Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols


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Methods and Protocols

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Citing Literature. Related Information. Close Figure Viewer. Browse All Figures Return to Figure. Previous Figure Next Figure. Email or Customer ID. Forgot password? Therefore, differences in protein glycosylation between humans and plants have to be taken into account and plant-specific glycosylation has to be eliminated to avoid adverse effects on quality, safety, and efficacy of the products.

The basal land plant Physcomitrella patens moss has been employed for the recombinant production of high-value therapeutic target proteins e. Being genetically excellently characterized and exceptionally amenable for precise gene targeting via homologous recombination, essential steps for the optimization of moss as a bioreactor for the production of recombinant proteins have been undertaken. Here, we discuss the glyco-engineering approaches to avoid non-human N- and O-glycosylation on target proteins produced in moss bioreactors.

Biopharmaceuticals are indispensable in modern medicine. In more than biopharmaceuticals were available on the market and around 10—20 more are approved every year Walsh, a. As the biggest group of biopharmaceuticals consists of pharmaceutical recombinant proteins, this term is often used as a synonym for the former.

The biochemical and pharmacological properties of a protein are not only determined by its amino acid sequence but also largely influenced by a palette of modifications that proteins undergo co- or posttranslationally Mann and Jensen, , usually grouped together and referred to as posttranslational modifications PTMs. Common PTMs found in pharmaceutical proteins are glycosylation, hydroxylation, carboxylation, amidation, sulfatation, disulfide bond formation, and proteolytic processing Walsh and Jefferis, The workhorse for the production of simple proteins is Escherichia coli , the best characterized expression system offering high product yields at low costs Walsh, a.

However, this microorganism is not able to perform some PTMs, which are indispensable for recombinant therapeutical proteins Kamionka, Consequently, mammalian cell lines are the preferred expression systems for the production of recombinant glycoproteins, as their protein glycosylation patterns largely resemble those of humans Schmidt, Among the mammalian cell lines, Chinese hamster ovary CHO cells comprise the leading host system for current biopharmaceuticals, even though several CHO-derived products presented non-human glycosylation Chung et al.

As higher eukaryotes, plants are able to synthesize complex multimeric proteins and perform PTMs in a similar manner as humans do. Therefore, plants and plant cell cultures are gradually gaining acceptance as production hosts for recombinant biopharmaceuticals. The host system for Elelyso TM , a recombinant glucocerebrosidase for the treatment of the lysosomal storage disease Morbus Gaucher, is a carrot-based cell line established by Protalix Shaaltiel et al. It is cultured in bioreactors based on disposable plastic bags. While other frequently used plant systems like alfalfa, tobacco, and Nicotiana benthamiana need to be grown in greenhouses, bioreactor cultivation is established for the aquatic plant Lemna minor and for the moss Physcomitrella patens Decker and Reski, ; Paul and Ma, ; Paul et al.

Within the following sections we will focus on the special features for biopharmaceutical production and achievements within glyco-engineering of the moss system. The non-seed plant P. It can be grown throughout its complete life cycle under contained conditions in vitro in a simple mineral medium Frank et al. The germination of the haploid spores leads to the growth of protonema Figure 1A , a branched filamentous tissue which comprises two distinct cell types, chloronema and caulonema.


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Every cell is in direct contact with the culture medium, allowing efficient nutrient uptake and product secretion Schillberg et al. This young tissue can be maintained in suspension cultures without any addition of phytohormones, only by mechanical disruption of the filaments. In contrast to immortalized or de-differentiated mammalian or higher-plant cell cultures, which are prone to instability or somaclonal variation Larkin and Scowcroft, ; Xu et al. In the next developmental step, buds differentiating from protonema cells give rise to the adult plant, the leafy gametophore, consisting of shoot-like, leaf-like, and root-like tissues Figure 1B.

After fertilization of the gametes, the sporophyte, the only diploid tissue in the life cycle of mosses, grows on and is sustained by the gametophore Reski et al. In vitro cultivation of all stages can be performed either on agar plates or as suspension cultures in liquid media. The availability of efficient protocols for protoplast isolation Figure 1C and transfection Rother et al.

The created moss strains can be preserved by cryo-conservation Schulte and Reski, , and thus can serve as Master Cell Banks. Physcomitrella patens in vitro cultivation and schematic representation of a knockout construct for gene targeting. A young filamentous tissue, protonema, ideal for suspension cultures; B adult leafy moss plant gametophore ; C protoplasts; D small scale liquid culture in flasks; E stirred tank bioreactor; F wave bioreactor image courtesy of greenovation Biotech GmbH ; G illustration of allele replacement via homologous recombination.

The regions homologous to the targeted gene, which are used for the knockout construct, are shown in gray, and the inserted selection cassette is depicted in white. Thick lines represent introns and rectangles exons. The employment of in vitro axenic plant cell or tissue cultures offers an environment in which contamination with human pathogens is rather unlikely Schillberg et al.

