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Biologics Services

Intact Molecular Weight Analysis

Advancements in the technology of modern mass spectrometry, such as electrospray ionization (ESI) and the time of flight (TOF) mass detector, have made it possible to measure the molecular weight of the protein with high accuracy (±0.01%). With this kind of mass determination accuracy, an intact molecular weight analysis of the protein could provide useful information on protein identity by comparing the measured mass with the expected. In addition to confirming the primary sequence of the protein, it is also very useful for detecting any post-translational modifications such as glycosylation, phosporylation, and oxidation of the protein.

Intact molecular weight analysis is used as a quick confirmation of protein identity and for detecting any possible post-translational modifications. It can be used as a fast method to profile the N-glycan distribution of IgG for cell line selection or cell culture optimization. Combined with some separation method (such as reverse phase or size-exclusion chromatography), intact molecular weight analysis can be a very powerful tool to characterize product-related impurities or degradants.

RMI employs a very advanced Q-TOF mass spectrometer to achieve the highest sensitivity and mass accuracy for intact molecular weight analysis.

Disulfide Bridge Analysis

The formation of disulfide bonds in proteins is a post-translational modification which is essential for stabilizing and maintaining the three-dimensional structure of proteins. A correctly disulfide bond linked protein is critical for its biological activity. Determination of disulfide bridges is an important step toward the understanding of the structural properties of a protein.

The general strategy for the determination of disulfide bridges is to use non-reduced peptide mapping. In non-reduced peptide mapping, the protein is digested with a suitable enzyme under non-reducing condition to generate disulfide linked peptides. The disulfide linked peptides are then separated on a reverse HPLC system and then identified by MS and MS/MS sequencing. Multiple disulfide-containing peptides are subjected to further analysis by partial reduction and alkylation to produce simpler disulfide-linked peptides suitable for unambiguous assignment. A reduced peptide mapping is generally performed to facilitate data interpretation.

Disulfide bridge determination is an important component of protein structural characterization for the Biologics License Application (BLA) as described in ICH Q6B guidelines.

N-terminal Sequencing

N-terminal sequencing can be accomplished by using peptide mapping. During the process, a suitable enzyme is used to digest the protein to generate the N-terminal containing peptide. The N-terminal containing peptide is then sequenced by MS/MS using a high performance accurate mass spectrometer. The procedure works with an N-terminal blocked protein (no de-blocking procedure needed). The turnaround time of this procedure is much faster than Edman degradation.

N-terminal sequencing can be used as an identity test for reference material characterization, to demonstrate comparability and consistency between batches. It can also be used at the discovery stage for de novo sequencing of unknown proteins.

C-terminal Sequencing

The assessment the C-terminal of the protein is a very important step for complete protein characterization. C-terminus heterogeneity is frequently observed during the monoclonal antibody production process. The C-terminal sequencing is usually accomplished by using peptide mapping and MS/MS sequencing. The protein is digested using a suitable enzyme to generate a C-terminal containing peptide. The C-terminal containing peptide is then sequenced by MS/MS using a high performance ion trap mass spectrometer.

C-terminal sequencing can be used as an identity test for reference material characterization and demonstration comparability and consistency between lots. It can also be used at the discovery stage for de novo sequencing of unknown proteins.

Peptide Mapping

Peptide mapping is the most powerful biochemical technique used to analyze the primary structure of a protein. In a peptide mapping analysis, the protein is digested with a suitable enzyme to generate peptides. The peptides are then separated by a reverse phase HPLC to generate a unique “fingerprint” map of the protein. An online mass spectrometer will be used to provide the MS/MS sequencing of the peptides.

For biologics characterization, peptide mapping has been widely used to confirm the primary sequence of the protein fermented from cells. It has also been used to detect any post translational modifications (such as deamidation, oxidation) of the products. At different stages of biologics development process, peptide mapping has been used to characterize the reference material, to demonstrate comparability and consistency between batches, and to release final products.

