The in-use stability study simulates the actual clinical application scenarios of biological products and investigates critical quality attributes including physical, chemical and biological parameters. It verifies that key quality indicators remain compliant with established specifications under the influence of environmental factors such as temperature, air exposure and light irradiation. The study aims to provide basis for prescribing preparation procedures, storage conditions and in-use shelf life for products requiring reconstitution or dilution in clinical settings, support the compilation of product insert and labeling information, and guide safe clinical medication.
Antibody drugs are composed of proteins with inherent physicochemical instability. Subjected to mechanical stress such as shaking, proteins are exposed to the gas-liquid interface, where hydrophobic regions adsorb onto the interface, triggering conformational changes and protein aggregation. The underlying mechanisms are as follows:
As biological macromolecules, proteins are susceptible to alterations under light exposure; multiple internal amino acid residues may undergo oxidation, thereby changing the conformational structure of protein monomers and inducing aggregation.
Elevated temperatures accelerate chemical reactions such as protein oxidation; high temperatures disrupt hydrogen bonds and cause protein unfolding, further leading to protein aggregation.
Formulation factors of pharmaceutical preparations, including pH value, ionic strength, protein concentration and excipients, also exert significant impacts on protein stability:
Changes in pH alter the net charge of proteins, while increased ionic strength affects electrostatic interactions between protein molecules, consequently influencing protein solubility and stability.
High protein concentration facilitates aggregate formation. Protein aggregates possess immunogenicity and can induce the production of anti-drug antibodies in the immune system, thereby compromising therapeutic efficacy.
Given the inherent characteristics of biological products, a comprehensive set of physical, chemical and biological analytical methods shall be adopted for in-use stability testing. Test items shall cover at least sensitive quality attributes that reflect product quality, safety and efficacy, and the investigation of these indicators serves as a guarantee for clinical medication safety.
Relevant analytical methods are introduced as follows.
Color
Color evaluation is a quality control procedure that assesses whether the color of pharmaceutical solutions complies with established specifications via standardized methodologies. It monitors product stability, purity variations and potential degradation risks to ensure the safety and efficacy of biological products.
Chemical Stability
Degradant formation: Color changes (e.g., yellowing, browning) may result from protein oxidation, glycation and aggregation.
Marker of light sensitivity: Certain biological drugs (e.g., monoclonal antibodies) are prone to photooxidation under light irradiation, and color variation serves as a direct indicator of light stability.
Safety
Abnormal color change acts as an alert signal for product deterioration.
Clarity
Clarity refers to the light scattering phenomenon caused by particulate matter (e.g., protein aggregates, degradation products or foreign impurities) in solution, reflecting the physical homogeneity of pharmaceuticals.
Chemical Stability
Oxidation may alter the refractive index of solutions or form insoluble particles, resulting in abnormal turbidity.
Physical Stability
Increased turbidity during storage usually indicates the occurrence of protein aggregation.
Visible Particulates
Visible particulates refer to unintended, insoluble particles (excluding air bubbles) that are visible to the naked eye and flow with the liquid in injections, such as metal scraps, glass fragments, fibers, rubber particles and precipitates. Entry of visible particulates into the human body may cause vascular embolism, phlebitis, granuloma, allergic reactions and even systemic infection, posing direct risks to patient medication safety.
Subvisible Particulates
Subvisible particulates are defined as invisible particles with a particle size below 50 µm. The average diameter of human capillaries ranges from 6 to 9 µm; hence, subvisible particulates exceeding a certain size or quantity may accumulate in the human body and cause irreversible harm.
Subvisible particulates are frequently detected during in-use stability testing for the following reasons: intravenous infusion via infusion pumps may increase particle generation during antibody administration; mechanical shock such as shaking and collision of infusion bags can induce particle formation of antibodies. Filters are therefore applied to remove particles generated during preparation and administration.
pH Value
Impact
pH value directly affects the three-dimensional conformation of proteins; deviation from the optimal pH range may lead to aggregation, degradation or irreversible denaturation.
Safety Risks
pH values deviating from the physiological range may trigger vascular irritation, pain or hemolytic reactions; massive infusion of drugs with abnormal pH may disrupt the acid-base balance of body fluids in patients. Common diluents for antibody drugs include 0.9% sodium chloride solution and 5% glucose solution. Clinically used glucose solution is generally acidic with a pH range of 3.2-6.5. Where low pH exerts adverse effects on the product, neutral 0.9% sodium chloride solution shall be selected as an alternative diluent.
Abnormal pH may induce the formation of subvisible particulates (e.g., proteins tend to aggregate and precipitate near their isoelectric points), which requires comprehensive analysis combined with clarity and visible particulate assessment.
Protein Content
Variations in protein content reflect drug degradation or adsorption tendency, directly affecting the biological activity and safety of pharmaceuticals.
