Biologics and Biosimilars Characterization

Biologics and Biosimilars Characterization

 

 Biopharmaceuticals analytical characterization solutions
 for improved safety, purity, potency  and compliance.

Biopharmaceuticals (pharmaceuticals manufactured by biotechnological methods) include monoclonal antibodies (mAbs), therapeutic proteins, fusion proteins, antibody drug conjugates and other biologics. Biopharmaceuticals tend to be highly complex molecules easily affected by changes within the manufacturing process. Something as simple as a temperature fluctuation can produce a change in structure that renders the mAb less active or inactive. Biologic drugs require highly sophisticated analytical workflows for their analysis and characterization with significant emphasis on GMP and regulatory compliance. Whether you are developing an original biotherapy or a biosimilar, product characterization studies are designed to ensure a robust and well specified biological drug. Before your mAb therapy reaches a patient, it is critical biopharmaceuticals comply with safety, potency and efficacy. We offer a full portfolio of products and services to support your biological drug characterization, method development, quality control (QC) for measuring impurities, microbial contamination, quality and identity of the biopharmaceuticals.

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Biologics, Biosimilars characterization and analysis

 
Identity

Intact mass determination

Determination of the intact molecular mass, often performed with SEC-MS and appropriate standards, is a necessary step in characterization of biologics, from clone selection to verification of the final product. Intact molecular mass analysis is commonly used to demonstrate the diversity of the protein/peptide products and verify the identity of the biologics. With optimization, it can also determine the intact molecular weight of all protein products, including bispecific monoclonal antibodies.

Instead of dealing with the sometimes unwieldly intact mass of a large protein biologic, a protein digestion analysis approach is used where the sample is first cleaved into just a few fragments which upon separation can be handled individually with specialized mass spec proteomic standards.

Titer measurement

Productivity of the cell line to deliver sufficient quantities of your mAb influences its commercial potential. Used as a baseline measurement, titer is measured using Protein-A affinity chromatography (HPLC). Early in the development of a mAb, a large number of harvest cell culture (HCC) samples must be screened for IgG titer. Affinity chromatography employing a Protein A ligand is often used to determine the mAb concentration as well as to purify it for downstream aggregate and charge variant analysis.

Amino acid analysis

Used to determine the amino acid composition of a mAb, amino acid analysis is a popular way to establish product identity. It is often performed alongside an extinction coefficient measurement, a method routinely employed to establish titer between different productions.

Certified Reference Materials with values assigned by metrologically valid procedures will be critical to minimize and control experimental variations in all steps of the amino acid analysis workflow including protein extraction, fractionation, enrichment, proteolysis and analysis.

Peptide mapping

To gain an indication of the identity of your mAb, comparison of simple, single enzyme peptide maps may be sufficient. More detailed therapeutic antibody characterization including N- or C-terminal sequencing, glycan characterization, and other aspects of antibody production, analysis, purification and fragmentation can better inform product engineering. Proteomic mass spectrometry provides additional structural information.

 

Post Translation Modification

N-glycan analysis

Highly variable glycosylation may impact mAb purity and produce variable function. Monoclonal antibodies carry N-glycans on their backbone that can be released using LC-MS to assess glycosylation patterns. Characterization of the N-glycans from a monoclonal antibody is necessary as part of a work package providing full structural details of the molecule under investigation. Understanding these N-glycan pathways is important because N-glycans affect many properties of glycoproteins including their conformation, solubility, antigenicity, and recognition by glycan-binding proteins.

Monoclonal antibodies (mAbs) higher order structure (HOS) comparison

Many factors influence HOS – the 3D structure of a mAb – ranging from the choice of cell line for mAb production to bioprocessing conditions such as temperature, pH and light exposure. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a method that allows for a detailed insight into the tertiary structure of a mAb.

mAb modification analysis

Many different post-translational modifications can occur during the mAb manufacturing process, which are greatly influenced by process parameters such as media, temperature, etc. To produce a consistent product, it is critical these modifications are reproduced during every round of mAb synthesis. Modification analysis includes disulfide bridge mapping, and evaluation of glycan basic structure. It is also wise to quantify sialylation, since sialic acid can have a negative effect on your mAb.

mAb charge distribution

As a result of post-translational modifications (PTMs) or due to chemical modifications, charge variants can have a considerable impact on the biological activity and pharmacokinetics of a mAb. A regulatory requirement for mAbs, charge variant analysis can be evaluated by techniques including cation exchange chromatography (CEX) and capillary isoelectric focusing (cIEF).

Data is available for HPLC analysis of monoclonal antibodies (mAb) with Charge Variance on Antibodix® WCX-NP10 as well as for the weak cation exchange (WCX) column TSKgel® CM-STAT.

TSK-GEL HPLC columns are routinely used in biopharmaceutical and mAb analysis including SEC, ion exchange chromatography (IEC), reversed phase (RPC) and hydrophilic interaction liquid chromatography (HILIC).

 

mAb Physical testing & size distribution

mAb physical testing

Used to characterize the appearance of a mAb, for example through particulate analysis, physical testing includes measurement of pH, osmolality and concentration. Packaging integrity is also assessed within physical testing protocol, for example using Karl Fischer moisture analysis or dye ingress to confirm closure integrity.

mAb size distribution

While a single mAb product is desired, the initial production material often contains size variants such as aggregates, fragments, and biomolecules exhibiting additional light chains. Since these have the potential to affect immunogenicity and potency, it is important to monitor their presence. Size exclusion chromatography (SEC) is the most commonly used method to evaluate size distribution. BIOshell™ HPLC and UHPLC columns deliver maximum speed and efficiency for the separation of biomolecules on both HPLC and UHPLC systems. In addition, the latest innovations in TSK gel column technology are focused on improving the quality of the purity analysis of therapeutic monoclonal antibody solutions and the determination of dimer and aggregate content. Variation in Column efficiency for quality control (QC) of therapeutic monoclonal antibodies is also a concern and is affected significantly by differing characteristics of the packing material chosen and how it was packed.

 

Sterility & impurities

Host-cell protein impurities

Host cell protein (HCP) impurities, present at PPM-levels in biotherapies, are a major immunogenicity risk because they can elicit an unpredictable immune response in patients. Their complex and diverse nature makes them challenging to detect or monitor. While most HCP impurities are effectively removed in typical downstream purification processes, a small population of HCPs are particularly challenging. Knockouts of a difficult-to-remove CHO HCP, lipoprotein lipase have been developed for improved polysorbate stability in monoclonal antibody formulations.

Manufacturing additives monitoring

A wide variety of manufacturing additives must be monitored during monoclonal antibodies (mAb) development and manufacturing including Detergents, Protein A, Transfection reagents, Antibiotics, Anti-foam agents, and Growth factors.

mAbs microbial testing

A variety of microbial testing procedures are necessary for GMP and compliant pharmaceutical development and manufacturing. Many mAbs are produced in microbial organisms to benefit from rapid growth rates and high yields, making it critical to monitor and control the presence of microbial contaminants. A component of the cell wall of Gram-negative bacteria, called endotoxin, produces responses ranging from fever and chills to fatal septic shock, making pyrogen detection (MAT in vitro testing) and subsequent removal from your mAb crucial and a regulatory requirement. Used to monitor the presence of potentially harmful microbial contamination, bioburden testing should be employed throughout the entire mAb manufacturing process. Sterility testing is also important to confirm the integrity of mAb production.

 

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