When working in biopharmaceutical or biologics analysis, you need as many tools as possible. Be it that you’re looking for the right instruments (like our new bioZen™ LC columns), the best resources, or the most insightful educational content, Phenomenex is here to help you at every step. This is why we put together the following biologics glossary.
We gathered the most important terms when it comes to biopharmaceutical and biologics analysis, and created an easy to use set of definitions and purposes. Explore the biologics glossary below!
Definition: Aggregation is the spontaneous formation of oligomerization and potential irreversible agglomeration of large molecules. Because proteins exist in equilibrium between folded and unfolded states, any change to environment, such as pH, temperature, or solvent composition, can disrupt the protein into a more unfolded state, thus leading to a cascade of aggregation.
Purpose: Quantitation of Aggregate is a critical quality attribute, as there is a high risk in aggregate causing immunogenicity. This is also a method for determination of higher order structure (quaternary structural determination of mAb) as well as an indicator of stability, as in formulation.
Definition: A biologic with the same sequence and similar function to an innovator biologic. Characterization of biosimilars requires the same techniques as its corresponding biologic.
Purpose: Analysis needs to show that the follow-on biologic is “similar to innovator.” Guidelines from FDA and EMA indicate the need for preclinical studies as well as extensive characterization.
Definition: Alterations of mAbs and proteins (i.e. post translational modifications) can lead to charge heterogeneity of the protein. These include acidic and basic variants.
Purpose: Because isoelectric points can indicate stability and/or efficacy concerns, determination of charge heterogeneity by IEX is a common analysis. This can also go along with stability studies/forced degradation. QC methods often use charge heterogeneity as a QC lot release method, though there are different orthogonal methods to determine this.
Drug–Antibody Ratio (DAR)
Definition: Drug to Antibody Ratio can mean two things: 1) The distribution of different attachments of payload (cytotoxic component) to an Antibody Drug Conjugate (ADC), 2) The average of different drug loads for an ADC- for cysteine conjugates, typically 3-4 is desirable.
Purpose: The average DAR or DAR distribution can have implications on ADC efficacy, safety clearance and pharmacokinetics. It is also necessary to characterize it as an “intentional chemical modification” performed on the protein.
Definition: Determination of glycoforms using HILIC or HILIC-MS; this can be performed either on the intact protein or enzymatically digested fragments (e.g. Fc/Fab)
Purpose: Glycosylation of proteins- specifically monoclonal antibodies- have indications in protein structure, as well as efficacy, clearance and immunogenicity. Glycosylation is arguably the most critical of all PTM’s.
Definition: Intact mass analysis involves using high resolution accurate mass (HRAM) instrument to confirm sequence and identify post-translational modifications like glycosylation. Using both highly efficient particles and optimized surface chemistries help for good peak shape as for HRAM scans across the keep. Performance under high temperature and using formic is needed for Zen-like mass spectra.
Purpose: Sequence confirmation and identification of post translational modifications. Also used as an orthogonal technique for abundance of glycoforms. For ADC’s with site-specific or lysine conjugations, intact mass gives average DAR and drug distribution.
Intact Reversed Phase
Definition: Intact reversed phase is an impurity analysis method. This is typically LC-UV, using trifluoroacetic acid (TFA). This ion pairing agent helps to further improve peak shape; even relatively small chemical changes like methionine oxidations can be separated on large proteins and protein fragments. A common approach with mAb’s is “middle down,” using reducing agents and/or enzymatic digestion to further identify PTM’s specific to a region of the therapeutic antibody.
Purpose: Intact reversed phase is commonly used for impurity analysis.
Definition: The most common method to identify different glycoforms/glycosylation patterns. It can be performed as early as cell line development/screening because glycosylation affects so many different attributes of the mAb or glycoprotein (including effector function, immunogenicity, clearance). N-glycan is a robust (if not labor intensive and long) method for absolute quantitation of glycoforms and is still the gold standard in glycosylation characterization.
Purpose: Glycosylation of proteins- specifically monoclonal antibodies– have indications in protein structure, as well as efficacy, clearance and immunogenicity. Glycosylation is arguably the most critical of all PTMs.
Definition: The digestion of a protein using a site-specific protease (typically a serine protease like trypsin which cleaves proteins at the at carboxy side of both lysine and arginine). This application can be used for primary sequence confirmation or used as a “conforms to standard” method.
Purpose: For protein characterization, peptide mapping is commonly used for identification of non-enzymatic, spontaneous post translational modifications, such as deamidation, oxidation, succinimide formation, pyroglutamate formation, and others. It can be used for confirming primary sequence, though more commonly for PTM identification.
Peptides – Natural
Definition: Peptides isolates from natural sources (immunosuppressants, collagen derived peptides, antimicrobials, etc.)
Purpose: Many natural peptides are the starting point for potential cytotoxic agents, antimicrobials, immunosuppressants, etc. These are often desired because of their low immunogenicity and high potency.
Peptides – Synthetic
Definition: Peptides synthesized by solid phase or liquid phase synthesis, using Fmoc/t-Boc chemistries
Purpose: Peptides synthesized by SPS allow for the flexibility of incorporation of unnatural amino acids and modifications that make them more favorable for PK (e.g. acylation)
Definition: Any disruption in primary or secondary structure- through post translational modifications- can lead to protein misfolding, which can be monitored closely with hydrophobic interaction. One common cause of misfolding is oxidation (methionine, tryptophan).
Purpose: Protein misfolding can occur and must be assessed in-process.
Definition: Proteins or peptides derived from host material, including Chinese Hamster Ovary (CHO), E. Coli, Yeast, and Human Embryonic Kidney (HEK 293)
Purpose: Protein therapeutics, especially those developed from either full-length antibody or engineered fragments, can be used
Synthetic DNA and RNA Oligonucleotides
Definition: Short oligonucleotides, commonly 18-24mer but ranging from 10-60mer, that are manufactured by solid phase synthesis using DMT chemistry
Purpose: DNA and RNA oligonucleotides are used in a range of biotherapeutics including gene expression, gene therapy, and cell therapy
Definition: Biologic or large molecule ranging in structure including viral vectors, recombinant proteins, peptides, and antibodies
Purpose: Vaccines are typically used to elicit an immune response, resulting in active acquired immunity.
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