Biopharmaceutical Engineering Research Articles

Development and implementation of the course entitled "Virtual Simulation Experiment of Recombinant Human Erythropoietin Manufacturing Process"

Considering the issues present in traditional learning methods of manufacturing process for biotechnology majors, this paper presents the development and implementation process of the course entitled "Virtual Simulation Experiment of Recombinant Human Erythropoiesis Manufacturing Process". The experiment combines modern biological manufacturing technology and three-dimensional information technology, with recombinant human erythropoiesis drug serving as the focal point. This paper elaborates on the teaching concepts, objectives, contents, implementation methods, experimental procedures, interactive steps, and assessment criteria used in the experiment. Through innovative experimental scheme design, teaching methodologies, and evaluation systems, this course aims to cultivate students' analytical and problem-solving skills in the field of biopharmaceutical engineering, while also broadening students' perspective and expanding their vision.

Genome‐scale metabolic models applied for human health and biopharmaceutical engineering

AbstractOver the last 15 years, genome‐scale metabolic models (GEMs) have been reconstructed for human and model animals, such as mouse and rat, to systematically understand metabolism, simulate multicellular or multi‐tissue interplay, understand human diseases, and guide cell factory design for biopharmaceutical protein production. Here, we describe how metabolic networks can be represented using stoichiometric matrices and well‐defined constraints for flux simulation. Then, we review the history of GEM development for quantitative understanding of Homo sapiens and other relevant animals, together with their applications. We describe how model develops from H. sapiens to other animals and from generic purpose to precise context‐specific simulation. The progress of GEMs for animals greatly expand our systematic understanding of metabolism in human and related animals. We discuss the difficulties and present perspectives on the GEM development and the quest to integrate more biological processes and omics data for future research and translation. We truly hope that this review can inspire new models developed for other mammalian organisms and generate new algorithms for integrating big data to conduct more in‐depth analysis to further make progress on human health and biopharmaceutical engineering.

Plant-based biopharmaceutical engineering.

Plants can be engineered to recombinantly produce high-quality proteins such as therapeutic proteins and vaccines, also known as molecular farming. Molecular farming can be established in various settings withminimal cold-chain requirements and could thus ensure rapid and global-scale deployment of biopharmaceuticals, promoting equitable access to pharmaceuticals. State of the artplant-based engineering relies on rationally assembled genetic circuits, engineered to enable the high-throughput and rapid expression of multimeric proteins with complex post-translational modifications. In this Review, we discuss the design of expression hosts and vectors, including Nicotiana benthamiana, viral elements and transient expression vectors, for the production of biopharmaceuticals in plants. We examineengineering ofpost-translational modifications and highlight the plant-based expression of monoclonalantibodies and nanoparticles, such as virus-like particles and protein bodies. Techno-economic analyses suggest a cost advantage of molecular farming compared with mammalian cell-based protein production systems. However, regulatory challenges remain to be addressed to enable the widespread translation of plant-based biopharmaceuticals.

BOPPPS teaching method-based effective online teaching strategies and practices for Biopharmaceutical Engineering

Due to the COVID-19 pandemic, the teaching of Biopharmaceutical Engineering course was carried out online and completed with satisfactory outcomes. In order to improve the efficiency of online teaching, ensure the substantive equivalence between online and offline teaching and achieve effective teaching, this article summarized the exploration and practical experience of online teaching, taking the Biopharmaceutical Engineering course as an example. This includes analysis of learner characteristics, selection of online teaching platform, development of teaching resources, optimization of teaching contents, BOPPPS teaching method-based design of teaching structure, and reflection of effective teaching. This paper is expected to provide a useful reference for online teaching.

(Invited) Using Cell Lensing and Nanosensor Chemical Cytometry to Characterize Immune Cell Populations

Recently, we developed a method called Nanosensor Chemical Cytometry (NCC), where an optical nanosensor array embedded within microfluidics can be used to interrogate chemical species efflux from individual cells. The cell itself was found to perform as an informative Gaussian lens, projecting both the nIR emission of the single-walled carbon nanotube sensors as well as various cellular physical properties. We found NCC to be able to profile immune heterogeneities at attomolar sensitivity in a completely non-destructive and real-time manner with rate of ~600 cells/hr.Macrophages are a critical part of the human immune response, and their collective heterogeneity is implicated in disease progression and prevention. Here, we use the NCC technique to profile the heterogeneity of inducible nitric oxide synthase (iNOS) responses from macrophage populations. Parameters measured nitric oxide (NO) efflux and refractive index (RI) changes at a single-cell level. Using NCC, NO was measured with (10−18 mol) sensitivity in a nondestructive and real-time manner with a throughput of exceeding the 200 cells/frame. With lipopolysaccharide (LPS) activation, NO efflux elevated from 342 (σ = 199) to 464 (σ = 206) attomol/cell·hr, in agreement with previously reported Griess assay results. This work provides an efficient strategy for chemical analysis of cell populations for manufacturing and biopharmaceutical engineering.

