Key Points for Quality Control and Regulatory Compliance of Recombinant Enzyme Proteins in In Vitro Diagnostic Reagents

Role of Recombinant Enzyme Proteins in In Vitro Diagnostic Reagents

Recombinant enzyme proteins are one of the core raw materials for in vitro diagnostic (IVD) reagents and are widely used in biochemical testing (e.g., enzymatic reagents for blood glucose and blood lipids), immunoassays (e.g., labeled enzymes HRP and ALP), and molecular diagnostics (e.g., hot-start polymerases and reverse transcriptases in PCR systems). Their purity, activity, and stability directly affect the sensitivity, specificity, and batch-to-batch reproducibility of reagents. In IVD reagent development, recombinant enzyme proteins are typically used as key raw materials, and complete traceability documents and performance validation data must be provided during registration submission.

Raw Material Management and Traceability Requirements

According to the Measures for the Administration of In Vitro Diagnostic Reagent Registration and accompanying guiding principles, when recombinant enzyme proteins are used as raw materials, their source (e.g., expression host, gene sequence origin), production process (fermentation and purification steps), and quality control standards (including purity, specific activity, and limits of residual impurities) should be clearly defined. It is recommended to establish a risk assessment file for raw materials, including control measures for potential viral contamination (from host cells). For imported enzyme raw materials, official or third-party inspection reports from the exporting country must also be provided. When selecting suppliers, companies should prioritize manufacturers certified to ISO 13485 or GMP and conduct regular on-site audits.

Establishment and Validation of Activity Assay Methods

The activity assay methods for recombinant enzyme proteins used in IVD reagents must match the intended use of the final reagent. For example, for recombinant alkaline phosphatase (ALP) used in chemiluminescent immunoassays, activity determination must be combined with the substrate system (e.g., AMPPD) and signal kinetics. The assay method should be validated, including linear range, precision, accuracy, and stability parameters. In reagent formulations, recombinant enzyme proteins often require the addition of stabilizers (e.g., glycerol, BSA, trehalose) to maintain long-term activity, and the effects of these excipients on enzyme activity must also be included in the validation scope. It is recommended to establish internal working standards and trace them to national or international reference materials (if available).

Stability Studies and Transport Validation

IVD reagents generally require long shelf life and in-use stability. For recombinant enzyme proteins as active components, stability studies should include accelerated testing (37°C, 25°C), real-time stability testing (2–8°C or -20°C), and repeated freeze-thaw/opening simulation tests. For lyophilized enzyme proteins, post-reconstitution stability must also be evaluated. During transport, extreme temperature conditions (e.g., high-temperature summer, low-temperature winter) and vibration conditions should be simulated to ensure that enzyme activity decay remains within the preset range. In accordance with YY/T 0287-2017 (ISO 13485), stability data should be submitted as part of the registration dossier.

Regulatory Compliance and Registration Submission Considerations

In China, IVD reagents containing recombinant enzyme proteins are classified as Class II or Class III medical devices. During registration, raw material performance study data, process validation data, stability study data, and clinical evaluation data must be submitted. Special attention should be paid to the following: absolute statements such as "100% accurate" are not permitted in submission materials, nor may specific clinical sensitivity/specificity data be fabricated. For recombinant enzyme proteins themselves, if sold as a separate component (e.g., raw material enzymes), they may fall under the category of biological reagents and must comply with the Measures for the Administration of Biological Products or the Requirements for the Management of Raw Materials for In Vitro Diagnostic Reagents. Companies should monitor the latest Classification Catalog for In Vitro Diagnostic Reagents and relevant review guiding principles issued by the National Medical Products Administration (NMPA) to ensure compliance.

Optimization Recommendations: Reducing Compliance Risk from the R&D Source

To reduce registration risk, it is recommended to introduce the Quality by Design (QbD) concept during the R&D stage of recombinant enzyme proteins. Establish associations between critical quality attributes (CQAs) and critical process parameters (CPPs), such as process parameters for removing endotoxins and host cell proteins during purification steps. At the same time, retain complete batch records and validation documents to respond quickly to deficiency letter requirements during review. By collaborating with regulatory consulting agencies, special risk points of recombinant enzyme proteins in IVD applications, such as cross-reactivity and non-specific adsorption, can be identified in advance.