Application of surrogate matrices in PCR-based method development and validation

SOT 2026 -- FDA guidance emphasizes ethical practices consistent with the principles of Replacement, Reduction and Refinement (3Rs), including New Approach Methodologies (NAMs). In PCR-based method development and validation, animal-derived biological matrices are frequently used to assess assay performance. Key biological matrices are often difficult to obtain, costly and require animal use. Use of comparable surrogate matrices, including non-animal alternatives, represents an effective strategy to reduce animal use while maintaining compliance with industry guidelines for analytical validation. Aqueous and vitreous humors are key biological matrices for ocular cell and gene therapies. The unique characteristics of ocular humors may affect test article recovery; therefore, the extraction efficiency should be assessed in PCR-based method validation. The objective was to determine if potential surrogate matrices, synthetic ocular humors and plasma, perform comparably to authentic ocular humors in a qPCR AAV DNA detection method targeting CMV promotor sequence.