Application of CDIO Mode in the Teaching of Biopharmaceutical Engineering

Due to the accelerated development of social science and technology, the development of modern science and technology is in urgent need of a large number of innovative scientific and technological talents, and the CDIO teaching mode based on the combination of practice and theory is receiving more and more attention in undergraduate teaching. CDIO teaching mode is a new education mode that has achieved remarkable results in the current higher engineering education. It always aims to train a new generation of international modern engineers. This paper introduces the proposal, background and application of CDIO, and embodies the effect of combining with the talent training program of biopharmaceutical engineering.

Solid-phase extraction methods for nucleic acid separation. A review.

The separation and purification of biomacromolecules such as nucleic acid is a perpetual topic in separation processes and bioengineering (fine chemicals, biopharmaceutical engineering, diagnostics, and biological characterization). In principle, the solid-phase extraction for nucleic acid exhibits efficient phase separation, low pollution risk, and small sample demand, compared to the conventional liquid-phase extraction. Herein, solid-phase extraction methods are systematically reviewed to outline research progress and explore additional solid-phase sorbents and devices for novel, flexible, and high-efficiency nucleic acid separation processes. The functional materials capture nucleic acid, magnetic and magnetic-free solid-phase extraction methods, separation device design and optimization, and high-throughput automatable applications based on high-performance solid-phase extraction are summarized. Finally, the current challenges and promising topics are discussed.

Nanosensor Chemical Cytometry for Characterizing the Efflux Heterogeneity of Nitric Oxide from Macrophages.

Macrophages are a critical part of the human immune response, and their collective heterogeneity is implicated in disease progression and prevention. A nondestructive, label-free tool does not currently exist for profiling the dynamic, antigenic responses of single macrophages in a collection to correlate with specific molecular expression and correlated biophysical properties at the cellular level, despite the potential for diagnosis and therapeutics. Herein, we develop a nanosensor chemical cytometry (NCC) that can profile the heterogeneity of inducible nitric oxide synthase (iNOS) responses from macrophage populations. By integrating a near-infrared (nIR) fluorescent nanosensor array and collagen layer with microfluidics, the cellular lensing effect of the macrophage was utilized to characterize both nitric oxide (NO) efflux and refractive index (RI) changes at a single-cell level. Using a parallel, multichannel approach, distinct iNOS heterogeneities of macrophages can be monitored at an attomolar (10-18 mol) sensitivity in a nondestructive and real-time manner with a throughput of exceeding the 200 cells/frame. We demonstrate that estimated mean NO efflux rates of macrophage populations are elevated from 342 (σ = 199) to 464 (σ = 206) attomol/cell·hr with a 3% larger increase in the heterogeneity, and estimated RI of macrophage decrease from 1.366 (σ = 0.015) to 1.359 (σ = 0.009) with trimodal subpopulations under lipopolysaccharide (LPS) activation. These measured values are also in good agreement with Griess assay results and previously reported measurements. This work provides an efficient strategy for single-cell analysis of macrophage populations for cellular manufacturing and biopharmaceutical engineering.

Insights into the cross-amyloid aggregation of Aβ40 and its N-terminal truncated peptide Aβ11-40 affected by epigallocatechin gallate

Inhibition of protein misfolding and aggregation is a great challenge in the field of biochemical and biopharmaceutical engineering. Alzheimer's disease (AD) is a protein-misfolding disease, and the interactions between 40-amino-acid-residue β-amyloid peptide (Aβ40) and its N-terminal truncated peptide Aβ11-40 demonstrate that Aβ11-40 may play an important role in the pathological process of AD. However, the effect of inhibitors on Aβ11-40 aggregation and on the cross-amyloid aggregation (co-assembly) between Aβ40 and Aβ11-40 has never been studied. Herein, coaggregation and seeding interactions between Aβ40 and Aβ11-40 as well as the effect of epigallocatechin gallate (EGCG), a small molecule inhibitor, on the cross-amyloid aggregation have been investigated by extensive analyses. It is found that Aβ11-40 participates in the aggregation of Aβ40 and leads to the formation of coaggregates that contain less β-sheet structures than pure Aβ40 aggregates. The aggregation kinetics along with morphologies and secondary structures of the coaggregates are also significantly affected by the Aβ40/Aβ11-40 ratio. EGCG accelerates the nucleation of Aβ40 but retards that of Aβ11-40 by affecting their elongation and secondary nucleation processes in solution and on solid surfaces. Meanwhile, EGCG makes the conformations of the seeding-induced Aβ aggregates more compact, especially for the homologous seedings. Isothermal titration calorimetry measurement indicates that hydrophobic interactions mainly contribute to the inhibition of the two Aβ isoforms by EGCG. The findings of this research have provided new insights into Aβ aggregation and the effect of an important inhibitor and the results would benefit in the development of potent inhibitors against co-assembly of different amyloid proteins.

