Special Issue "Recombinant Glycoproteins"

A special issue of Bioengineering (ISSN 2306-5354).

Deadline for manuscript submissions: closed (28 February 2017).

Special Issue Editors

Prof. Dr. Stephan Hinderlich
Website
Guest Editor
Beuth University of Applied Sciences Berlin, Department of Life Sciences and Technology, Berlin, Germany
Interests: biochemistry of glycans; chemical biology; glycan engineering; glycan analysis; glycobiotechnology; sialic acids; therapeutic glycoproteins
Prof. Dr. Hans Henning Von Horsten
Website
Guest Editor
Hochschule für Technik und Wirtschaft Berlin—University of Applied Sciences Life Science Engineering, Berlin, Germany
Interests: bioprocess engineering; carbohydrate interactions; cell line engineering; glycan engineering; glycobiology; intracellular N-glycan processing; sugar nucleotide metabolism; therapeutic glycoproteins

Special Issue Information

Dear Colleagues,

Recombinant glycoproteins have emerged as a prominent class of pharmaceutically active molecules with an ever increasing number of new therapeutic and preventive indications. Therapeutic antibodies, hormones, cytokines and glycosylated vaccines are produced by mammalian cell culture, a process depending on living cells that is notoriously difficult to control and validate and also inherently prone to cell line and process induced product variations. Of all posttranslational product modifications, glycosylation is the most complex and is well known for its contribution to overall product microheterogeneity. Changes in the glycosylation profile of a product have a prominent impact on drug safety, pharmacodynamics and pharmacokinetics. Therefore, it comes as no surprise that the entire field of glycobiology has gained so much attention from industry.

The current Special Issue emphasizes technical advances in this important field of recombinant glycoproteins, including advances in the field of glycoprotein engineering, process impact on glycoprotein integrity, in-process glycan analysis as well as discoveries pertaining to glycan functional activities. This may include engineering of the protein backbone as well as the attached glycans of a recombinant glycoprotein. Creation of novel recombinant glycoproteins with improved or completely new features, as well as studies aimed at achieving chemically homogenous glycosylation, are also of interest.

We look forward to receiving your contributions to these state-of-the-art issues.

Prof. Dr. Stephan Hinderlich
Prof. Dr. Hans Henning von Horsten
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Bioengineering is an international peer-reviewed open access quarterly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1000 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Glycan engineering
  • Biomanufacturing/bioprocessing
  • Cell line engineering
  • Therapeutic glycoproteins
  • Recombinant monoclonal antibodies
  • Antibody–drug conjugates

Published Papers (6 papers)

Order results
Result details
Select all
Export citation of selected articles as:

Research

Open AccessArticle
In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor
Bioengineering 2017, 4(3), 70; https://doi.org/10.3390/bioengineering4030070 - 15 Aug 2017
Cited by 1
Abstract
Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus [...] Read more.
Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences. Full article
(This article belongs to the Special Issue Recombinant Glycoproteins)
Show Figures

Figure 1

Open AccessArticle
Comparative N-Glycosylation Analysis of the Fc Portions of a Chimeric Human Coagulation Factor VIII and Immunoglobulin G1
Bioengineering 2017, 4(2), 44; https://doi.org/10.3390/bioengineering4020044 - 17 May 2017
Cited by 2
Abstract
Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion [...] Read more.
Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion to Fc-immunoglobulin (Ig)G that mediate protection from clearance or degradation via binding to the neonatal Fc receptor. While human-like N-glycosylation of recombinant FVIII is known to be crucial for the clotting factor’s quality and function, the particular glycosylation of the fused Fc portion has not been investigated in detail so far, despite its known impact on Fcγ receptor binding. Here, we analyzed the N-glycosylation of the Fc part of a chimeric FVIII-Fc protein compared to a commercial IgG1 purified from human plasma. Fc parts from both samples were released by enzymatic cleavage and were subsequently separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Corresponding protein bands were referred to PNGase F in-gel digestion in order to release the respective N-glycans. Analysis via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed structural differences of both N-glycan patterns. Labeling with 2-aminobenzamide (2AB) and analysis via hydrophilic interaction liquid chromatography (HILIC) allowed a quantitative comparison of the respective N-glycosylation. Observed variations in Fc glycosylation of the chimeric FVIII fusion protein and human plasma-derived IgG1, e.g., regarding terminal sialylation, are discussed, focusing on the impact of the clotting factor’s properties, most notably its binding to Fcγ receptors. Full article
(This article belongs to the Special Issue Recombinant Glycoproteins)
Show Figures

