5′ Capping Efficiency
5′ Capping Efficiency is a Critical Quality Attribute (CQA) for messenger RNA (mRNA) drug substance and drug product.
Quality Attributes
Appearance
Appearance is a qualitative assessment of a parenteral drug product’s color, clarity/opalescence, and absence of visible particles.
Assay (Strength) / Concentration
Concentration/Strength must be reported as an absolute numerical value (e.g., mg/mL, μg/mL, vp/mL, or gc/mL), with units clearly stated in all regulatory filings.
Bioburden: Pre-Sterilization Microbial Control (IPC)
Bioburden is the number of viable microorganisms present in a process stream prior to sterilization (e.g., unfiltered bulk solution)
Capsid Aggregation
Capsid Aggregation is the process where individual Adeno-Associated Virus (AAV) particles self-associate into larger, multi-particle complexes.
Capsid Charge Heterogeneity
For Adeno-Associated Virus (AAV) vectors, Capsid Charge Heterogeneity refers to the presence of viral particles with varying net surface charges, despite identical primary amino acid sequences.
Capsid Empty/Full Ratio
The Empty/Full Capsid Ratio is a measure of product purity for AAV gene therapies. It defines the proportion of viral capsids that successfully encapsulate the therapeutic genome (“full”) versus those that do not (“empty”).
Capsid Post-Translational Modifications (PTMs)
Post-Translational Modifications (PTMs) are covalent chemical changes that occur to proteins after they are synthesized.
Capsid Protein Ratio (VP1/VP2/VP3)
The Adeno-Associated Virus (AAV) capsid is a protein shell composed of 60 subunits made from three distinct structural viral proteins: VP1, VP2, and VP3.
Capsid Serotype Identity
Capsid Serotype Identity is the definitive confirmation of the specific Adeno-Associated Virus (AAV) serotype (e.g., AAV2, AAV8, AAV9) that forms the viral vector’s protein shell.
Capsid Thermal Stability
Capsid Thermal Stability is the measure of an Adeno-Associated Virus (AAV) capsid’s structural integrity and its resistance to unfolding or dissociation when subjected to increasing temperature.
Capsid Titer
For Adeno-Associated Virus (AAV) gene therapy products, Capsid Titer is the quantitative measurement of the total number of viral capsid particles in a given volume
Container Closure Integrity (CCI)
Container Closure Integrity (CCI) is the ability of a primary packaging system (e.g., vial, stopper, and seal; or a pre-filled syringe) to prevent the ingress of contaminants and the loss of product content.
dsRNA Impurity
Double-stranded RNA (dsRNA) is a critical process-related impurity in messenger RNA (mRNA) drug substance and drug product.
Encapsidated DNA Impurities
Encapsidated DNA impurities are non-target, product-related DNA fragments from the host cell genome or production plasmids that are inadvertently packaged inside the AAV capsid during manufacturing.
Endotoxin
Endotoxin (lipopolysaccharide, LPS) is a Gram-negative bacterial cell wall component and a potent pyrogen.
Extractable Volume
Extractable Volume is the verified quantity of solution that can be consistently withdrawn from a final drug product container.
Fill Volume
Fill Volume is the volume of drug product dispensed into the final container (e.g., vial or prefilled syringe).
Functional RNA Content
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Identity
Identity is the quality attribute that confirms a drug contains the intended active and exhibits structural (and, where applicable, immunochemical/biological) features characteristic of that product.
Lipid Identification & Content
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mRNA Concentration (Total)
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mRNA Integrity / Size Heterogeneity
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mRNA Purity
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Mycoplasma Contamination
Mycoplasma are a genus of small, simple bacteria that lack a cell wall, making them resistant to many common antibiotics and difficult to detect by standard microbiological methods.
Osmolality
Osmolality is a measure of the total solute concentration in a solution and is a fundamental physicochemical property of a biologic drug product.
pH (potential of hydrogen)
pH measures the acidity or alkalinity of an aqueous solution.
Poly(A) tail length, distribution, and heterogeneity
The poly(A) tail, is a long sequence of adenine nucleotides (~50-250 A’s) added to the 3′ end of an mRNA molecule.
Potency
Potency is the quantitative measure of a product’s ability to elicit its intended biological effect.
Replication Competent AAV (rcAAV)
Replication-Competent AAV (rcAAV) are rare, undesirable viral particles that can arise from recombination during AAV vector manufacturing, resulting in genomes containing both rep and cap genes flanked by ITRs.
Residual Benzonase
Benzonase is a genetically engineered endonuclease used during the manufacturing of many biologics, particularly AAVs, to digest and remove unwanted DNA
Residual Host Cell DNA (hcDNA)
Residual hcDNA consists of fragments from the host cell line (e.g., CHO, HEK293) remaining after purification.
Residual Host Cell Proteins (HCPs)
Host Cell Proteins (HCPs) are a complex mixture of proteins generated by the host cells (e.g., CHO, HEK293, E. coli) used during the manufacturing of a biopharmaceutical.
Residual Plasmid DNA
Residual Plasmids are DNA fragments originating from the plasmid vectors used to transfect host cells during the manufacturing of certain biologics, most notably AAV gene therapies.
Residual Process Impurities (NTPs, cap analogs, buffer components)
Residual Protein (IVT enzymes)
Residual IVT enzymes are process-related impurities that remain in the mRNA drug substance after the in vitro transcription (IVT) reaction.
Sterility
Sterility is the absence of viable microorganisms.
Sub-Visible Particles (SVP)
Sub-visible particles (SVPs) are particles present in parenteral drug products that are not visible to the naked eye, typically ranging in size from 1 to 100 micrometers (µm).
Vector Genome Identity
Vector Genome Identity confirms that the packaged therapeutic genome has the correct nucleotide sequence.
Vector Genome Integrity
Vector Genome Integrity measures whether the DNA payload within an AAV vector is full-length, intact, and free from major truncations or modifications.
Vector Genome Titer (Concentration/Strength)
Genome Titer measures the concentration of viral particles carrying the vector genome, usually reported as vector genomes per milliliter (vg/mL).
Visible Particulates
Visible particulates are mobile, undissolved particles—other than gas bubbles—unintentionally present in an injectable product.