For discovery research, peptide mapping with MS/MS sequencing is the most powerful technique for de novo sequencing of unknown proteins. It has been used to determine the disulfide bridges, to locate the sites of glycosylation (both N- and O- linked) and phosphorylation.

At RMI, we employ a reverse phase HPLC with a narrow bore or capillary column for the highest sensitivity. For MS/MS sequencing, a high performance mass spectrometer is used with the most advanced ion trap technology. Our senior staff has more than 18 years of biopharmaceutical industry experience in protein analysis and characterization using mass spectrometry. We offer the highest quality peptide mapping and are ready to solve any challenges in the area of protein characterization for discovery and development.

Glycosylation Analysis

After post translation, proteins can be further modified by being attached to carbohydrate groups by glycosidic bonds via glycosylation. There two types of glycosidic bonds that can occur in the process, called N-linkage and O-linkage. During the biologics manufacturing process, the glycosylation of the protein can be affected by the cell-line and bioreactor conditions. The variation in glycosylation of the protein could potentially have an impact on the bioactivity, safety, and efficacy of the product. As stated in the ICH Q6B guideline, the glycosylation (glycosylation sites, carbohydrate content, and carbohydrate structure) of a therapeutic glycoprotein should be characterized to the extent possible.

Glycosylation Site Analysis

The goal of the analysis is to determine the glycosylation sites (both N-linked and O-linked) of a glycoprotein. The information regarding the occupancy and oligosaccharide structure of the site can be obtained through the analysis. The LC/MS/MS peptide mapping is used as a major tool for glycosylation analysis. In order to generate the desirable glycopeptides easy for identification, different or combination of the enzymes (trypsin, Lys-C, Asp-N, Glu-C) could be used for digestion. The glycopeptides will be fragmented by a high performance ion trap mass spectrometer for structural analysis.

The glycosylation site analysis is an important part of the complete characterization of the product for BLA filing, establishing reference material, and demonstration of comparability between batches.

Site-Specific Oligosaccharide Profile Analysis

The traditional oligosaccharide profile analysis is to use PNGase F to remove the N-glycans from all N-linked sites within a glycoprotein and then to analyze the released N-glycans. However, if a glycoprotein contains more than one N-linked site, the specific N-glycan population profile at each site would be lost through this approach. As the FDA/EMEA is demanding more site-specific oligosaccharide profile information for the existing and future biological products, RMI has developed a site-specific oligosaccharide profile analysis to fulfill these requests.

Site-specific oligosaccharide profile analysis is to determine the overall population profile of the N-glycans or O-glycans at each individual glycosylation site within a glycoprotein. The key for site-specific oligosaccharide profile analysis is to generate the glycopeptides which only contain one glycosylation site. The glycopeptides are then separated by a reverse phase HPLC and detected by a high performance mass spectrometer. The oligosaccharide population is then quantified using the extracted ion chromatograms from the mass spectrometric data for each oligosaccharide species.

Site-specific oligosaccharide profile analysis is an important assay in response to FDA/EMEA requirements for the well characterized biologics. It can be used to characterize the reference material, demonstrate comparability and consistency between batches, and release final products

Characterization of Impurities and Degradants for Biologics

Impurities, which can be product related or non-product related, and degradants in the biological products could have a severe impact on the bioactivity, safety, and efficacy of the final product. Therefore, it is extremely important to characterize any impurities and degradants in the product during all stages of the manufacturing process. It is a key step toward defining well characterized biologics and demonstrating comparability and consistency between batches.

Reversed phase or size-exclusion chromatography will be used to analyze the impurities and degradants in the products. Usually, these separation techniques are combined with mass spectrometric analysis for identification purposes. Intact molecular weight analysis, peptide mapping, and N-terminal sequencing could be used for the characterization of the impurities and degradants.

Our senior staff has more than 18 years of biopharmaceutical industry experience in trouble shooting with protein impurities and degradants. With our state-of-the-art mass spectrometry capacities and our extremely experienced staff, RMI is ready to solve the most challenging problems of biologics manufacturing.