|Send to printer »|
Feature Articles : Jan 1, 2005 ( )
Exploiting Post-Translational Modifications
Assessing Strategies to Meet Demand!--h2>
By 2010, glycoprotein and antibody drugs are expected to exceed $68 billion in worldwide sales. Meeting the demand for such therapeutics is generating challenges for both production and manufacturing.
Chief among these is accurately and reproducibly mimicking natural post-translational modifications. Identifying and characterizing such modifications impact half-life, quality, cost, and potential undesired immunogenicity of the therapeutic.
The post-translational modification of glycosylation customizes a broad spectrum of proteins. Addition of carbohydrates helps establish the three-dimensional structure and thus, inherently affects function (efficacy) of a therapeutic. But carbohydrates can form branching structures with sophisticated architecture.
It is estimated that the nine common human monosaccharides could form more than 15 million possible simple tetrasaccharides. Additionally, cells differ in their complement of glycosylation enzymes. Thus, different host cells can produce different glycosylation patterns on expressed recombinant proteins.
IBC's "Third Annual Post-Translational Modifications" conference was held last November. Presenters shared their perspectives for effectively dealing with such issues. Strategies discussed include using arrays to accelerate glycan analysis, modifying glycosylation of monoclonal antibodies to enhance potency, chemically synthesizing proteins, and better monitoring of surreptitious protein aggregates.
In an attempt to more exactly match human patterns of glycosylation, most recombinantly produced therapeutic glycoproteins are expressed in mammalian cells, particularly Chinese hamster ovary (CHO) cells.
Unfortunately, CHO cells engineered to make large batches may glycosylate proteins in a nonhuman manner. This could produce potentially immunogenic proteins. Additionally, incorrect glycosylation may reduce half-life resulting in increased incidence of side effects as well as amplified costs for therapy.
Characterization of glycoproteins becomes of paramount importance. "Most proteins are mixtures of glycoforms," says Johanna Griffin, Ph.D., CCO and president, Procognia (Philadelphia). "There is no genetic code for glycans. So, analyzing them can be difficult especially if multiple different glycans are present. Today, traditional glycan analysis is laborious, complex, and can take weeks to complete."
Standard methods include isoelectric focusing (IEF), acid hydrolysis (to determine monosaccharide composition), chemical or enzymatic cleavage, and mass spectrometry (MS) to identify glycans.
Procognia uses a lectin-based array to speed up glycan analysis. Dr. Griffin notes, "It takes only about three hours to analyze up to 20 different samples of the same protein and this can be done in real time without sample purification. So, all samples are analyzed in parallel with controls."
The biochip contains about 30 different lectins in multiple concentrations. "This is sufficient to analyze all features in a mixture. We run samples such as culture supernatants directly and detect using a labeled probe, usually a polyclonal antibody specific for the protein.
"We also perform an intensity scan of sample bound to the biochip. This is analyzed in detail with our algorithms, software, and databases. Using these new methods, one can get a highly specific fingerprint obtained in a rapid fashion that is easy to interpret."
A common and variable type of post-translational modification in glycoproteins is that of O-glycosylation. In this case, sugar groups are linked to the hydroxyls of serine or threonine residues.
"Historically, the industry has placed more emphasis on characterizing N-glycosylation of bioproducts rather than O-glycosylation," says Mark R. Hardy, Ph.D., senior scientist, characterization and analytical development, Wyeth BioPharma (Andover, MA). "This may give the erroneous impression that N-glycosylation is more important than O-glycosylation."
Dr. Hardy feels that O-glycosylation needs to play a more prominent role in the minds of biotech scientists. "Part of the difficulty in analyzing O-glycans is that they are less tractable (than N-linked glycans). There are no generic enzymes to release O-linked oligosaccharides. This post-translational modification occurs in the Golgi apparatus where sugar attachment is catalyzed by an array of specific glycosyltransferases.
"As a result, the type of sugars added varies depending on the cell expressing the glycoprotein, the state of the cell, and competition between enzymes, substrates, and other factors. So, biosynthesis isn't straightforward, it's a rather chaotic pathway."
Although a number of traditional tools are available (such as reductive alkaline cleavage or "beta elimination"), a more holistic approach can be taken using peptide mapping, liquid chromatography, and mass spectrometry.
"These exploit the selectivity of reverse-phase HPLC proteolytic fragment separation and resolution. Site occupancy and forms of glycosylation can be determined. You can also do a combination of these assays. Although this is not necessarily a quantitative technique, it is valuable for characterization and comparisons."