In clinical in-use stability studies, drugs may adsorb to contact materials and filter membranes. For diluted low-concentration products, it is necessary to minimize the number of administration components and pipeline length, and evaluate filter compatibility to guarantee the effective drug dosage. If protein concentrations of low-, medium- and high-concentration samples collected at sampling points (effluent from infusion sets) comply with predefined quality specifications without significant changes, it indicates that disposable clinical infusion sets exert no obvious adsorption effect on the product.
As a core indicator for stability studies, protein concentration is combined with other analytical methods to ensure drug safety:
SEC-HPLC is used to monitor the ratio of monomer to aggregate, and combined with protein concentration data to evaluate physical stability and prevent reduced efficacy caused by irreversible aggregation.
Reduction of excipient concentration during dilution may disrupt antibody conformation, which requires combined monitoring of protein concentration and biological activity.
Purity Analysis
Purity is a critical quality attribute (CQA) of biological drugs, directly determining product safety and therapeutic efficacy. It reflects the homogeneity of active pharmaceutical ingredients, and the presence of impurities impairs biological activity and safety. Chromatographic methods are commonly applied for purity detection: Ion Exchange High Performance Liquid Chromatography (IEX-HPLC) or Imaging Capillary Isoelectric Focusing (icIEF) are used to detect charge variants, while Size Exclusion High Performance Liquid Chromatography (SEC-HPLC) is adopted for the determination of high molecular weight variants.
SEC-HPLC
SEC-HPLC is a separation technology based on protein molecular size. It employs a porous gel column: larger protein molecules cannot enter the porous structure of the gel and elute faster, while smaller impurity molecules penetrate into the gel pores and elute at a slower rate. The absorbance signal eluted from the column is monitored to obtain the molecular size distribution of protein samples, so as to evaluate product purity.
CE-SDS
nrCE-SDS (Non-reduced CE-SDS): Under non-reduced conditions (without addition of β-mercaptoethanol or DTT), antibodies retain intact disulfide bond structures. Coated uniformly with Sodium Dodecyl Sulfate (SDS), molecules are separated based on molecular weight differences, which is mainly used for the detection of intact antibodies and low molecular weight variants.
rCE-SDS (Reduced CE-SDS): Under the action of reducing agents such as DTT, interchain disulfide bonds of antibodies are broken, dissociating into heavy chains, light chains and non-glycosylated heavy chains. Separation and detection are performed via capillary electrophoresis to assess chemical degradation such as fragmentation and free toxin release.
Charge Variant Detection Methods
Common methods include Cation Exchange High Performance Liquid Chromatography (CEX-HPLC) and electrophoretic technology (icIEF).
CEX-HPLC
Separation is achieved based on the specific charge interaction between cation exchangers and molecules with different charge properties. After sample pretreatment and loading onto the CEX column, acidic and basic variants bind differently to the stationary phase cation exchangers due to distinct charge characteristics. As the mobile phase flows through the column, variants elute at different rates, realizing effective separation.
icIEF
A stable pH gradient is established inside the capillary. Biological molecules (e.g., monoclonal antibodies, fusion proteins) migrate to their isoelectric points (zero net charge) under an electric field and focus into distinct bands for charge variant separation. Compared with conventional Capillary Isoelectric Focusing (CIEF), icIEF adopts full-column dynamic imaging technology to monitor the entire focusing process of the capillary in real time via a high-sensitivity CMOS sensor.
Biological Activity
Biological activity/potency is a core indicator to evaluate the pharmacological efficacy of biological drugs. Based on the mechanism of action, antigen-binding activity (e.g., ELISA) and/or cell functional activity (e.g., reporter gene assay) are generally selected for potency determination. The relative biological potency is calculated by comparison with standardized reference materials.
ELISA (Enzyme-Linked Immunosorbent Assay)
Based on the principle of antigen-antibody interaction, ELISA quantifies target molecules via enzyme-substrate colorimetric reaction. Common formats for activity determination include double-antibody sandwich method and competitive ELISA.
Reporter Gene Assay
Target cells are constructed and incubated with antibody drugs to activate reporter gene expression. The expression level of luciferase is detected to reflect the degree of cell activation, so as to determine drug biological activity.
Conclusion
Test items for clinical in-use stability cover key quality attributes reflecting product quality changes, safety and efficacy, which are also core release specifications of biological products. For BLA submission, in-use stability testing shall adopt fully validated analytical methods. However, as the sample concentration and buffer matrix for in-use stability differ from those of drug substance and finished product, supplementary method validation or even redevelopment and subsequent validation are required. For the IND stage, analytical methods are still in the early development phase; only appropriate method applicability assessment is needed to ensure suitability for sample detection at corresponding concentrations (especially low-concentration samples) and buffer matrices.
If all test items of in-use stability meet predefined acceptance criteria, it can be confirmed that the product remains stable under specified conditions throughout the in-use period. If the results fail to meet stability requirements, root cause analysis shall be conducted, and repeat tests shall be performed after adjusting experimental conditions until compliance is achieved. In-use stability studies not only guarantee the safety of clinical medication, but also provide reference for storage conditions during product use based on research findings.
