Recent Trends and Technological Developments in Biopharmaceuticals Manufacturing

A special issue of Life (ISSN 2075-1729). This special issue belongs to the section "Pharmaceutical Science".

Deadline for manuscript submissions: closed (30 July 2021) | Viewed by 26909

Special Issue Editors


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Chief Guest Editor
CICECO - Aveiro Institute of Materials, Chemistry Department, University of Aveiro, Aveiro, Portugal
Interests: biopharmaceuticals; recombinant production; fermentation; downstream processing; aqueous biphasic systems; chromatography; ionic liquids; biopharmaceuticals stabilization

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Guest Editor
CICECO - Aveiro Institute of Materials, Chemistry Department, University of Aveiro, Aveiro, Portugal
Interests: separation processes; ionic liquids; deep eutectic solvents; (bio)pharmaceuticals; biomarkers; purification; stability
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Guest Editor
CICS-UBI—Health Science Research Centre, University of Beira Interior, 6200-506 Covilha, Portugal
Interests: biopharmaceuticals; recombinant production; downstream processing; chromatography; gene therapy; gene silencing; health biotechnology
Special Issues, Collections and Topics in MDPI journals

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Guest Editor
Centre for Mechanical Engineering, Materials and Processes, Department of Chemical Engineering, University of Coimbra, 3030-790 Coimbra, Portugal
Interests: biopharmaceuticals; upstream and downstream processing; nucleic acids; chromatography; formulation; gene tehrapy; gene regulation; molecular biology; biological activity

Special Issue Information

Dear Colleagues, 

The advent of biopharmaceuticals in current medicine brought enormous benefits to the treatment of life-threatening human diseases (e.g., cancer, diabetes and neurodegenerative disorders), and improved the well-being of many people worldwide. The global portfolio of these therapeutic products include proteins and antibodies, nucleic acids, and cell-based products, and continues to expand at a rapid pace - approvals in the period 2015-2018 essentially double the typical five-yearly historical approval pace (G. Walsh, Nat. Biotechnol., 36:1136-1145, 2018) -, representing a significant share of the entire market of pharmaceuticals.

Innovation in the (bio)pharmaceutical industry has been driven towards the development of cost-effective manufacturing processes, envisaging the delivery of products in high quantity, with superior quality (purity), and high specificity, with the ultimate goal of benefiting patients. Progress in this direction have resulted from the application of novel technologies in the upstream stage (high-throughput, single-use devices, statistical optimization of media and fermentation conditions, QbD, and continuous processing), while at the downstream level, chromatography has evolved through the development of new resins and ligands, coupled with advances in process modelling, operating and control strategies.

An emerging trend is the application of alternative solvents such as ionic liquids and deep eutectic solvents, in which their structure and physicochemical properties can be tuned to address unmet needs in (bio)pharmaceutical research. These compounds may be derived from natural and reneawable sources and hold great promise in the development of efficient, sustainable and cost-effective biopharmaceuticals purification processes.

This Special Issue of MDPI Life aims to provide the latest progresses achieved in pharmaceuticals bioprocessing. We welcome submissions of original research, comprehensive reviews and perspectives, including, but not limited, to the following fields:

- Upstream processing (genetic engineering, systems biology, difficult-to-express proteins, expression conditions, Quality by Design approaches, process analytical technologies);

- Chromatographic purification methods (process modelling and control, continuous bioprocessing, design and characterization of resins and ligands, new formats);

- Alternative purification methods (aqueous biphasic systems, filtration, crystallization, precipitation);

- Application of neoteric solvents in upstream and downstream stages;

- Analytical characterization of biopharmaceuticals (stability, post-translational modifications, biological activity, immunogenicity); 

Dr. Augusto Quaresma Henriques Pedro
Prof. Dr. Mara Guadalupe Freire Martins
Prof. Dr. Fani Pereira de Sousa
Dr. Patrícia Alexandra Nunes Pereira
Guest Editors

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Keywords

  • biopharmaceuticals
  • technological advances
  • process control
  • new solvents and ligands/resins
  • purification
  • stability

Published Papers (6 papers)