The potential of ionic liquids in biopharmaceutical engineering

• General criterions for ILs in biopharmaceutical applications are discussed. • Stabilization roles and mechanisms of ILs in biopharmaceuticals are described. • Application of ILs in transdermal and oral delivery of biopharmaceuticals are summarized. • Potential applications of ILs in the design of vaccine adjuvants are prospected. • Challenges of ILs in biopharmaceutical engineering are discussed. Biopharmaceuticals, such as proteins, peptides, nucleic acids and vaccines, bring about great hopes for the prevention and treatment of various diseases, but the industrialization of these products still faces challenges such as structural instability, inefficient bioactivity and low bioavailability. Ionic liquids (ILs), the marvelous solvent media with inimitable and tunable properties, may provide alternative solutions to overcome the above problems of biopharmaceutical industry. Progress has gradually been made through studies by combination of ILs with biomacromolecules. The applications involved the stabilization, protection, and delivery of biopharmaceuticals. Recent trends are being forwarded to using ILs in vaccines and nucleic acid drugs. However, challenges remain on the toxicity and safety issues. Besides, the cost of adding ILs to the benefits of biopharmaceuticals need to be considered.

Flow & Segregation of Granular Materials Out of Storage and Flowing Vessels: Application in Food Science & Technology and in Bio-Pharmaceutical Engineering

Flow & Segregation of Granular Materials Out of Storage and Flowing Vessels: Application in Food Science & Technology and in Bio-Pharmaceutical Engineering

HMEJ-mediated efficient site-specific gene integration in chicken cells

BackgroundThe production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species.ResultsHere we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system in chicken DF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in in chicken PGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 in chicken PGCs through the insertion of a fluorescent protein in the frame at the 3′ end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chicken eggs, suggested its good promise for the mass production of protein with pharmaceutical importance using the chicken oviduct system.ConclusionsTaken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration in chicken PGCs, which holds great potential for the biopharmaceutical engineering of chicken cells.

Forced Degradation Testing as Complementary Tool for Biosimilarity Assessment.

Oxidation of monoclonal antibodies (mAbs) can impact their efficacy and may therefore represent critical quality attributes (CQA) that require evaluation. To complement classical CQA, bevacizumab and infliximab were subjected to oxidative stress by H2O2 for 24, 48, or 72 h to probe their oxidation susceptibility. For investigation, a middle-up approach was used utilizing liquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS). In both mAbs, the Fc/2 subunit was completely oxidized. Additional oxidations were found in the light chain (LC) and in the Fd’ subunit of infliximab, but not in bevacizumab. By direct comparison of methionine positions, the oxidized residues in infliximab were assigned to M55 in LC and M18 in Fd’. The forced oxidation approach was further exploited for comparison of respective biosimilar products. Both for bevacizumab and infliximab, comparison of posttranslational modification profiles demonstrated high similarity of the unstressed reference product (RP) and the biosimilar (BS). However, for bevacizumab, comparison after forced oxidation revealed a higher susceptibility of the BS compared to the RP. It may thus be considered a useful tool for biopharmaceutical engineering, biosimilarity assessment, as well as for quality control of protein drugs.

Evaluation of Polyphenolic Compounds and Pharmacological Activities in Hairy Root Cultures of Ligularia fischeri Turcz. f. spiciformis (Nakai).

A considerable amount of bioactive compounds have been used for the biopharmaceutical engineering to help human health and nutrition. Hairy root culture (HRC) or transgenic root is a favourable alternative technique for phytochemical production. Ligularia fischeri is a significant source of pharmaceutically important active compounds with an enormous range of health care applications. HRC of L. fischeri was developed using Agrobacterium rhizogenes for the production of polyphenolic compounds with antioxidant, antimicrobial, antidiabetic, anticancer and anti-inflammatory pharmaceutical activities. Hairy roots (HRs) were selected by morphological assessment, genetic and molecular analyses. The maximum accumulation of fresh mass (94.15 g/L) and dry mass (9.45 g/L) was recorded in MS liquid medium supplemented with 30 g/L sucrose at 28 days. Furthermore, HRs successfully produced numerous polyphenolic compounds, including six hydroxycinnamic acids, seven flavonols, seven hydroxybenzoic acids, vanillin, resveratrol, pyrogallol, homogentisic, and veratric acids, which were identified by UHPLC analysis. HRs produced higher total phenolic (185.65 mg/g), and flavonoid (5.25 mg/g) contents than non-transformed roots (125.55 mg/g and 3.75 mg/g). As a result of these metabolic changes, pharmaceutical activities were found higher in HRs than non-transformed roots (NTRs). The present study indicates that HRC has the potential to increase the content of beneficial polyphenolic compounds with higher potential pharmaceutical activities. To the best of our knowledge, the present study is the first report on enhancing the production of polyphenolic compounds with pharmaceutical activities from the HRCs of L. fischeri.

