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Plugin glycosylation literature survey ricky connolly 091211-1 Plugin glycosylation literature survey ricky connolly 091211-1 Document Transcript

  • Glycosylation forbiopharmaceutical drugsRicky ConnollyBiotechnology, Dublin City University; email: ricky.connolly2@mail.dcu.ieAbstractSince the mid 2000s, the patents for many blockbuster drugs have begun to expire. Thisis the driving force behind the current great development in the biopharmaceuticalindustry, the ‘second generation’ biopharmaceuticals. This new trend is focussed onmodifying existing protein therapeutics in order to enhance their pharmacological,biological, and structural properties. These modifications include but are not limited to:generation of fusion conjugates, incorporation of chemical modifications such aspegylation, and, crucially for this paper, modification of glycosylation profiles. The goalof this paper is to outline the chemical and biological basis of protein glycosylation, toexamine the pharmacological and structural implications of glycosylation, to compareand evaluate the current production strategies, and to survey the current range ofanalytical methods available to characterise glycoprotein therapeutics.
  • Table of ContentsIntroduction ................................................................................................................................. 3 Overview ...................................................................................................................................... 3 Glycan synthesis ........................................................................................................................... 4Stability .......................................................................................................................................... 6 Aggregation .................................................................................................................................. 6 Crosslinking ................................................................................................................................. 7 Proteolysis .................................................................................................................................... 7 Oxidation ..................................................................................................................................... 7 pH ................................................................................................................................................ 7 Kinetic Denaturation ................................................................................................................... 8 Chemical Denaturation ............................................................................................................... 8 Temperature ................................................................................................................................ 8Pharmacology .............................................................................................................................. 9 Receptor Binding ......................................................................................................................... 9 Circulatory lifetime .................................................................................................................... 10 Bioavailability ............................................................................................................................. 10 Distribution................................................................................................................................ 11 Clearance rates ........................................................................................................................... 12 Antibody function ...................................................................................................................... 12 Immunogenicity ......................................................................................................................... 12Glycosylation and host cells .................................................................................................. 13 Mammalian Cells ....................................................................................................................... 14 Yeasts.......................................................................................................................................... 15 Bacteria....................................................................................................................................... 16Analytical methods ................................................................................................................... 18 Mass spectrometric .................................................................................................................... 18 Chromatographic ....................................................................................................................... 19 Electrophoretic .......................................................................................................................... 20 Bioaffinity methods .................................................................................................................... 20Bioinformatics ........................................................................................................................... 22 Complexity of glycan structures ................................................................................................ 22 Prediction tools .......................................................................................................................... 22 Glycobiology databases .............................................................................................................. 23References................................................................................................................................... 24 Abstract | DCU