Other reasons for characterizing O-glycosylation include verifying the consistency of manufactured bioproducts and validating function. "Alterations in O-glycosylation can potentially affect potency, half-life, clearance, and immunogenicity, etc.," notes Dr. Hardy.
At least 10 therapeutic monoclonal antibodies (Mabs) have made it to the marketplace. Many more have failed due to issues such as toxicity, cost effectiveness, lack of efficacy, or safety. Modifying the glycosylation of Mabs holds promise as a next-generation antibody technology.
According to Nobuo Hanai, Ph.D., president and CEO, BioWa (Princeton, NJ), "Complex changes in glycosylation often are a problem because they increase the heterogeneities of antibody molecules. We found that one way to overcome this problem is to lower the content of fucose in the N-glycans of Mabs. We accomplish this by expressing Mabs in cells engineered to have a reduced capacity to add fucose.
"That is, we use a fucosyl transferase gene-knock-out technology. This produces Mabs that are more potent, especially for antibody-dependent-cell-mediated cytotoxicity (ADCC), in which leukocytes such as NK cells and monocytes kill target cells such as cancer cells.
"As a result you can reduce the Mab dose administered and still achieve efficacy. Aside from reducing side effects, this also produces a significant cost savings as there are no issues of manufacturability of the modified Mabs."
The company's Potelligent technology can produce Mabs with a 100-fold enhancement in ADCC. Dr. Hanai notes, "This is especially important since 6070% of Caucasians are poor responders to antibody medicines using ADCC such as Rituximab.
"Potelligent technology represents an improvement to overcome this genotype problem that is related to the Fc receptor. Deletion of fucose enhances the binding of Fc regions to the Fc gamma receptor IIIa in these patients, resulting in very high ADCC. Another advantage is that the technology does not require any changes to current production systems."
Chemically Synthesized Proteins
Why not skirt around the issue of appropriate addition of post-translational modifications by making a custom constructed synthetic product? That's what scientists at Gryphon Therapeutics (S. San Francisco) are doing.
Gerd Kochendoerfer, Ph.D., director, R&D, says, "Recent advancements in the total chemical synthesis of proteins allows designing and producing polymer-protein constructs of defined covalent structure and function.
"Basically, we just use standard peptide synthesis and link peptides together. Polymers can be built to be linear or branched. Also, any modification can be introduced, for example, fatty acid, phosphorylation, sugars, etc. Although the current size limitation is 200250 amino acids for synthesis, you can enlarge that by combining a recombinant protein that is, say, two-thirds expressed and then link to it the synthetically made peptide. This has been done up to ~800 residues.
"Chemical synthesis is a powerful alternative to the recombinant production of pharmaceutical proteins. The main advantages are specificity in that you target exactly where you want a modification, and much more diversity of attachment is possible since any post-translational modification can be added."
Gryphon's lead product (Synthetic Erythropoiesis Protein, or SEP) is a 166 amino acid protein that was built by total chemical synthesis. It is being evaluated as an anemia treatment.
Protein aggregates are one undesirable post-translational modification, according to John Philo, Ph.D., director of biophysical chemistry, Alliance Protein Laboratories (Camarillo, CA).
"There is heightened concern and regulatory scrutiny now in the industry regarding aggregation. Companies are seeking better methodologies to monitor aggregation, which is one of the most common ways biopharmaceutical products degrade. Not only is this a stability issue, but it's also of concern because patients may inadvertently generate an immune response to a therapeutic because of small amounts of aggregates."
Dr. Philo cites erythropoietin and beta interferon as examples where aggregates are thought to produce immunogenicity, raising safety concerns and affecting market share for multibillion dollar products.
The standard methodology for monitoring aggregation is via size exclusion chromatography (SEC). According to Dr. Philo, "SEC can fail to detect aggregation because the aggregates may simply be filtered out or dissociate due to dilution. Analytical ultracentrifugation and light scattering have advantages for measuring aggregation relative to SEC.
"For sedimentation velocity, essentially you do a hard spin, pellet the protein, and the optical system measures the rate of movement. Aggregates move faster so you can quantitate and size them as well. Also, you can do this using the formulated product, without changing into a buffer system that may artificially increase or decrease aggregation."
Coupling SEC with multi-angle laser light scattering (MALLS) detectors provides an improved way to detect aggregation because it can determine the stoichiometry of any formed complexes. Additionally, batch-mode dynamic light scattering can detect trace amounts (<0.01%) of very large aggregates, with no dilution or column-filtering effects.
Dr. Philo notes, "We are a contract research lab specializing in these and other biophysical techniques. Researchers can visit our website for information about all these methodologies (www.ap-lab.com)."
© 2016 Genetic Engineering & Biotechnology News, All Rights Reserved