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Research

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16 pages, 1436 KiB  
Article
Initial Screening of Poly(ethylene glycol) Amino Ligands for Affinity Purification of Plasmid DNA in Aqueous Two-Phase Systems
by Nuno R. da Silva, Paula Jorge, José A. Martins, José A. Teixeira and João C. Marcos
Life 2021, 11(11), 1138; https://doi.org/10.3390/life11111138 - 26 Oct 2021
Cited by 4 | Viewed by 2222
Abstract
Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, [...] Read more.
Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEG–amine) or conjugation with positively charged amino acids (PEG–lysine, PEG–arginine, and PEG–histidine) were studied to increase the selectivity of PEG–dextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEG–dextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination. Full article
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16 pages, 17579 KiB  
Article
Efficient Isolation of Bacterial RNAs Using Silica-Based Materials Modified with Ionic Liquids
by Patrícia Pereira, Augusto Q. Pedro, Márcia C. Neves, João C. Martins, Inês Rodrigues, Mara G. Freire and Fani Sousa
Life 2021, 11(10), 1090; https://doi.org/10.3390/life11101090 - 15 Oct 2021
Cited by 4 | Viewed by 2170
Abstract
High quality nucleic acids (with high integrity, purity, and biological activity) have become indispensable products of modern society, both in molecular diagnosis and to be used as biopharmaceuticals. As the current methods available for the extraction and purification of nucleic acids are laborious, [...] Read more.
High quality nucleic acids (with high integrity, purity, and biological activity) have become indispensable products of modern society, both in molecular diagnosis and to be used as biopharmaceuticals. As the current methods available for the extraction and purification of nucleic acids are laborious, time-consuming, and usually rely on the use of hazardous chemicals, there is an unmet need towards the development of more sustainable and cost-effective technologies for nucleic acids purification. Accordingly, this study addresses the preparation and evaluation of silica-based materials chemically modified with chloride-based ionic liquids (supported ionic liquids, SILs) as potential materials to effectively isolate RNAs. The investigated chloride-based SILs comprise the following cations: 1-methyl-3-propylimidazolium, triethylpropylammonium, dimethylbutylpropylammonium, and trioctylpropylammonium. All SILs were synthesized by us and characterized by solid-state 13C Nuclear Magnetic Resonance (NMR), Scanning Electron Microscopy (SEM), elemental analysis, and zeta potential measurements, confirming the successful covalent attachment of each IL cation with no relevant changes in the morphology of materials. Their innovative application as chromatographic supports for the isolation of recombinant RNA was then evaluated. Adsorption kinetics of transfer RNA (tRNA) on the modified silica-based materials were investigated at 25 °C. Irrespective to the immobilized IL, the adsorption experimental data are better described by a pseudo first-order model, and maximum tRNA binding capacities of circa 16 µmol of tRNA/g of material were achieved with silica modified with 1-methyl-3-propylimidazolium chloride and dimethylbutylpropylammonium chloride. Furthermore, the multimodal character displayed by SILs was explored towards the purification of tRNA from Escherichia coli lysates, which in addition to tRNA contain ribosomal RNA and genomic DNA. The best performance on the tRNA isolation was achieved with SILs comprising 1-methyl-3-propylimidazolium chloride and dimethylbutylpropylammonium chloride. Overall, the IL modified silica-based materials represent a more efficient, sustainable, and cost-effective technology for the purification of bacterial RNAs, paving the way for their use in the purification of distinct biomolecules or nucleic acids from other sources. Full article
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13 pages, 2289 KiB  
Article
Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells
by Shang Xiao, Waqas Ahmed, Ali Mohsin and Meijin Guo
Life 2021, 11(9), 945; https://doi.org/10.3390/life11090945 - 10 Sep 2021
Cited by 5 | Viewed by 3206
Abstract
Chinese hamster ovary (CHO) cells are the most important host system used for monoclonal antibody (mAb) expression. Moreover, the fed-batch culture mode is the most widely used method to increase mAb expression in CHO cells by increasing the amount of feed. However, a [...] Read more.
Chinese hamster ovary (CHO) cells are the most important host system used for monoclonal antibody (mAb) expression. Moreover, the fed-batch culture mode is the most widely used method to increase mAb expression in CHO cells by increasing the amount of feed. However, a high amount of culture feed results in the production of metabolic byproducts. In this work, we used a continuous feeding strategy to reduce metabolic byproducts and improve mouse–human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 expression in Chinese hamster ovary-K1 cells. Moreover, the effects of the feeding strategy on the cell culture and monoclonal antibody production were evaluated in chemically defined suspension cultures of recombinant CHO-K1 cells. Compared with bolus feeding methods, the continuous feeding method did not have any advantages when the feeding amount was low, but with a high feeding amount, the continuous feeding method significantly reduced the concentrations of lactate and NH4+ in the later culture stage. At the end of the culture stage, compared with bolus feeding methods, the lactate and NH4+ concentrations under the continuous feeding mode were reduced by approximately 45% and 80%, respectively. In addition, the antibody C12 expression level was also increased by almost 10%. Compared to the bolus feeding method, the antibody C12 produced by the continuous feeding method had a lower content of high-mannose glycoforms. Further analysis found that the osmolality of the continuous feeding method was lower than that of the typical fed-batch bolus feeding method. Conclusively, these results indicate that the continuous feeding method is very useful for reducing metabolic byproducts and achieving higher levels of mAb production. Full article
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23 pages, 47986 KiB  
Article
Strategies for the Production of Soluble Interferon-Alpha Consensus and Potential Application in Arboviruses and SARS-CoV-2
by Felipe Grabarz, Alexandre Paulo Yague Lopes, Flávia Ferreira Barbosa, Giovana Cappio Barazzone, Jademilson Celestino Santos, Viviane Fongaro Botosso, Soraia Attie Calil Jorge, Ana Lucia Tabet Oller Nascimento, Renato Mancini Astray and Viviane Maimoni Gonçalves
Life 2021, 11(6), 460; https://doi.org/10.3390/life11060460 - 21 May 2021
Cited by 3 | Viewed by 4030
Abstract
Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all [...] Read more.
Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2. Full article
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Review

