Proteins & Peptides Biosimilarity Studies

Biosimilarity studies, a key step to ensure the similarity between biosimilars and reference or original drugs, are also important for regulatory agencies to approve biosimilars for marketing. These studies involve establishing and confirming the similarity between biosimilars and original drugs, including physicochemical properties, conformation, and biological efficacy. CD Formulation deploys a series of orthogonal strategies to demonstrate changes in critical quality attributes (CQAs) of protein/peptide biosimilar products to provide highly relevant early characterization and later comparative data.

Purpose of Biosimilarity Studies

Biosimilarity studies involve comparing the biochemical and biological properties of biosimilars and original drugs and evaluating their similarities in terms of in vivo activity, pharmacokinetics, immunogenicity, etc. These studies can be conducted through in vitro experiments and animal tests to determine the degree of similarity between biosimilars and original drugs. Through similarity studies, it can be ensured that biosimilars have the same efficacy and safety as original drugs, thus providing guarantees for clinical use. At the same time, biosimilarity studies are also an important basis for regulatory agencies to approve biosimilars for marketing. The conduct of biosimilarity studies is of great significance in promoting the development and popularization of biosimilars.

Fig.1 Orthogonal analytical platforms for different Critical Quality Attributes (CQAs) are used in analytical similarity assessment. (Nupur N, et al., 2022)

Explore Our Proteins & Peptides Biosimilarity Studies

Confirmation of biosimilar similarity relies on detailed analysis of physicochemical, structural and bioefficacy attributes, as well as appropriate pharmacokinetic, immunogenicity and pharmacodynamic biological evaluations. Drawing on decades of experience in protein/peptide biopharmaceuticals and biosimilars, CD Formulation implements a strategic approach to biosimilar similarity studies that provides highly relevant early characterization and late-stage comparative data.

Our biosimilarity study team develops fully integrated plans and utilizes a range of analytical techniques to perform similarity analyses on all key attributes of your protein/peptide product, including physicochemical properties, identity, purity, molecular weight, primary structure, higher order structure, aggregates, cell-based and animal-based bioefficacy, and immunogenicity. These studies are conducted based on the criteria outlined in ICH Q6B.

Available similarity study services include:

Amino Acid Sequence Confirmation

Sequence coverage is one of the key tests to confirm the identity of a biosimilar. Our scientists perform sequence confirmation by MS-based peptide mapping and LC-MS/MS with Edman degradation.

• Peptide Mapping and LC-MS/MS: Proteins are cleaved into peptides by enzymes (usually trypsin, chymotrypsin, Lys-C). The peptides are separated by chromatography. In the mass spectrometer, the molecular weight of the peptides is determined and fragment spectra are acquired. Individual peptides are then assigned to the spectra using appropriate software.
• Edman Degradation Sequencing: This technique is more suitable for sequence analysis without prior knowledge of the sequence and can also distinguish between leucine and isoleucine.

Isoelectric Point Analysis

The isoelectric point is the pH value at which a protein does not carry any charge. Isoelectric point analysis helps identify if there are differences in the molecular structure because the isoelectric point will change if there are differences in the molecular structure. Our scientists perform isoelectric point analysis using capillary electrophoresis, or cIEF.

Post-translational Modification (PTM)

PTMs, such as deamidation, oxidation and deoxidation, N-terminal amino acid cyclization, aspartate isomerization, C-terminal lysine loss or methylation, etc., are key factors affecting protein/peptide pharmacodynamics and pharmacokinetics. Incorrect translation can reduce drug activity and even increase the risk of immunogenicity. Our PTM services are based on peptide mapping and LC-MS/MS detection. Modified peptides differ from native peptides in retention time, mass-to-charge ratio, and fragmentation spectrum. Using appropriate software, the relative level of modification can be determined.

Molecular Weight Confirmation

Molecular weight confirmation is one of the most important tests to confirm the identity of protein and peptide molecules, the profile of glycans and modifications, and the molecular structure. We use high-resolution mass spectrometers for molecular weight determination.