  • IntroductionThe human genome contains at least 30,000 protein-coding genes, and throughalternative mRNA splicing, these give rise to over 100,000 proteins (Venter, 2001). Thediversity of the human proteome is furthermagnified by post-translational modification ofproteins. At least 50 percent of mammalianproteins are glycosylated (Zafar, 2011). Morethan two thirds of the 200 or so biopharma-ceutical products licenced for sale in theUnited States and European Union arerecombinant human proteins (Li, 2010). In2010, five of the top ten selling pharmaceuticalproducts were recombinant human proteinsand this is projected to rise to eight of the topten by 2014 (Hirschler, 2010). Additionally,over 70 percent of the therapeutics currently inclinical trials are glycosylated human proteins (Sethuraman, 2006).OverviewGlycosylation is defined as the covalent attachment of oligosaccharide moieties (glycans)to the side chains of amino acid residues of proteins. There are at least five differentclasses of glycosylation, each defined bythe amino acid the glycan in linked to andthe type of linkage used. By far the mostcommon of these are N-linked and O-linked glycosylation (Apweilier, 1999).This review will focus solely on N-linkedglycosylation because it is more commontype found in human therapeutic proteintherapeutics (Pandhal, 2010). N-linkedglycosylation involves the enzymatic attachment of the N-Acetylglucosamine residue atthe terminal end of the glycan to the amide group of an asparagine residue by means of aβ1 glycosidic linkage (Geyer, 2006). DCU | Introduction
  • The asparagine must be located within the three residue sequence Asparagine-X-Serine/Threonine, where X represents any amino acid except proline, which inhibitsattachment of the N-glycan through stearic hindrance caused by its rotationally-lockedside group (Shyama, 2010). There are further structural requirements in addition to theAsp-X-Ser/Thr sequon, mostly concerning the three-dimensional localisation andaccessibility of the sequon within the folded protein (Apweilier, 1999).Glycan synthesisN-glycosylation takes place in the endoplasmic reticulum, at the same sites as proteintranslation. The first step in glycan synthesis involves the addition of two GlcNAcresidues and five mannose residues to the membrane-anchored dolichol-PP. Thedolichol-PP-Man5GlcNAc2 group is then flipped to the inner membrane side of theendoplasmic reticulum by the enzyme RFT1 (Pandhal, 2010).The core oligosaccharide (Glc3Man9GlcNAc2) is constructed on the GlcNAc residue bya series of glycosyltransferases. After this, the glycan is transferred to a sequon-labelledasparagine residue on the protein chain as it emerges from the ribosome (Pandhal, 2010). Introduction | DCU
  • The glycan is trimmed by glucosidase I and II to remove the α1,2-linked (Shailubhai,1987) and α1,3-linked (Saxena, 1987) glucose residues, respectively. This leaves us with aMan9GlcNAc2 structure. Next, α1,2-mannosidase removes one of the α1,2-linkedmannose residues. The Man8GlcNAc2 glycan is then exported to the Golgi apparatus.Here, more α1,2-mannosidases remove several mannose residues, producingMan5GlcNAc2. After this, the glycosylation pathway diverges into different patterns ofresidue trimming and addition, catalysed by a series of glycosidases andglycosyltransferases (Pandhal, 2010).This results in three distinct prototypic classes of glycan structure into which themajority of glycans fall: complex, hybrid, and oligomannose (Kornfeld, 1985).Oligomannose glycans contain only mannose residues in addition to their core structures.Hybrid glycans can contain a diverse range of residues including galactose, glucoronicacid, and xylose (in plants). Complex glycans resemble hybrid glycans but they comewith varying degrees of fucosylation and sialylation (Pandhal, 2010). Sialylation is oftenvital for correct protein function, this will be discussed later.Glycans are constructed from monosaccharide units linked together with glycosidicbonds. Unlike peptides and nucleic acids, one monosaccharide unit can be linked tomultiple other units. This is called branching. Biantennary glycans are the most common,but triantennary and tetrantennary glycans are not unheard of (Campion, 1989). Anothercommon structural feature is to have a single (β1,4) GlcNAc residue linked to the firstbranching point mannose unit. This bisecting GlcNAc residue is often involved insignalling (Yoshimura, 1998). The molecular structures, branching types and families of DCU | Introduction
  • glycans attached to a protein describe its microheterogeneity in terms of glycosylation.This is distinct from the macroheterogeneity, which is described by the total number ofglycans attached to the protein and their positions.StabilityA protein instability is defined as a physical or chemical vulnerability that is prone toaltering the structural conformation or activity in a detrimental way. Although they havehigh therapeutic efficacy, the main drawback to pharmaceutical use of proteins is theirinherent structural and chemical instabilities. These impose limitations on the proteinproduction and purification stages, product formulation, storage and transport, and half-life in the body. Glycosylation has been shown to ameliorate many of these instabilities.In this section, the major protein instabilities will be outlined, and the efficacy ofglycosylation on these will be investigated.AggregationProteins are very complex molecules with hundreds if not thousands of exposedfunctional groups. This makes non-native aggregation a very common problem forprotein biopharmaceuticals. Aggregation can be caused by relatively small changes in theprotein structure (Saluja, 2008). Aggregation can drastically reduce the biologicalfunction of a protein (Philo, 2009) and in some cases can induce an immunogenicreaction in patients (Rosenberg, 2006). Glycosylation has been shown to prevent theformation of protein aggregates (Kayser, 2011). The likely explanation is that the bulkyspatial nature of glycan moieties provides a level of stearic repulsion between proteins(Imperiali, 1999), reducing the likelihood of aggregation (Hoiberg-Nielsen, 2006).PrecipitationPrecipitation is the ultimate effect of protein aggregation. Eventually, the proteins formaggregates so large that they become insoluble (Chi, 2003). Precipitation is a majorproblem for biotherapeutic product formulation because it is desirable to store proteinsolutions at relatively high concentrations, but at high enough concentrations mostproteins will form aggregates and precipitate out of solution (Wang, 2005).Glycosylation decreases the propensity to form insoluble precipitates for many proteins(Kayser, 2011). It has been shown that the decrease in insolubility is directly Stability | DCU
