Biopharma product development is a multi-step process. During process development, characterization of the product at each step can help in improving and ensuring that a high-quality product is consistently manufactured. Defining critical quality attributes for the complex biological molecule is difficult, however, analysis of physicochemical properties and linked structural & functional characterization can directly define the quality of the product. Many regulatory agencies also suggest that before seeking approval, product linked impurity and modifications should also be assessed thoroughly. Complex nature of biopharma product necessitates that a multidisciplinary approach is adopted for their in-depth characterization.
At Shantani, we utilize several advanced biophysical, biochemical and cell-biology tools to characterize these products. In the line of product development, we divide these characterizations into three categories and provide the characterization services, both on the project and on ‘a-la-carte’ basis. For biosimilar comparability, these studies are carried out along with the reference compound.
Minimum characterization is required to confirm the intended profile of the product
Even a slight variation in manufacturing and sample storage process can have a huge impact on the quality of the product. Along with the structural characterization, products are analyzed for probable oxidation, deamidation, and change in isoform pattern to ensure that their therapeutic potential is not altered.
In-depth characterization of primary, secondary & tertiary structure and function of the product is carried out
Typically carried out using ESI-Q-TOF or MALDI-TOF/TOF using Standard Operating Protocols (SOPs). Currently, for biological samples > 60 kDa, ESI-Q-TOF based intact mass analysis can provide a resolution up-to 20 ppm. We have characterized intact mass of proteins ranging from 4 kDa to 230 kDa.
Single and/or multi-protease ‘in-gel’ or ‘in-solution’ digestions of proteins are carried out using standardized protocols. Peptides are separated using RPLC and infused into ESI-Q-TOF set up for MS and MS/MS analysis. Obtained masses are compared with the theoretical digest of the proteins using bioinformatic tools and peptide-maps are prepared. For biosimilar comparability studies, protease digested peptides can also be analyzed using a HPLC-UV based method.
Utilizing standardized method, N-Glycans are extracted using PNGase F treatment, labelled with 2-AB, cleaned and analyzed using Liquid-Chromatography-Fluorescence (LC-Flur) based workflow. If required, glycans are identified using mass-spectrometry.
Utilizing standardized method, N-Glycans are extracted using PNGase F treatment, labelled with 2-AB, cleaned and analyzed using Liquid-Chromatography-Fluorescence (LC-Flur) based workflow. Further, based on the predicted glycan profile, N-Glycans are sequentially digested using Sialidase, Galactosidase, Fucosidase and additional enzymes in different combinations. LC-Flu chromatographic patterns are utilized in establishing the Glycan Linkages. Additionally, if required, digested glycans are identified using mass-spectrometry.
Charge Variants are analyzed using Liquid-Chromatography-strong-cation exchange chromatography (LC-SCX) using standard operating protocols. Based on the product, either, a salt gradient, or a combination of mobile-phase that provide linear pH-Gradient, is utilized to elute different variants. Variants are detected using in-line Ultra-Violet absorption (UV) or fluorescence detectors. Alternatively, variants can be analyzed using off-line mass-spectrometers.
In a standardized method, samples are de-formulated using Gel-Filtration-Chromatography and then analyzed using CD (near and/or Far-UV) and Fluorescence Spectroscopy. Data is analyzed and interpreted in line of establishing secondary structure components and folding pattern of the protein. Tryptophan Fluorescence Quenching experiments can also be applied for additional analysis.
Using well-optimized protocols, product is digested with proteases in reduced and non-reduced conditions and obtained peptides are analyzed using LC-UV or LC-MS based workflows.
Using well-optimized protocols, product is digested in an ultrafast mode with a defined protease. Peptides are separated using RPLC and infused into ESI-Q-TOF set up for MS and MS/MS analysis. Obtained masses are compared with the theoretical digest of the proteins using bioinformatic tools. For Global Analysis of Oxidation and Deamidation – intensities of all the Modified peptides, containing Methionine (for oxidation) and/or Asparagine & Glutamine (for deamidation), are compared with unmodified peptides. For Targeted Analysis – system suitability studies are performed and then the intensities of targeted modified and unmodified peptides are compared in establishing Oxidation and Deamidation of the product.
Redox indicator dye is used to study cytotoxicity levels in different type of cell lines. End point measurements are done using both colorimetry and fluorimeter. The assay is scalable and can be performed in different plate (6/12/24/96 wells) formats.