Primary Structure

The primary structure of a therapeutic protein or peptide is the linear sequence of amino acids in the polypeptide chain, including the location of disulfide bonds. Regulations require that the primary structure of a protein or peptide must be characterized to identify batch-to-batch differences and ensure product safety. Our team of protein experts can conduct in-depth and highly sensitive analysis of the primary structure of any type of therapeutic protein or peptide using a series of analytical technologies, including amino acid sequence analysis, Edman degradation, de novo sequencing, peptide mapping, etc.

Higher Order Structure (HOS)

HOS is the general term for the secondary, tertiary, and quaternary structures of proteins. Correct HOS is critical to ensure the normal function, activity, and stability of biopharmaceutical products. Our protein scientists apply various analytical methods to obtain HOS data about proteins and peptides to guide subsequent formulation development, process development, stability studies, etc.

• For secondary structure, circular dichroism (CD) spectroscopy and Fourier transform infrared (FTIR) spectroscopy are used.
• For tertiary/quaternary structure, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, cryo-electron microscopy (cryo-EM), and hydrogen-deuterium exchange (HDX) are used.

Charge Variants

Charge variant monitoring allows for rapid comparative evaluation of proteins in reference products and biosimilars. Our scientists use ion exchange chromatography to monitor charge variants in your reference product and biosimilar proteins to support biocompatibility studies.

Glycan Analysis

Proteins can undergo both N- and O-glycosylation. Some glycans can be immunogenic, causing adverse effects in patients, while other glycans can affect activity. Typically, we use mass spectrometry or MALDI-TOF for glycan analysis.

Aggregate Characterization

Aggregates are a key factor affecting the activity of protein/peptide biopharmaceuticals. It is very important to determine the level of aggregates. First, aggregated molecules are inactive. Second, they can cause immunogenicity and tissue degradation. Our scientists use different measurement techniques depending on the size of the particles.

• For small aggregates, size exclusion chromatography (SEC) is used.
• For larger particles, techniques based on light scattering measurements are used for analysis, such as DLS, MALS, or even analytical ultracentrifugation (AUC).

Bioactivity Assays

Comparing the bioactivity between two products is the most important aspect of similarity studies. Our scientists can tailor solutions based on the mechanism of action of your protein/peptide molecule. ELISA and surface plasmon resonance (SPR) are used to detect binding to receptors or antigens. Cell-based tests and animal model-based tests are used for functional assays.

Typical Proteins & Peptide Similarity Research Programs

Why Choose Us for Proteins & Peptides Biosimilarity Studies?

• Our extensive experience and deep industry knowledge enable us to meet the challenges of any biosimilar development and biosimilarity confirmation studies.
• Our similarity study programs include all relevant quality attributes involved in evaluating your protein/peptide therapeutics, from analytical testing and bioassays (in vitro or in vivo) to animal pharmacokinetics (PK) and/or pharmacodynamics (PD) and toxicity assessments.
• We have established advanced technology platforms, such as ligand binding assays, and enzymatic and cell-based assays, to perform PK, PD, immunogenicity, and efficacy study assays.
• We provide industry-leading bioanalytical support for PK/PD as well as efficacy and safety studies throughout the preclinical development of protein/peptide biotherapeutics.
• We provide non-GLP studies that are strictly customized according to regulatory requirements (FDA, EMA, and OECD GLP regulatory standards) and your needs for biosimilar development.
• We provide flexible testing options and customized solutions to meet different research needs.

Publication

As a leading global provider of protein/peptide biosimilarity studies, CD Formulation applies detailed analytical knowledge to evaluate the critical quality attributes of your protein therapeutic products throughout your product development lifecycle, from early to late development and ongoing manufacturing, to help you ensure that manufacturing process changes do not affect product quality, efficacy or safety and meet regulatory requirements. Please feel free to contact us if you are interested in our services.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.