  • proportional to the size and number of glycans present (Tams, 1999). One possibleexplanation for this phenomenon is that glycans are more soluble than peptides, and theyconfer some of this solubility to the glycoprotein as a whole.CrosslinkingCrosslinking, also known as polymerisation-induced inactivation, occurs primarily whenthe methionine residues of two different proteins form a disulphide linkage. This leadsto the formation of protein oligomers which have diminished biological activity (Wang,1999). Glycosylation has been shown to prevent chemical crosslinking between proteins(Runkel, 1998). The mechanism is probably the same as that of aggregation, the mutualstearic repulsion between glycosylated proteins.ProteolysisProteolysis is the degradation of the peptide backbone through hydrolytic reactions.These reactions are carried out by the ubiquitous protease enzymes found in all tissues.They are a major problem from a drug administration perspective because protease-sensitivity can drastically reduce the level of therapeutic that reaches the site of action(Tang, 2004). Glycosylation has been shown to inhibit proteolytic degradation (Vegarud,1975). The most likely mechanism is through stearic blockage of the protease cleavagesites by the glycan (Russell, 2009).OxidationProteins are susceptible to oxidative degradation at the level of their primary structure.This is of particular concern for proteins rich in the more reactive amino acids such asHis, Met, Cys, Tyr and Trp (Manning, 2010), which are prone to accepting free radicals.Since free radicals are basically unavoidable at all stages of the pipeline from cell cultureto drug administration (even exposure to stray light can be harmful (Kerwin, 2007)),oxidation poses a serious threat to the stability and functionality of therapeutic proteins.Glycosylation has been shown to reduce the effects of oxidative damage in at least onecommercial therapeutic (Uchida, 1997). One proposed mechanism for this protection isthat the glycans are ‘soaking up’ the radicals, thus preventing them from reaching theprotein itself (Pristov, 2011).pHProteins are only stable within a limited pH range. pH denaturation begins when the ionbalance disrupts the hydrogen- and ion-bonding capacity of the amino acids. This affectsthe tertiary structure of the protein and leads to the formation of non-native chemical DCU | Stability
  • bonds as the peptide reconfigures into a more thermodynamically-stable state (Solá,2009). Glycosylation has been shown to improve the pH stability of protein therapeuticsby up to a 13-fold increase (Masarova, 2001). This marked improvement is due to thefact that attached glycans decrease the solvent accessible surface area of the protein,acting as a molecular buffer between the electrostatic forces of the solvent and those ofthe protein.Kinetic DenaturationDue to their complex nature, protein molecules have several three-dimensional states inwhich they are thermodynamically stable, but usually have only state in which they arefunctionally active. This means that proteins can undergo kinetic inactivation even atlow temperatures by ‘flipping’ to another stable but inactive state (Arakawa, 2001). It hasbeen demonstrated that glycosylation increases the kinetic stability of proteins.Specifically, the level of stability conferred seems to be correlated to the number ofglycans present, their positioning on the protein, and their size (Solá, 2007). This is mostlikely because a folded, glycosylated protein has a lower free-energy profile compared toboth the unfolded protein and to the folded-unglycosylated protein (Shental-Bechor,2008).Chemical DenaturationChemical denaturation can be defined as the loss of structural integrity of a protein inresponse to exposure to a chemical agent. One of the primary reasons for chemicaldestabilisation is that the protein often has high Van der Waals affinity to the denaturant.This allows the chemical molecule to intrude into the tertiary structure of the protein,disrupting the global conformation and reducing functionality (Hual, 2008).Glycosylation has been shown to promote conformational stability in opposition tochemical denaturants (Sytkowski, 1991). The probable explanation for this effect is thatglycosylation essentially ‘compacts’ the protein, increasing the strength of its internalionic, hydrogen and Van der Waals bonds and reducing the peptide’s affinity for outsidechemical molecules (Solá, 2007).TemperatureAll of the bonds within a protein are sensitive to thermal fluctuations. Outside of a smalltemperature range, these bonds will break or form non-native bonds, destroying thebiological activity of the protein (Vogt, 1997). The effects of freezing are less wellstudied compared to those of heating, but some comprehensive studies of thephenomena exist (Bhatnagar, 2007). Glycosylation has been shown to improve the Stability | DCU
  • thermal stability range for several therapeutically important proteins including EPO,alpha 1-antitrypsin, interferon-β, and follicle-stimulating hormone (Solá, 2009). It islikely that these increases in stability are due to the constraint of peptide mobility causedby glycan attachment (Wormald, 1999). The magnitude of thermal stabilisation isproportional to the number and size of the glycan attachments (Wang, 1996).PharmacologyPharmacology is divided into two fields of study: pharmacokinetics andpharmacodynamics. Pharmacokinetics examines the action of drugs within the body overa given period of time; profiling distribution, metabolism, and excretion.Pharmacodynamics studies the mechanisms of action of drugs within the body, studyingthe drug-receptor interactions and dose/response profiles. In other words,pharmacokinetics studies what the body does to the drug, while pharmacodynamicsstudies what the drug does to the body (Benet, 1984).Proteins tend to have poor pharmacokinetic profiles because they are very quicklycleared by proteolytic degradation pathways, hepatic and renal elimination, andreceptor-mediated endocytosis (Tang, 2004). They have sharp pharmacodynamicsprofiles due to their exceptionally high binding affinities with receptors compared tosmall molecule drugs and have high turnover rates of their substrates. Glycosylationstrongly affects the pharmacological properties of a protein. In this section, the majorintrinsic pharmacokinetic and pharmacodynamic limitations of proteins are reviewed.For each, the impact of glycosylation on these limitations are examined.Receptor BindingOne of the most characteristic traits of proteins is the extraordinarily high affinity withwhich they bind to their receptors. This is both as blessing and a curse. A high receptorassociation rate allows the design of therapeutics with strong biological efficacy, but itoften means the protein has a very blunt therapeutic response curve (Solá, 2010). Thisleads to dosage schemes that require multiple injections per day, with widely fluctuatinglevels of drug in the body over the course of the day. For this reason, it is often desirableto ‘smooth out’ the response curve to a protein therapeutic. In practise, this meansreducing receptor affinity of the protein. DCU | Pharmacology