Figure 1

Open AccessArticle
Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy
Bioengineering 2017, 4(2), 42; https://doi.org/10.3390/bioengineering4020042 - 08 May 2017
Cited by 3
Abstract
IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG [...] Read more.
IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich) and hematological (CD20) cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated. Full article
(This article belongs to the Special Issue Recombinant Glycoproteins)
Show Figures

Figure 1

Open AccessArticle
Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans
Bioengineering 2017, 4(2), 38; https://doi.org/10.3390/bioengineering4020038 - 28 Apr 2017
Cited by 6
Abstract
Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one [...] Read more.
Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy. Full article
(This article belongs to the Special Issue Recombinant Glycoproteins)
Show Figures

Figure 1

Open AccessArticle
Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC
Bioengineering 2017, 4(2), 34; https://doi.org/10.3390/bioengineering4020034 - 15 Apr 2017
Cited by 3
Abstract
There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a [...] Read more.
There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a mucin which the organism exhibits a distinct tropism for. Stable clonal populations of cells expressing a construct encoding for a truncated version of MUC5AC containing N- and C-termini interspersed with two native tandem repeat sequences (N + 2TR + C) were generated. Binding of H. pylori to protein immunoprecipitated from cell lysates and supernatants was assessed. High molecular weight mucin could be detected in both cell lysates and supernatants of transfected cells. Recombinant protein formed high molecular weight oligomers, was both N and O glycosylated, underwent cleavage similar to native MUC5AC and was secreted from the cell. H. pylori bound better to secreted mucin than intracellular mucin suggesting that modifications on extracellular MUC5AC promoted binding. Lectin analysis demonstrated that secreted mucin was differentially glycosylated compared to intracellular mucin. H. pylori also bound to a recombinant C-terminus MUC5AC protein, but binding to this protein did not inhibit binding to the N + 2TR + C protein. This study demonstrates the feasibility of using recombinant mucins containing tandem repeat sequences to assess microbial mucin interactions. Full article
(This article belongs to the Special Issue Recombinant Glycoproteins)
Show Figures

Graphical abstract

Open AccessArticle
Biotinylated N-Acetyllactosamine- and N,N-Diacetyllactosamine-Based Oligosaccharides as Novel Ligands for Human Galectin-3
Bioengineering 2017, 4(2), 31; https://doi.org/10.3390/bioengineering4020031 - 05 Apr 2017
Cited by 5
Abstract
Galectin inhibitor design is an emerging research field due to the involvement of galectins in cancer. Galectin-3, in particular, plays an important role in tumor progression. To generate inhibitors, modifications of the glycan structure can be introduced. Conjugation of hydrophobic compounds to saccharides [...] Read more.
Galectin inhibitor design is an emerging research field due to the involvement of galectins in cancer. Galectin-3, in particular, plays an important role in tumor progression. To generate inhibitors, modifications of the glycan structure can be introduced. Conjugation of hydrophobic compounds to saccharides has proven to be promising as increased binding of galectin-3 can be observed. In the present study, we report on neo-glycans carrying hydrophobic biotin as novel ligands for human galectin-3. We modified N-acetyllactosamine- and N,N-diacetyllactosamine-based tetrasaccharides at the C6-position of the terminal saccharide unit using selective enzymatic oxidation and subsequent chemical conjugation of biotinamidohexanoic acid hydrazide. These neo-glycans were much better bound by galectin-3 than the unmodified counterparts. High selectivity for galectin-3 over galectin-1 was also proven. We generated multivalent neo-glycoproteins by conjugation of neo-glycans to bovine serum albumin showing high affinity for galectin-3. Compared to non-biotinylated neo-glycoproteins, we achieved high binding levels of galectin-3 with a lesser amount of conjugated neo-glycans. Multivalent ligand presentation of neo-glycoproteins significantly increased the inhibitory potency towards galectin-3 binding to asialofetuin when compared to free monovalent glycans. Our findings show the positive impact of 6-biotinylation of tetrasaccharides on galectin-3 binding, which broadens the recent design approaches for producing high-affinity ligands. Full article
(This article belongs to the Special Issue Recombinant Glycoproteins)
Show Figures

Graphical abstract

Back to TopTop