can be successfully used for stable expression of one or several GOIs.

Evaluation of lentiviral vector stability and development of ion exchange purification processes

Abstract: In this manuscript, we employ parallel batch stability and chromatographic screens in concert with linear and step gradient experiments to develop a high yield, HCP clearance anion exchange capture process for lentiviral vector (LVV) purification. An initial broad resin screen is carried out to determine anion exchange‐based resins that exhibit high recovery of LVV. LVV stability is then evaluated and conditions are established where the vector exhibits good stability, namely phosphate buffer at pH 6.5–7.5, with low to moderate salt concentrations. A subsequent high‐throughput batch screen is then carried out with a subset of resins selected from the first screen under stable conditions to identify optimal wash and elution steps to further improve product yield and protein clearance. Linear gradient experiments are also conducted in mini‐column format to refine the operating conditions and final step gradient processes are established that exhibit greater than 70% yield of infectious LVV while also achieving up to 2.89 log reduction values (LRV) of HCPs during the process. The large set of stability and chromatographic data provided in this work represent an important contribution to knowledge in the field about the chromatographic efficacy of a wide range of resins for LVV bioprocessing under stable conditions.