  • Glycosylation has been used to reduce the receptor affinity for several commerciallyimportant protein therapeutics. Darling et al showed that EPO-IRS (the standard formof human erythropoietin recognized European Pharmacopoeia) showed a 20-fold lowerreceptor association rate compared with artificially deglycosylated EPO (Darling, 2002).Similarly, another study compared the relative receptor binding affinities of severalisoforms of erythropoietin. The EPO isoforms are defined by the total number of sialicacid residues found on their glycocomponent. The experiment involved measuring thequantity of each isoform needed to displace inactive EPO from receptors expressed onthe surface of human erythroleukemia cells. There was a direct inverse relationshipbetween sialic acid content and receptor binding affinity (Egrie, 2001).The most likely mechanisms by which this reduction in binding affinity can be explainedis that electrostatic repulsion between the sialic acid residues of the glycans and thereceptor serve to decrease the liklihood of receptor binding (Elliott, 2004).Circulatory lifetimeGlycosylation can have a dramatic effect on the circulatory lifetime of a therapeuticprotein. Comparing the serum half-life of the enzyme ceruloplasmin in it’s a natively-glycosylated state with that of an artificially deglycosylated variant, the circulatorylifetime of the deglycosylated variant was found to be an order of magnitude lower(Morell, 1968). Similarly, the addition of extra glycans (hyperglycosylation) has beenshown to decrease the clearance rate of proteins.Perlman et al (2003) observed a 4-fold increase in serum half-life of a variant of humanfollicle stimulating hormone (FSH) with two addition N-linked glycosylation sites. Thesialic acid content of attached glycans has been shown to be of high importance toextending the serum half-life of a protein. Sialic residues have a net negative charge atbiological pH. This electrostatic repulsion confers protection from both renal (Kanwar,1984) and hepatic (Morell, 1971) clearence mechanisms.BioavailabilityToday the vast majority of protein therapeutics are delivered by parenteral injection(Soltero 2001). This contrasts with small molecule drugs, which are typically deliveredthrough oral routes (Nandita, 2003). From a clinical standpoint, it would be verydesirable to have oral-administered protein therapeutics. Unfortunately, there are severalbarriers to making this technology a reality. Pharmacology | DCU
  • Unless injected directly into the bloodstream, drugs must pass through severalmembrane barriers before they can begin systemic circulation, and if the target of thedrug is intracellular, the peptide must pass through the lipid membrane of the cell.There are four mechanisms by which a protein therapeutic may pass through amembrane: passive and facilitated diffusion, active transport, and receptor mediatedendocytosis (Kopacek, 2011). The rate at which a drug absorbs in the body is determinedby the rate at which the drug is transported across these barriers.Glycosylation has been shown to improve protein absorption in several cases. Egleton etal (2001) demonstrated that the addition of an O-linked glycan to an opioid peptideicreased its blood brain barrier permeability from 1.0 μl/(min·g) to 2.2 μl/(min·g), with acomparable increase in measured analgesic activity. Albert et al (1993) produced aglycosylated, orally-active version of octreotide, a regulatory peptide that inhibits theproduction of somatropin and other growth hormones, which had ten times greater oralbioavailability compared to the parent molecule. Nomoto et al (1998) significantlyimproved intestinal uptake of a peptide by the sodium ion-dependent D-glucosetransporter through the addition of a small glycan moiety.DistributionDrug distribution throughout the tissues of the body is controlled by blood perfusion,plasma protein and tissue binding affinity, pH, and membrane permeability (Kopacek,2011). Controlling the distribution of a drug is vital to ensuring that the drug reachesthe target tissue and that it does not end up in the wrong tissue, where it will have no (oreven adverse) biological effects.Glycosylation has been shown to improve the tissue distribution of therapeutic drugs.Sasayama et al (2000) chemically glycosylated human interleukin-1ɑ with an N-acetylneuraminic acid moiety and monitored the in vivo tissue distribution in ratsfollowing intraperitoneal (IP) injection. Up to five-fold greater levels of the glycosylatedvariant were observed in the kidney, spleen, lung, and blood. Similarly, Ceaglio et al(2008) created a mutant version of interferon-ɑ with four additional N-glycans whichdisplayed a ten-fold increase in distribution half-life (t1/2β) after ten hours post-injection,and levels remained detectable 96 hours after injection. The most likely explanations forthe increase in tissue distribution are that the glycoproteins are protected fromproteolytic and immune-inactivation pathways or that the more soluble glycans reducethe hydrophobicity of the peptide, increasing the rate of transport. DCU | Pharmacology