Moreover, only in these systems culture conditions can be precisely controlled and standardized Hohe and Reski, , which is essential for the production of pharmaceuticals according to good manufacturing practice GMP guidelines Fischer et al. Various scales of highly controllable cultivation devices were developed for Physcomitrella, ranging from simple shaking flasks Figure 1D and 5 L aerated flasks to diverse forms of photobioreactors, including stirred glass tank bioreactors with a volume of up to 15 L Figure 1E ; Hohe and Reski, and a modular tubular bioreactor with a working volume of up to L reviewed in Decker and Reski, , More recently, disposable wave-bag reactors Figure 1F were employed for high-density protein production purposes under full cGMP compliance www.

Several pharmaceutically interesting proteins have been synthesized in moss bioreactors, among them the growth factors vascular endothelial growth factor VEGF; Baur et al. In addition, proteins with a function in immune responses like IgGs Schuster et al. Furthermore, two products for enzyme-replacement therapies, human alpha-galactosidase and glucocerebrosidase are expected to reach clinical trial phases by the end of www.

The use of Physcomitrella as a production host for recombinant biopharmaceuticals was facilitated by well-developed molecular toolboxes. Heterologous as well as endogenous promoters were characterized for their suitability to achieve high levels of recombinant product Horstmann et al. In addition, several moss-derived signal peptides were evaluated for improved secretion of the recombinant product to the surrounding medium Schaaf et al. The moss genome sequence is available since Rensing et al. The internet resource www.

This resource was very convenient for the identification of genes involved in the glycosylation of recombinant proteins synthesized in moss bioreactors. However, the main driver for moss functional genomics approaches in general and glyco-engineering in particular was the unique accessibility of this organism for gene targeting approaches via homologous recombination. Displaying an exceptionally high rate of homologous recombination in mitotic cells Strepp et al.

Undesirable gene functions can be completely eliminated by targeted knockout approaches.

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The knockout construct used for the transfection of moss protoplasts regularly consists of — bp genomic DNA homologous regions flanking each side of a selection cassette, which interrupts or replaces the target gene when indicated Figure 1G. Glyco-engineering of moss was successfully accomplished by various gene targeting approaches see below. Protein glycosylation is a complex and heterogeneous modification which can be classified in two main categories, N- and O-glycosylation. O -glycans, on the other hand, are attached to the hydroxyl group of serine S , threonine T , hydroxylysine or hydroxyproline Hyp; Varki et al.

In contrast to N-glycosylation, consensus sequences for O-glycosylation in mammals are not well defined or non-existing Hansen et al. N-glycosylation in animals is a largely cell-type and species-specific feature Raju et al. Moreover, potential glycosylation sites on a given protein can be either unmodified or occupied by varying glycan structures which result from the maturation of the glycan throughout the endoplasmic reticulum ER and the Golgi apparatus GA , leading to microheterogeneity of glycoproteins Kolarich et al. Compared to other higher eukaryotes, plants display more conserved glycan patterns between different species and a less diverse palette of N -glycans Bosch et al.

As higher eukaryotes, plants are able to produce N -glycans of the complex type with the core sugar structure Man3GlcNAc2 consisting of two N -acetylglucosamine and three mannose residues that is identical to humans Lerouge et al. Up to two terminally attached GlcNAc residues are also common between plant and bi-antennary mammalian complex-type glycoprotein oligosaccharides reviewed in Gomord et al. These sugar structures are common for all land plants analyzed so far, including mosses as the evolutionary oldest group Koprivova et al. Their presence raised concerns about plant-produced biopharmaceuticals as they were shown to induce antibody formation in mammals van Ree et al.

The consequence of an immune response against the pharmaceutical can lead to antibody-mediated reduction of product efficacy as well as to severe clinical complications Schellekens, Consequently, first plant glyco-engineering approaches aimed at targeting the glycosyltransferases responsible for the addition of these two residues. However, lack of this residue proved to be advantageous for the efficacy of antibodies targeting tumor cells Shields et al. The underlying phenomenon, antibody-dependent cellular cytotoxicity ADCC , comprises receptor binding and activation of a natural killer cell by an antigen—antibody complex on the target cell surface resulting in lysis of the target cell.

However, Le a epitopes were described on recombinant proteins produced in plants Petruccelli et al. Although Le a epitopes are found in humans as part of the Lewis-positive histo-blood groups Henry et al.

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Furthermore, antibodies against Le a epitopes are frequent Wilson et al. Out of these, exclusively one gene galt1 was shown to be responsible for the synthesis of Le a in moss. The disruption of galt1 alone resulted in the absence of the complete Le a epitope, not only of the galactose residue but also of the terminal fucose, both in the total moss N -glycan pool and on the moss-produced rhEPO Parsons et al. The lack of GalT1 activity did not affect the moss growth rate. The homogeneity of the rhEPO glycosylation achieved in the moss galt1 knockout line was remarkable, with almost only one glycosylation form, the aimed core structure with terminal GlcNAc residues Parsons et al.