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19 pages, 2598 KiB  
Review
Chromatography-Free Purification Strategies for Large Biological Macromolecular Complexes Involving Fractionated PEG Precipitation and Density Gradients
by Fabian Henneberg and Ashwin Chari
Life 2021, 11(12), 1289; https://doi.org/10.3390/life11121289 - 24 Nov 2021
Cited by 3 | Viewed by 3059
Abstract
A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these [...] Read more.
A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis. As the faithful function of macromolecular complexes is essential for cell survival, their mis-function leads to the development of human diseases. Furthermore, detailed mechanistic interrogation of the function of macromolecular machines can be exploited to develop and optimize biotechnological processes. The purification of intact macromolecular complexes is an essential prerequisite for this; however, chromatographic purification schemes can induce the dissociation of subunits or the disintegration of the whole complex. Here, we discuss the development and application of chromatography-free purification strategies based on fractionated PEG precipitation and orthogonal density gradient centrifugation that overcomes existing limitations of established chromatographic purification protocols. The presented case studies illustrate the capabilities of these procedures for the purification of macromolecular complexes. Full article
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21 pages, 6625 KiB  
Review
Bioprocess Control: Current Progress and Future Perspectives
by Anurag S. Rathore, Somesh Mishra, Saxena Nikita and Priyanka Priyanka
Life 2021, 11(6), 557; https://doi.org/10.3390/life11060557 - 13 Jun 2021
Cited by 43 | Viewed by 10480
Abstract
Typical bioprocess comprises of different unit operations wherein a near optimal environment is required for cells to grow, divide, and synthesize the desired product. However, bioprocess control caters to unique challenges that arise due to non-linearity, variability, and complexity of biotech processes. This [...] Read more.
Typical bioprocess comprises of different unit operations wherein a near optimal environment is required for cells to grow, divide, and synthesize the desired product. However, bioprocess control caters to unique challenges that arise due to non-linearity, variability, and complexity of biotech processes. This article presents a review of modern control strategies employed in bioprocessing. Conventional control strategies (open loop, closed loop) along with modern control schemes such as fuzzy logic, model predictive control, adaptive control and neural network-based control are illustrated, and their effectiveness is highlighted. Furthermore, it is elucidated that bioprocess control is more than just automation, and includes aspects such as system architecture, software applications, hardware, and interfaces, all of which are optimized and compiled as per demand. This needs to be accomplished while keeping process requirement, production cost, market value of product, regulatory constraints, and data acquisition requirements in our purview. This article aims to offer an overview of the current best practices in bioprocess control, monitoring, and automation. Full article
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