  • Clearance ratesDrug clearance studies measure the rate of removal of drug from circulation. Thismainly takes place in the liver and kidneys. The drugs are carried along by the flow ofblood until they reach the liver, where they are taken up by hepatocytes and are brokendown by nonspecific proteases, before the amino acids are recycled back into the body’smetabolic cycle (Kahn, 2011). It is advantageous to attempt to reduce the clearance ratesof protein biopharmaceuticals to a minimum so that expensive drug is not wasted. Anyeffective reduction of the level of clearance of a therapeutic will be valuable from aclinical and economic point of view, and glycosylation has shown to be an efficientstrategy to achieve this reduction.A novel hyperglycosylated variant of human EPO, darbepoetin alfa (DA), wasengineered to display two additional N-linked glycans. In a double-blind, randomized,cross-over clinical trial in humans, DA showed a 2.5-fold lower rate of clearance (1.6mL/h·kg versus 4.0 mL/h·kg) (Macdougall, 1999). The reduction in clearance rateobserved for hyperglycosylated proteins has been attributed to the increased sialic acidcontent of the introduced glycans (Egrie, 1993).Antibody functionIgG antibodies have two large biantennary N-linked carbohydrate moities attached tothe Fc effector region . The structure of these glycans has an effect on the chainorientation, chain spacing and surface residue exposure (Kaneko, 2006). Thesedifferences in structure can alter antibody effector function (Burton, 2006). It has beenshown that IgG antibodies engineered to remove fucose residues from their glycancomponent have a much stronger antibody-dependent cell-mediated cytotoxicity profilecompared to wild-type IgGs (Yamane-Ohnuki, 2004). This is of clinical importancebecause higher activation of immune system cells can stimulate the body to fight a widerange of conditions, including cancer (Satoh, 2006).ImmunogenicityGlycosylation has been shown to help prevent the generation of neutralizing antibodiesagainst therapeutic drugs. There are several theories which attempt to explain this effect.One explanation is that, as outlined earlier, glycosylation inhibits the formation ofprotein aggregates. Antibodies are often raised more efficiently against aggregates thanindividual proteins, so it stands to reason that glycosylation prevents the development ofan immune reaction by inhibiting aggregation of the drug in circulation (Moore 1980).Another possible explanation of the inhibition of immune response is that the bulky Pharmacology | DCU
  • nature of the carbohydrate moieties provide a degree of stearic hindrance, shielding thepeptide chain below from immune cells (Casadevall, 2002). A further theory is that theterminal sialic acid residues of glycans provide a degree of electrostatic repulsion, againpreventing immune cell surface receptors from binding (Fernandes, 2002).Glycosylation and host cellsIt is estimated that well over 50 percent of human proteins are glycosylated (Apweiler,1999), although this figure is disputed (Khoury, 2011). Nevertheless, almost 40 percentof all approved biopharmaceutical 2% 55% a.products are glycosylated (Walsh, 2% 2%2010). Therefore, for an organism to Mammalian 10%become widely used as a producer E. colistrain, it must be able to perform Yeastglycosylation to some extent. Between2006 and 2010, 58 new Insectbiopharmaceuticals gained approval. Animals32 of these were produced in 29% Syntheticmammalian cell lines and of these, 24 CHOwere produced in Chinese hamster 3% 3% 75% b.ovary (CHO) cells. It is clear that 6% NS0 3%CHO cells are still the workhorse of Sp2/0 3%the biopharmaceutical industry. 7% rat-mouse hybrid hybridomaOther common host cell systems Murine hybridomainclude the various mouse myeloma Murine myelomastrains, including NS0 and sp2/0, as Immortalizedwell as yeast cells, mostly Pichia humanpastoris and Saccharomyces cerevisiae.Despite its lack of mammalianglycosylation machinery, E. coli is still widely used for smaller proteins and antibodyfragments, and E. coli-produced biopharmaceuticals accounted for 30 percent of newlyapproved products during the period from 2006 to 2010 (Walsh, 2010). DCU | Glycosylation and host cells
  • Mammalian CellsThe obvious choice for producing recombinant human proteins are mammalian cells.The major advantage of mammalian cells is that they possess the ability to produceproteins with fully human-type glycosylation (Butler, 2005). Additionally, they are ableto produce large molecular weight proteins, and natively possess the machinery toconduct correct folding, quality control, and secretion (Demain, 2009).There are several major limitations to mammalian cells as a producer strain. Mammaliancells have extraordinarily high cost of culture, mostly due their fastidious mediarequirements. A fully validated production process using these cells can cost $2-4 millionper year in growth media alone (Demain, 2009). They also have a slower growth cyclethan microbial producer strains, with doubling times of 12-20 hours, approximately tentimes slower than that of E. coli strains (Sunstrom, 2000).Mammalian cell lines are susceptible to infection by viruses which are potentiallypathogenic to humans. Infection of a production process can be disastrous and can causethe entire production facility to be shut down by regulators (Berting, 2010). Wholebatches of product have been lost to viral contamination (Bethencourt, 2009). Theirmedia requirements are notoriously fastidious. Mammalian cells produce their ownglycosylated proteins, which makes downstream separation of the target glycoproteinmuch more difficult.Historically, mammalian cell culture processes have suffered from low product yields,but through advances in media and production optimization, gene amplification andplasmid engineering, this productivity has increased markedly, often reaching expressionlevels of 10-15 g/L for some products in CHO cells (Huang, 2010).In addition to these advances, new producer strains have been developed. The PER.C6cell line is derived from human retinal tissue and looks to have a future as a majorproducer cell line. PER.C6 cells can tolerate very high cell densities in culture; up to 150x 106 cells/mL have been reported (Golden, 2009), they show robust scale upcharacteristics (Xie, 2003), and can produce recombinant protein at very high titres of atleast to 25 g/L (Schirmer, 2010). Glycosylation and host cells | DCU