Curriculum design for training applied students of biopharmaceutical engineering based on the product life cycle

Biopharmaceutical engineering is a newly established undergraduate degree in China, and a lot of issues regarding students’ training remain to be explored and refined, particularly the curriculum reflecting the industrial needs for students with applied (hands-on) skills, which has been limited. To narrow the gap between industrial demands and students’ training and to provide undergraduates with rational curriculum, in the present investigation, we proposed to reconstruct the curriculum system for applied (hands-on) training of undergraduates of biopharmaceutical engineering based on the life cycle of medicinal products for the first time. A total of 52 subjects on the curriculum were selected and included in different stages of the medicinal products life cycle. After surveying from a recruitment website and biopharmaceutical enterprises, eight applied positions, including drug screening engineer, cell culture engineer, molecular biology technician, fermentation engineer, microorganism engineer, protein purification engineer, protein analysis technician and bio-preparation engineer, were then selected. According to the requirements of each position, 31 subjects were finally chosen from the above-mentioned 52 subjects that are involved in the life cycle of medicinal products. These subjects belong to four clusters and constitute the basis of the curriculum system for applied training of undergraduates of biopharmaceutical engineering. The results obtained from this work are expected to provide evidence for the training of biopharmaceutical students as well as the implementation of collaborative education between university and industry.

Optimization of a Coupling Process for Insulin Degludec According to a Quality by Design (QbD) Paradigm.

This case study described a successful application of the quality by design (QbD) principles to a coupling process development of insulin degludec. Failure mode effects analysis (FMEA) risk analysis was first used to recognize critical process parameters (CPPs). Five CPPs, including coupling temperature (Temp), pH of desB30 solution (pH), reaction time (Time), desB30 concentration (Conc), and molar equivalent of ester per mole of desB30 insulin (MolE), were then investigated using a fractional factorial design. The curvature effect was found significant, indicating the requirement of second-order models. Afterwards, a central composite design was built with an augmented star and center points study. Regression models were developed for the CPPs to predict the purity and yield of predegludec using above experimental data. The R2 and adjusted R2 were higher than 96 and 93% for the two models respectively. The Q2 values were more than 80% indicating a good predictive ability of models. MolE was found to be the most significant factor affecting both yield and purity of predegludec. Temp, pH, and Conc were also significant for predegludec purity, while Time appeared to remarkably influence the yield model. The multi-dimensional design space and normal operating region (NOR) with a robust setpoint were determined using a probability-based Monte-Carlo simulation method. The verified experimental results showed that the design space was reliable and effective. This study enriches the understanding of acetylation process and is instructional to other complicated operations in biopharmaceutical engineering.

The Leaves of Aronia melanocarpa L. and Hippophae rhamnoides L. as Source of Active Ingredients for Biopharmaceutical Engineering

Abstract Linking Europe and Central Asia, Romania is considered to be an important meeting point between different biogeographic regions and ecosystems. In this context, Romanian flora is truly an exceptional source of biological active phytocompounds, basis of new biopharmaceuticals. Data indicate that in Romania there are about 3600 higher plants (ferns and flowering plants) that grow wild and other 800 hybrid species. Amongst these, about 800 are medicinal species, some of them unique in Europe. In order to demonstrate their potential use as active ingredients in biopharmaceutical engineering, this work was aimed at revealing chemical composition, particularly polyphenols profile (HPTLC method) and antioxidant activity (DPPH method) face to potential dermal benefits (single and repeated dose tolerance test in rabbits) of two (ethanolic) extracts prepared from some less studied plant pieces of two valuable phytomedicines; leaves of chokeberries (Aronia melanocarpa L.) and leaves of sea-buckthorn (Hippophae rhamnoides L.). Analytical studies have shown the occurrence of numerous polyphenols species, quercetin, gallic and caffeoylquinic acid derivates, known to have a high antioxidant potency. DPPH assay has confirmed high antioxidant activity of both vegetal extracts, even stronger than that of references, rutin and gallic acid phenolics (IC50 ranging from 2.66 to 5.08 μg / mL). In vivo, pharmacological studies have indicated good dermal tolerance and healthy, supple and reepitelized tegument after being treated with both vegetal extracts, which has revealed their potential use as antioxidant and healing ingredients for (topic) biopharmaceuticals. Thus, while Aronia melanocarpa L. as well as Hippophae rhamnoides L. are known for their valuable fruit and seed derived products, this study proves that the (ethanolic) extracts prepared from their leaves pieces should also be taken into consideration.