SBIR-STTR Award

Biotherapeutics currently constitute a $70 billion market, but their clinical efficacy is often compromised by limitations arising from proteolytic degradation, uptake by cells of the reticuloendothelial system, renal removal, and immunocomplex formation. This can lead to difficulties in reaching and maintaining effective therapeutic concentrations in the blood. The most popular approach to lengthen the active life of a protein therapeutic has been conjugation to polyethyleneglycol (PEGylation). However, PEG is not eliminated via normal detoxification mechanisms in the body and the administration of PEGylated proteins can even generate anti-PEG antibodies. An emerging alternative to PEGylation is polysialylation which involves attachment of polymers of polysialic acid (PSA) to a protein. PSA is being developed for clinical use and polysialylated versions of insulin and erythropoietin have displayed improved tolerance and pharmacokinetics. PSA is synthesized in the body on neural cell adhesion molecule and, unlike PEG, is metabolized as a natural sugar molecule by sialidases. Unfortunately, as with PEGylation, the PSA conjugation process is technically complex and expensive. The multi-step, in vitro process of PSA conjugation is further complicated by the fact that standard chemical conjugation of PSA results in products with random attachment patterns and undesirable heterogeneity. Glycobia specializes in glycoengineering bacteria for use as an expression platform for the stereospecific biosynthesis of therapeutic glycoproteins. The specific hypothesis behind the current proposed studies is that glycoengineered E. coli can be used to produce PSA-conjugated proteins in a single fermentation without the need for in vitro chemical modification. Based on these observations, the objective of this proposal is to generate PSA-conjugated recombinant protein in glycoengineered E. coli by: cloning and expressing the genetic machinery for PSA synthesis in glycoengineered E. coli (Aim1) and conjugating PSA to recombinant human insulin in the periplasm of glycoengineered E. coli (Aim 2). Such an expression platform will represent a stereospecific, directed, rapid, and cost-effective process for the production of PSA-conjugated biotherapeutics that will bring the production process of PSA-conjugated proteins in concert with their tremendous therapeutic potential. , ,

Public Health Relevance:
The efficacy of protein drugs is often compromised by premature elimination from the blood, which results in unacceptably short therapeutic windows and costs that are prohibitive to the healthcare consumer. The chemical attachment of polysialic acid to therapeutic proteins results in improved tolerance and pharmacokinetics, but the process of polysialic acid conjugation is technically challenging and expensive. These proposed studies focus on producing polysialic acid-conjugated proteins in Escherichia coli fermentation without the need for in vitro chemical modification.

Thesaurus Terms:
Asp-1;Abscission;Address;Amination;Amines;Anabolism;Antibodies;Asparaginase Ii;Asparagine;Asparagine Deaminase;Bacteria;Biologic Therapy;Biological Response Modifier Therapy;Biological Therapy;Biosynthetic Proteins;Blood;Blotting, Western;Body Tissues;C-Terminal;Caps;Cachectin;Cachectin-Tumor Necrosis Factor;Capsules;Care, Health;Cells;Chemicals;Clinical;Cloning;Colaspase;Colony Stimulating Factor 3;Complex;Coupling;Drug Kinetics;Drug Metabolic Detoxication;Drugs;E Coli;Ecsf;Erbb2;Erbb2 Gene;Engineering;Engineerings;Epoetin;Erythropoietin;Escherichia Coli;Excision;Extirpation;Fermentation;Genes;Genes, Her-2;Genes, Her2;Genes, Erbb-2;Genes, Neu;Genetic;Glycans;Glycoproteins;Granulocyte Colony-Stimulating Factor;Her -2;Her-2;Her2;Her2/Neu;Half-Life;Half-Lifes;Healthcare;Heterogeneity;Human;Human Egf Receptor 2 Gene;Human, General;Humulin R;Hydration;Hydration Status;Ifn;Immune System;In Vitro;Insulin;Insulin (Ox), 8a-L-Threonine-10a-L-Isoleucine-30b-L-Threonine-;Insulin, Regular;Interferons;Kidney;L Asparagine Amidohydrolase;L-Asp;L-Asparaginase;L-Asparagine;Laboratories;Lcf-Asp;Lead;Life;Link;Lipids;Location;Man (Taxonomy);Man, Modern;Marketing;Medication;Mercaptans;Mercapto Compounds;Metabolic Detoxication, Drug;Metabolic Detoxification, Drug;Metabolic Drug Detoxications;Metabolic Glycosylation;Metabolism Of Toxic Agents;Modification;N-Acetylneuraminic Acid;N-Acetylneuraminic Acids;Nkh-1;Neural Cell Adhesion Molecules;Novolin R;O Antigen, Bacterial;O Antigens;O-Specific Polysaccharides;Pattern;Pb Element;Peptides;Periplasmic Space;Pharmaceutic Preparations;Pharmaceutical Preparations;Pharmacokinetics;Plasmids;Pluripoietin;Polymers;Polysaccharides;Polysialic Acid;Process;Production;Property;Property, Loinc Axis 2;Proteins;Recombinant Proteins;Recombinants;Removal;Reticuloendothelial System;Reticuloendothelial System, Blood;Serasa;Sialic Acid;Sialic Acids;Site;Structure;Sulfhydryl Compounds;Surgical Removal;Tkr1;Tnf (Unspecified);Tnf Receptor Ligands;Tnf-Alpha;Technology;Therapeutic;Thiols;Time;Tissues;Tumor Necrosis Factor;Tumor Necrosis Factor Family Protein;Tumor Necrosis Factor-Alpha;Tumor Necrosis Factors;Urinary System, Kidney;Western Blotting;Western Blottings;Western Immunoblotting;Asparaginase;Base;Biosynthesis;Biotherapeutics;Biotherapy;Body System, Allergic/Immunologic;C-Erbb-2;C-Erbb-2 Genes;C-Erbb-2 Proto-Oncogenes;Capsule (Pharmacologic);Chemical Standard;Clinical Efficacy;Cost;Detoxification;Drug/Agent;Erythrocyte Colony Stimulating Factor;Gene Product;Glycosylation;Granulocyte Colony Stimulating Factor;Heavy Metal Pb;Heavy Metal Lead;Hematopoietin;Immunogenicity;Improved;In Vivo;Nano Particle;Nanoparticle;Organ System, Allergic/Immunologic;Periplasm;Periplasmic;Premature;Protein Blotting;Public Health Relevance;Renal;Resection;Residence;Stoichiometry;Sugar;Sulfhydryl Group;Therapeutic Protein;Tumor Necrosis Factor (Unspecified);Uptake