  • YeastsLike humans, yeasts belong to the eukaryotic domain. This being so, many of themetabolic pathways and molecular machinery are conserved between both species,including much of the glycosylation apparatus (Rich, 2009). The two most commonstrains used for industrial production of recombinant proteins are Saccharomycescerevisiae and Pichia pastoris. Both strains have had their genomes thoroughlycharacterized (Goffeau, 1996) (De Schutter, 2009).Yeasts have properties that make them attractive as a biopharmaceutical productionsystem for several reasons. Yeast cells enjoy both the fast growth rates of microbes andthe complex enzymatic machinery of eukaryotes (Verma, 1998). They offer reasonableyields of recombinant product, yields of up to 9 g/L have been reported (Valdivieso-Ugarte, 2006). Their growth kinetics and nutritional requirements are well characterized,so scale-up from lab bench to industrial fermentation is a relatively simple procedure(Hamilton, 2007).Yeasts can tolerate very high cell densities, as high as 130 g/L in some strains (Gellison,1992), magnifying the product yield attainable per litre of culture volume (Cregg, 2009).Additionally, yeasts can be induced to secrete expressed recombinant therapeutics intothe culture media, simplifying the downstream processing, which typically accounts for50-80 percent of the total cost associated with a production process (Roque, 2004). Yeastcells are capable of carrying out N-linked glycosylation at the Asn-X-Ser/Thr motif, butthere are differences between native yeast-produced N-glycans and those produced inhuman cells.Both yeast and human cells share the same initial steps in their N-linked glycosylationpathways. The Glc3Man9GlcNAc2 glycan precursor is constructed on a membranebound dolichol-PP on the endoplasmic reticulum. This precursor is then transferred toan Asn residue on the peptide as it emerges from the ribosome (Jigami, 2008). This iscarried out by an oligosaccharyltransferase complex consisting of eight subunits; whichdiffer structurally between humans and yeast, but which carry out the same function(Kelleher, 2006). After this, the glycan is trimmed by three glucose units and onemannose unit, leaving a Man8GlcNAc2 structure, which is then exported to the Golgiapparatus (Jigami, 2008). DCU | Glycosylation and host cells
  • It is after this step that human and yeast N-glycosylation diverge. In human cells, theglycan is trimmed and extended with a series of glycosidases and glycosyltransferases toyield the final glycan structure, which is often capped with sialic acid residues (Hamilton,2007). In yeast cells, the core Man8GlcNAc2 is extended, adding numerous mannoseunits, leaving the glycan comparatively hypermannosylated (Gemmill, 1999).The major technical hurdle with using yeast cells as a host for producing recombinanthuman therapeutics is the inactivation of the endogenous machinery that leads tohypermannosylation and the introduction of glycosyltransferases and glycosidases thatlead to the production of human-like glycans. The first objective has been achieved in S.cerevisiae by eliminating the Och1 gene, which codes for the first enzyme in themannose-extension pathway, thereby preventing hypermannosylation (Nakanishi-Shindo, 1993).On the second objective, progress has been made towards producing human-typeglycans. By introducing the Mns-II and Gnt-II genes, minimal human complex-typeglycans (GlcNAc2Man3GlcNAc2) have been produced in P. pastoris (Hamilton, 2003).Combined, these advances pave the way towards the use of yeast cells to producefunctionl, homogeneous human therapeutic glycoproteins.BacteriaAlthough once dismissed as lacking the ability to perform complex glycosylation, it isnow known that many bacterial strains can produce even more complex and diverseglycan structures than even mammalian cells (Abu-Qarn, 2008). This opens the door tothe possibility of using bacterial cells as production vectors. This is an extremelydesirable goal from an economic standpoint for several reasons. Bacteria have muchshorter generation times than the currently used CHO cells. Given optimal growthconditions, an E. coli population can double in as little as 18-20 minutes (Irwin, 2010),while a mammalian culture will take 12-20 hours (Nakahara, 2002) (Sunstrom, 2000).Bacterial hosts tend to be quite easy to modify genetically, and there are strains availableto meet any number of specific culture and production conditions (Bachmann, 1972). Glycosylation and host cells | DCU
  • They can grow to quite high cell densities in culture (20 to 175 g/l dry biomass) (Lee,1996), with recombinant target protein accounting for up to 30 percent of total cellularprotein by weight (Suzuki, 2006). Yields as high as 0.5 mg/mL have been achieved byoptimizing many conditions simultaneously (Sivashanmugam, 2009).One of the main intrinsic limitations to bacterial expression systems is the tendency ofrecombinant proteins to crash out into aggregates and inclusion bodies. This is partlybecause bacteria lack the specific charaponins which aid in the folding of mammalianproteins. This problem has been theoretically mitigated by coexpressing the chaperonewith the target protein (Lin, 2001).There are large differences between bacterial and mammalian glycosylation patterns.The mammalian core glycan is attached to the asparagine residues by an N-acetylglucosamine-β(1-4) linkage, whereas C. jejuni, the model organism for studyingbacterial glycosylation, utilises an N-acetylgalactosamine-α(1-3)-bacillosamine linkage(Stanley, 2009) (Young, 2002). Bacillosamine is a rare amino sugar found in bacteria.Additionally, the recognition sequence differs between human and bacterial systems.Human cells utilise the three residue Asn-X-Ser/Thr sequon, while bacteria use a longerfive residue Asp/Glu -X-Asp-X-Ser/Thr sequon (Rich, 2009), where x represents anyamino acid except proline, which inhibits attachment of the N-glycan through stearichindrance (Shyama, 2010). Additionally, human glycosylation occurs during proteintranslation as the peptide emerges from the ribosome, while bacterial glycosylationoccurs after translation and folding as a true post-translational modification (Kowarik,2006).Taking into account these differences, a putative roadmap can be built. First, it will benecessary to manipulating the bacterial host cells into utilising the mammalian GlcNAcpeptide linkage. Then it will be necessary to deal with the sequon problem. This can besolved in two ways. The bacterial machinery can be modified to utilise the human-typesequon or the target protein sequence can be modified to include the bacterial N-glycosylation sequon. Ideally, the glycosylation apparatus will need to be transferred intoa more ideal production host such as E. coli or B. subtilis. DCU | Glycosylation and host cells