Glycopeptides of moss-produced rhEPO from two glyco-engineered moss lines. Salt adducts are marked with asterisks. The glycosylation patterns are schematized with sugar symbols above each peak. Le a structures are totally absent in the triple KO line. Further terminal elongation of plant N -glycans has been demonstrated for N. This will be a future task for moss glyco-engineering.

Outstanding success has been achieved so far by engineering plant N-glycosylation patterns for the production of humanized glycoproteins. In contrast, the issue of adverse O-glycosylation in PMPs has not been addressed in the same detail. Concerning plant O -glycan engineering, recombinant proteins displaying human so-called mucin-type O-glycosylation were generated recently Daskalova et al.

In contrast to rather conserved N-glycosylation patterns, plant-typical O-glycosylation differs fundamentally from the typical human mucin-type O-glycosylation reviewed by Gomord et al. In plants, the main attachment site for O-glycosylation is 4- trans -Hyp reviewed by Showalter, , while no glycosylation of Hyp occurs in animals Gorres and Raines, Hyp is generated posttranslationally by prolyl 4-hydroxylases P4H via hydroxylation of proline.

Prolyl-hydroxylation is a very common modification both in mammals and in plants, though recognition sequences differ. In plants, the target motif for O-glycosylation after P4H-catalyzed hydroxylation, the so-called glycomodules present on Hyp-rich glycoproteins HRGPs , are defined Kieliszewski and Lamport, and validated Tan et al.

In fact, undesired plant-typical prolyl-hydroxylation and in some cases even non-human O-glycosylation of biopharmaceuticals was reported Karnoup, ; Weise et al.


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After systematic disruption of each of the six p4h genes in Physcomitrella , targeted deletion of p4h1 resulted in the complete elimination of the previously reported prolyl-hydroxylation of moss-produced rhEPO Parsons et al. As prolyl-hydroxylation and further glycosylation of plant extracellular matrix and cell wall proteins play important roles for growth, cell differentiation, and stress adaption Lamport et al. The ease of gene targeting in moss enabled glyco-engineering approaches for the elimination of any plant-typical immunogenic residues.

This provides a plant-based system offering the stable production of safe protein therapeutics. Eva L. Decker, Juliana Parsons, and Ralf Reski were involved in gathering and interpretation of data, writing the manuscript and revising the work. Ralf Reski is co-inventor of the moss bioreactor and co-founder of greenovation Biotech. He currently serves as advisory board member of this company. Decker, Juliana Parsons, and Ralf Reski are co-inventors of patents and patent applications related to the topic discussed here. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Anne Katrin Prowse for proof-reading of the manuscript. Apweiler, R. Acta , 4—8. Bailey, L. Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture. CrossRef Full Text. Bardor, M. Glycobiology 13, — Baur, A. Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens.

Plant Biotechnol. Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin. Glycobiology 14, — Bosch, D. N-glycosylation of plant-produced recombinant proteins. Production of biologically active recombinant human factor H in Physcomitrella. Castilho, A. Engineering of sialylated mucin-type O-glycosylation in plants.

N-Linked Glycosylation Part 1

Generation of biologically active multi-sialylated recombinant human EPOFc in plants. Chung, C. Cove, D. Mosses as model systems for the study of metabolism and development. Plant Biol. Cox, K. Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Croset, A. Daskalova, S. Engineering of N. BMC Biotechnol. Decker, E. Moss bioreactors producing improved biopharmaceuticals. Current achievements in the production of complex biopharmaceuticals with moss bioreactors.

Glycoprotein production in moss bioreactors. Plant Cell Rep. Fischer, R. GMP issues for recombinant plant-derived pharmaceutical proteins.

Glyco-Engineering

N-glycans harboring the Lewis A epitope are expressed at the surface of plant cells. Plant J. Frank, W. Molecular tools to study Physcomitrella patens. Gavel, Y. Protein Eng. Gomord, V. Plant-specific glycosylation patterns in the context of therapeutic protein production. Gorres, K. Prolyl 4-hydroxylase. Hansen, J. NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility.

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Lewis histo-blood group system and associated secretory phenotypes. Vox Sang. Hohe, A. An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens. Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts.

Plant Sci. Control of growth and differentiation of bioreactor cultures of Physcomitrella by environmental parameters. Plant Cell Tissue Organ Cult. Horstmann, V. Quantitative promoter analysis in Physcomitrella patens : a set of plant vectors activating gene expression within three orders of magnitude. Hossler, P. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 19, — Huether, C. Glyco-engineering of moss lacking plant-specific sugar residues.

Epub Glycosylation Engineering Of Biopharmaceuticals Methods And Protocols

Jez, J. Expression of functionally active sialylated human erythropoietin in plants. Jin, C.

Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols
Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols
Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols
Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols
Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols
Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols

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