Glycoengineering is a clinically-validated strategy to enhance the therapeutic properties of protein and peptide drugs. This strategy involves the attachment and manipulation of carbohydrates (i.e., glycans) to improve the stability, solubility, serum half-life, and activity of these drugs. A key factor in most glycoengineering is the inclusion of terminal sialic acid residues on glycans by a process known as sialylation. Sialic acid is large and carries a negative charge which serves to improve stability, decrease aggregation, slow clearance, and impede immune response. Nearly all examples of glycoengineering require eukaryotic cell culture and/or the in vitro conjugation of glycans. Unfortunately, eukaryotic cell culture can be expensive, time consuming, and can result in inconsistent and incomplete sialylation. Although in vitro glycosylation can result in similar effects, the process is expensive, difficult, and has not been scalable to a commercial level. Glycoengineering would be greatly improved if a simple host cell such as Escherichia coli was used for production of sialylated therapeutic proteins. Glycobia specializes in genetically engineering bacteria for the bottom up glycoengineering (BUG) of therapeutic glycoproteins. Since E. coli lacks native protein glycosylation pathways of any kind, BUG can produce tailored glycan structures that can be site-specifically conjugated to target proteins. The specific hypothesis behind this proposal is that glycoengineered E. coli can produce enhanced therapeutic proteins by sialylation in a short, single fermentation. In Phase I of this project we engineered E. coli to attach humanlike, sialyl-T glycans to recombinant proteins. The sialyl-T glycan is a sialylated Thomsen- Friedenreich antigen that can be found on erythrocytes in the human body. This type of glycosylation is simply not possible in any other known expression host. We also show that bacterial glycosylation improves the in vitro stability of therapeutic proteins expressed in E. coli. We anticipate that our BUG expression platform will be capable of producing sialylated proteins in a controlled, rapid, cost-effective manner. The objective of this proposal is to synthesize and advance our first drug targets from glycoengineered E. coli into preclinical testing by: (i) expressing, purifying, and characterizing glycosylated drug candidates from E. coli and (ii) testing stability, pharmacokinetics, and immunogenicity of these drug candidates in animal models. We will compare their performance to aglycosylated and asialylated versions of these same drugs to isolate the effects of sialylation. The benchmark of success for this project is the generation of positive preclinical validation data to further advance commercialization of this technology. This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of generating cost-effective glycoengineered protein drugs for both companies and patients.

Public Health Relevance Statement:


Public Health Relevance:
Sialylation is a glycoengineering strategy to improve the therapeutic properties of protein and peptide drugs by the addition of sialic acid residues. Sialic acid improves drug stability, decreases aggregation, slows clearance, and hinders immune response. The proposed studies focus on, for the first time, producing sialylated recombinant therapeutics in the simple bacterium Escherichia coli and advancing them into preclinical testing.

Project Terms:
Address; Anabolism; Animal Model; Animals; Aranest; asparaginase; Bacteria; base; Benchmarking; Biological Assay; Biomanufacturing; Bioreactors; Biotechnology; Blood; Capital; Carbohydrates; Cell Culture Techniques; Cells; Charge; chemical stability; Chemicals; Clinical; Collaborations; commercialization; cost effective; Data; design; dolichyl-diphosphooligosaccharide - protein glycotransferase; drug candidate; Drug Kinetics; Drug Stability; Drug Targeting; Engineering; Enzymes; Erythrocytes; Erythropoietin; Escherichia coli; Eukaryotic Cell; Excision; Feedback; Fermentation; Generations; Genetic Engineering; Glycolipids; Glycopeptides; Glycoproteins; glycosylation; Goals; Half-Life; Human; Human body; Immune response; Immunoassay; immunogenicity; improved; In Vitro; Kidney; Lead; Link; link protein; Marketing; Modeling; Mus; neutralizing antibody; Pathway interactions; Patients; Peptides; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; phase 1 study; Polysaccharides; pre-clinical; Preclinical Testing; Process; Production; Property; Protein Glycosylation; Proteins; public health relevance; Rattus; Recombinant Proteins; Recombinants; Relative (related person); Renal clearance function; Reticuloendothelial System; Serum; Sialic Acids; sialylation; Site; Solubility; Solutions; Somatropin; stability testing; Structure; success; Technology; Therapeutic; therapeutic protein; Thompson-Friedenreich Antigen; Time; uptake; Validation