  • All three of these goals have been achieved to an extent. By transferring pglB, anoligosaccharyltransferase with relaxed substrate specificity, into bacterial expressionsystems, human-type glycans with GlcNAc at their non-reducing can be linked toproteins (Wacker, 2006).Considering the problem of consensus sequences, Kowarik (2006) et al were able toengineer additional Asp/Glu-X-Asp-X-Ser/Thr sequons into AcrA, a component of amulti-drug efflux complex, from C. jejuni, and achieved functional glycosylation at thesesites. Thus there is no theoretical reason why human therapeutic proteins could not beengineered to replace their N-gylcosylation sequons with bacterial versions, or to addadditional sequons to noncatalytic loops, where they are less likely to interfere withprotein function.Considering the host strain issue, once the mechanics of C. jejuni glycosylation wereelucidated in detail, it was relatively easy to transfer this system into other bacterialspecies, including E. coli (Wacker, 2002). Furthermore, this system has been used toproduce functional, correctly folded, glycosylated murine single-chain antibodyfragments, proteins with high therapeutic potential (Lizak, 2011).Analytical methodsTo analyse a pool glycan structures extracted from a cell or protein sample, two steps areneeded. Firstly, the glycans must be separated and secondly, the individual glycans mustbe identified. To date, three major classes of glycan analysis have emerged;electrophoresis, chromatography and mass spectrometry (MS) (Pabst, 2011). Note thanin practise, many of these techniques are used in tandem to achieve higher-resolutionseparation or more comprehensive analysis.Mass spectrometricWithin mass spectrometry, there are two methods which have dominated. Matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF) is suited to theanalysis of biological structures because the method of ionization is less hard, meaning Analytical methods | DCU
  • fragile biological structures are less likely to fragment. The glycans are embedded in amatrix composed of crystallised low molecular weight molecules. 2,5-dihydroxy benzoicacid (DHB) and its derivatives are the most widely-used matrices (Harvey, 2005). Thematrix is irradiated with a high-powered UV laser, causing some of the matrix moleculesto vaporise into a hot cloud of gas. Through a series of reactions, the glycans becomebound to charged species, usually sodium cations (Morelle, 2007). The charged glycansare then sent to a mass analyser.The mass analyser generates a defined electric filed which accelerates the glycanstowards a detector. This ‘time of flight’ is determined by the mass-to-charge ratio (m/z)of the glycan. The analyser outputs a mass spectrum, where each peak represents acharged fragment of the original glycan (Morelle, 2009). By comparing the outputspectrum to a database of such spectra, the glycan can be identified.The other major MS technique is electrospray ionization (ESI).Instead of embedding thesample in a molecular matrix; the ions are formed directly from solution (Chait, 2011).The solution containing the glycans is forced through a thin capillary tube. At the tip ofthe tube, a strong electric field is applied. As the glycans leave the tube, they are exposedto this field and ionized. This creates a spray of charged particles. The ions are thendetected by a TOF analyser, or other equivalent apparatus (Loo, 2000).MS techniques can be coupled into tandem arrays. These can be used, for example, tofirst separate a pool of glycans by their mass charge ratio, then selecting a set of glycanswithin a specific range of (m/z) vales to go on to a second stage where they arefragmented by collision-induced dissociation (CID) and analysed again, providingfurther structural information (Harvey, 2000).ChromatographicIn chromatographic methods, the glycan solution is pumped through a densely-packedstationary phase. The glycans are slowed down on their passage through the column bythe stationary phase particles. The samples elute from the column at different times, thisis the retention time. The retention time is directly dependant on the glycan mass, sowith a properly calibrated column the mass of each glycan can be determined from theretention time. DCU | Analytical methods
  • Within the glycobiology field, several chromatographic techniques have dominated.High pH anion exchange chromatography (HPAEC) has been one of the most widely-used techniques to date (Townsend, 1991). HPAEC is sensitive to branching patternsand oligosaccharide compositions. The glycans bind to the stationary phase which isloaded with negatively-charged functional groups. The glycans are then displaced andeluted by the introduction of a positively charged eluent (Dionex, 1997).Another method used for smaller proteins is reverse-phase high-performance liquidchromatography (RP-HPLC). RP-HPLC uses a non-polar stationary phase. Theglycans are derivatised (tagged) with a highly hydrophobic molecule that allows theglycan to be selectively eluted (Wuhrer, 2005). This method can provide detailedinformation about glycan structure and isomeric configuration (Gillmeister, 2009).Another advantage of HPLC-based systems is that there exist large databases of peakpositions for glycans, making identification much easier (Campbell, 2008).ElectrophoreticThe major electrophoretic method used in the characterization of glycans is capillaryelectrophoresis (CE). Like all electrophoretic methods, CE separation is based on thesize-to-charge ratio of a sample. Protein glyco-isoforms tend to be structurally verysimilar. The differences may be difficult to detect using chromatographic orspectrometric methods (Zamfir, 2008). The major advantage of CE is that it hasunrivalled separation power (Mechref, 2009). The sample is introduced into a chamberwhere it is taken up by a capillary tube. An electric field is applied over the tube. As theglycans travel through the tube, they are separated out by the slight differences in theirmass-to-charge ratios (Landers, 1995).Bioaffinity methodsBioaffinity methods are the newest breakthrough in the field of glycobiology applications.It is defined as the separation of molecules based on their reversible interaction withbiological macromolecules. These separations involve highly-specific interactionsbetween the glycoligand and a carbohydrate-binding protein (Tetala, 2010). Theadvantage of bioaffinity chromatography is that it allows highly-selective one step Analytical methods | DCU
