Analytical Procedures

Analyzing the 5′ cap by enzymatic digestion and Liquid Chromatography-Mass Spectrometry (LC-MS) is commonly used method for confirming the mRNA 5′ end structure.

AF4–MALS is an increasingly adopted, column-free, size-based separation coupled with absolute light-scattering detection that complements SEC/AEX/SV-AUC by resolving aggregate populations and—when combined with UV/dRI and appropriate assumptions—supporting composition assessments for AAV and LNP programs.

Alkaline agarose gel electrophoresis (AAGE) is a denaturing electrophoretic method run at high pH (typically NaOH) that disrupts base-pairing so DNA migrates largely as single-stranded nucleic acid according to length.

Analytical ultracentrifugation (AUC) is a first-principles, solution-phase technique for characterizing macromolecules and nanoparticles without a stationary phase (“matrix-free”).

Anion-exchange chromatography (AEX-HPLC/UPLC) separates analytes by differences in net negative charge interacting with a positively charged stationary phase.

AVI primarily assesses visible particulates and container/closure defects, ensuring each unit meets compendial ‘essentially free of visible particles’ requirements

The Bacterial Endotoxins Test (BET) is a compendial safety assay that detects and quantifies Gram-negative bacterial endotoxins (LPS) in parenteral drugs and devices using Limulus Amebocyte Lysate (LAL) or recombinant Factor C (rFC), ensuring endotoxin levels remain below regulatory limits.

The Bicinchoninic Acid (BCA) Assay is a widely used laboratory method for determining the total concentration of protein in a solution.

The Benzonase ELISA is a specific immunoassay that quantifies residual Benzonase.

(Coming Soon..) Confirms identity through the highly specific binding interaction between the drug product and a targeted antibody, proving the presence of the correct molecule. Uses antibodies specific to a particular AAV serotype (e.g., AAV9) to confirm the identity of the viral capsid.

Bio-Layer Interferometry (BLI) is a label-free, real-time optical technique used to measure biomolecular interactions.

CE-SDS is a high-resolution, automated electrophoretic technique that separates proteins by molecular weight, enabling quantitative assessment of purity, identity, and stability.

Capillary Isoelectric Focusing (cIEF) is a high-resolution electrophoretic technique that separates proteins and other amphoteric molecules based on their isoelectric point (pI), which is the specific pH at which a molecule carries no net electrical charge.

While other methods infer identity based on physicochemical properties, mass spectrometry provides unambiguous confirmation by directly measuring the molecular weight and amino acid sequence of the capsid proteins (VP1, VP2, VP3).

Cell-based potency assays are bioassays that quantitatively measure a drug’s biological activity in living cells, designed to reflect the product’s mechanism of action (MOA).

Charge Detection Mass Spectrometry (CDMS) measures the mass of individual ions in the gas phase.

Cryo-Transmission Electron Microscopy (cryo-TEM) is high-resolution imaging technique used to visualize the structure of biomolecules and nanoparticles in their native, hydrated state.

Differential Scanning Calorimetry (DSC) is a powerful thermoanalytical technique that directly measures the heat changes associated with the thermal unfolding of a molecule in solution.

Differential Scanning Fluorimetry (DSF), also known as a thermal shift assay, is a rapid and high-throughput analytical technique that monitors the thermal unfolding of a protein.

Digital PCR (dPCR) is an advanced form of the polymerase chain reaction that provides absolute quantification of nucleic acid sequences without the need for a standard curve.

Detecting and quantifying double-stranded RNA (dsRNA) is a critical analytical challenge in mRNA therapeutic development.

Dynamic Light Scattering (DLS) is a rapid analytical technique used for the low-resolution screening of particle size and the detection of aggregates.

The Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used immunoassay that combines high sensitivity and specificity with a flexible plate-based format.

Enzyme activity assays measure the functional activity of a therapeutic product.

The Extractable Volume Test is a compendial, physical test performed on parenteral drug products to confirm that the volume of solution that can be withdrawn from the final container meets or exceeds the labeled volume.

Flow Imaging Microscopy (FIM) is a particle analysis technique that combines the direct imaging capabilities of microscopy with the high-throughput analysis of flow cytometry.

The Host Cell Protein (HCP) ELISA is a multi-analyte sandwich immunoassay

The characterization of Host Cell Proteins (HCPs) by Mass Spectrometry (MS) is an advanced proteomics workflow used for the identification and quantification of individual protein impurities that originate from the host cell line used in production.

High-Performance Liquid Chromatography (HPLC) and its modern evolution, Ultra-Performance Liquid Chromatography (UPLC), are core analytical platforms used to separate, identify, and quantify the individual components within complex mixtures.