  • purification of glycoproteins, meaning less sample needs to be used. It can be used foranalytical, preparatory and diagnostic applications.Lectins are proteins involved in carbohydrate-based recognition. Given their high-specificity for distinct oligosaccharide epitopes, lectins are the logical solution for thestationary-phase ligand. They are able to select for not only overall glycan structure, butalso for the configuration of the linkages between the monosaccharide units (Mechref,2002). This allows them to isolate a target from a pool of glycans or glycoproteins.Several lectin-affinity based techniques have been developed. The first is the standardchromatographic column approach, where the lectins are immobilised onto a stationaryphase and the pool of glycoproteins are washed through the column (Tetala, 2010). Thetarget glycoproteins bind to the lectins and can be eluted and fractionated. Thisapproach can be improved by adding multiple lectins to the same column, allowingsimultaneous selection of multiple targets at one time (Yang, 2004). Another approach isto immobilize the lectins onto the surface of a microtiter plate. This allows simultaneousanalysis of an even greater number of glycoproteins in a rapids and high-throughputfashion (Kuno, 2005).A modification of this approach is the enzyme-linked lectin assay (ELLA) (Wu, 2009).Similar to the standard ELISA assay, the target glycoprotein is bound to the surface of amicrotiter plate, before a blocking solution is added to prevent nonspecific binding.Then lectins are added to the wells, and bind to the target glycoprotein, if present. Thelectins are detected by the addition of labelled antibodies raised to bind to antigens onthe lectin. The label is detected and the quantity of label is proportional to the quantityof target glycoprotein.A similar technique is the carbohydrate array, in which glycans themselves areimmobilized to the plate and their interaction with a target protein is assayed (Oyelaran,2007). DCU | Analytical methods
  • BioinformaticsThe primary goal of glycomics is to profile the expression and activity of all of theglycosyltransferases, glycosidases, and other glycosylation apparatus, as well as the entireglycan component within a cell under specific conditions (Aoki-Kinoshita, 2008). Theglycoprofiles of cells under different conditions can be compared. From this, we willbegin to deduce to conditions which lead to specific glycosylation events and, ultimately,have the ability to rationally design the glycosylation machinery of producer cell lines.Bioinformatic methods will be the key to achieving this goal.Complexity of glycan structuresComputationally speaking, glycomics is a much more daunting field than proteomics andgenomics. Unlike genes and proteins, the glycoprofile of a cell is not encoded directlyby the genome but indirectly through the compliment of glycosylation enzymes active inthe cell. This means that to predict the glycosylation state of a newly-translated protein,one would need to have full knowledge of the entire spectrum of glycosylation enzymesexpressed at the time as well as their substrate specificities, kinetic rates, their cofactorsand inhibitors, and a plethora of other variables.Additionally, glycans themselves are structurally highly complex molecules. Unlike thelinear sequence of nucleic acids or amino acids which describe genes and proteins,glycans are composed of sugar monosaccharaides. These can be linked together bydifferent types of bonds, and a residue can be linked to more than one other residue(branching), and each branch can be linked in a number of different ways. Otherstructural variables include anomeric configuration, epimeric configuration, andreducing terminal attachments (Laine, 1994).These structural traits magnify enormously the number of possible unique structuresthat can be built from a given set of residues. To put this into perspective, the four DNAbases can give rise to 256 possible four-unit combinations, and the twenty amino acidscan give rise to 160,000 possible four-unit arrangements, while a four-unit glycan canpotentially be assembled in 15 million different combinations (Von der Leith, 2004).Prediction toolsN-glycosylation only occurs at sites which carry the specific Asn-XSer/Thr motif. If thismotif occurs in a given peptide sequence, it represents a potential glycosylation site. Bioinformatics | DCU
  • Bioinformaticians have taken advantage of this knowledge to uncover information aboutthe ubiquity of glycoproteins in nature. Zafar et al (2011) used a computer algorithm toscan all of the sequence data available on the ExPASy protein database and flag anysequences which contained the signature motif. They found that more than 50 percentof all proteins (prokaryotic and eukaryotic) contain at least one copy of the motif. Thisoverturns previously held assumptions about the exclusivity of glycosylation machineryto eukaryotes (Nothaft, 2010).A similar experiment conducted by Thanka et al applied statistical analysis to thesequences of 992 experimentally-confirmed O-linked glycoproteins in an effort todiscover a signature O-linked motif analogous to the Asn-XSer/Thr motif of N-glycosylation (Christlet, 2001). They found that the presence of a proline residue ateither the + or -1 position relative to the serine/threonine site strongly promotes 3glycosylation and that aromatic amino acids near the site strongly inhibit glycosylation.Glycobiology databasesTo aid to experiments like these, several online tools and databases have been developedto identify signature motifs for different types of glycosylation within an uploadedsequence (Kamath, 2011). NetNGlyc is an artificial neural network trained not only tofind N-glycosylation sequence motifs, but to look at them in the context of thesurrounding amino acids whose influence on the local topology and physiochemicalproperties may affect the glycosylation state of the biding site (Gupta, 2004).Similarly, NetOGlyc parses the local sequence surrounding serine/threonine sites to findprobable O-linked glycosylation sites (Julenius, 2005). Several databases have emergedwhich attempt to catalogue and document glycoproteins. Each has a different specialty.GlycoBase records the HPLC elution data for N-linked glycans (Campbell, 2008), whileGlycosuiteDB contains over 3200 unique entries from 245 different species,documenting the glycan structure, peptide linkage type and host protein (Cooper, 2003).O-GlycBase contains detailed information on O- and C- linked glycans and was thedataset used to train the NetOGlyc neural net mentioned above.An enormous amount of data is being produced by glycobiology labs around the worldand the bottleneck has now shifted to the computational analysis and interpretation ofthis data. To fully take advantage of the possibilities that this field offers, it will benecessary to build and utilise new bioinformatics tools, algorithms and databases. DCU | Bioinformatics
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