Capillary Electrophoresis with Laser-Induced Fluorescence (CE-LIF) is a high-resolution separation technique used to assess mRNA integrity and size heterogeneity in drug substance characterization and stability testing.

Analysis by Liquid Chromatography-Mass Spectrometry (LC-MS) following enzymatic digestion is is the definitive industry standard for characterizing the 5′ cap of mRNA drug substances.

Harmonized across USP <788>, Ph. Eur. 2.9.19, and JP 6.07 under the Pharmacopoeial Discussion Group (PDG).

Mass Photometry (MP) is a label-free technique that measures the mass of individual molecules in their native state directly in solution.

Mass spectrometry (MS), typically coupled with liquid chromatography (LC–MS/MS), is one of the most versatile and powerful analytical platforms. 

Membrane Filtration is the compendial standard for quantifying the number of culturable microorganisms (bioburden) in a process stream. 

Mycoplasma detection is a critical safety test for any biopharmaceutical product produced in cell culture.

Nanoparticle Tracking Analysis (NTA) is a high-resolution analytical technique used to visualize, track, and size individual nanoparticles in a liquid suspension on a particle-by-particle basis.

Nanopore sequencing is a third-generation technique, works by passing a single nucleic acid strand through a biological pore (a “nanopore”) and measuring the characteristic changes in an electrical current as each nucleotide passes through.

Next-Generation Sequencing (NGS) refers to a set of high-throughput platforms that allow the simultaneous sequencing of millions of DNA molecules. 

The primary method used in the biopharmaceutical industry is Freezing Point Depression Osmometry, as defined in USP General Chapter 〈785〉.

The definitive “gold standard” for confirming a protein’s primary structure by generating a unique peptide “fingerprint” that is compared to a reference standard.

Poly(A) Tail Analysis via Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful “bottom-up” method used for the detailed characterization of the mRNA poly(A) tail.

The measurement of pH by potentiometry is a fundamental physicochemical test that quantitatively determines the acidity or alkalinity (hydrogen ion activity) of an aqueous solution.

USP General Chapter <61> Microbial Enumeration Tests provides harmonized, validated methods for quantifying viable aerobic microorganisms.

Quantitative PCR (qPCR), also known as Real-Time PCR, is the industry-standard method for detection and quantification of nucleic acids. While newer technologies like Digital PCR (dPCR) are gaining traction for viral titering, qPCR remains a widely accepted GMP method for impurity testing – particularly for Residual Host Cell DNA (resDNA) – and is commonly used […]

Quantitative PCR (qPCR), also known as real-time PCR, is a molecular biology technique used to amplify and simultaneously quantify a targeted DNA molecule.

Rapid Microbiological Methods (RMMs) are advanced technologies designed to provide faster detection and enumeration of microorganisms compared to traditional, growth-based methods like membrane filtration.

Please check digital PCR and ELISA posts.

The Replication Competent Adeno-Associated Virus (rcAAV) Assay is a critical safety test designed to detect the presence of inadvertently generated, replication capable AAV particles in a gene therapy product.

A classic identity test that uses enzymes to cut the vector genome at specific locations, generating a unique pattern of DNA fragments that serves as a fingerprint.

Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) is a chromatographic technique that separates molecules based on their hydrophobicity.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a sensitive and specific technique used to detect and amplify RNA sequences. 

Sanger sequencing is a well-established, high-accuracy method for determining the nucleotide sequence of targeted DNA regions.

Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) couples a size-exclusion chromatography (SEC) system with a MALS detector.

The SoloVPE system enables rapid, accurate UV-Vis concentration measurements of proteins and nucleic acids without dilution, using Variable Pathlength Technology (VPT) and Slope Spectroscopy.

Instrumental color measurement is a quantitative procedure that provides objective data to support the broader Critical Quality Attribute of Appearance, which also includes assessments of clarity and opalescence.

The Sterility Test is a compendial microbiological method, defined in USP <71> and other major pharmacopeias, to determine whether a parenteral drug product contains viable contaminating microorganisms (bacteria, yeast, or fungi).

Transgene Expression Assays are a critical category of in vitro cell-based bioassays used to evaluate the biological activity (potency) of a gene therapy product.

Transmission Electron Microscopy (TEM) is a high-resolution imaging technique that uses electrons to visualize specimens at the nanoscale.

Turbidimetry and Nephelometry are instrumental techniques used to quantitatively measure the clarity, or lack thereof (opalescence/haze), of a liquid drug product.

UV-Vis Spectrophotometry measures how much light a substance absorbs at specific wavelengths in the ultraviolet (UV) and visible portions of the electromagnetic spectrum.

Overall, MVI remains the universally accepted compendial method for ensuring parenteral drug products are essentially free of visible particulates. Despite subjectivity and throughput challenges, it serves as the regulatory reference against which emerging automated systems are qualified.