PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations

Abstract

Recombinant adeno-associated viral vectors (rAAV) represent a gene therapy tool of ever-increasing importance. Their utilization as a delivery vehicle for gene replacement, silencing and editing, among other purposes, demonstrate considerable versatility. Emerging vector utilization in various experimental, preclinical and clinical applications establishes the necessity of producing and characterizing a wide variety of rAAV preparations. Critically important characteristics concerning quality control are rAAV titer quantification and the detection of impurities. Differences in rAAV constructs necessitate the development of highly standardized quantification assays to make direct comparisons of different preparations in terms of assembly or purification efficiency, as well as experimental or therapeutic dosages. The development of universal methods for impurities quantification is rather complicated, since variable production platforms are utilized for rAAV assembly. However, general agreements also should be achieved to address this issue. The majority of methods for rAAV quantification and quality control are based on PCR techniques. Despite the progress made, increasing evidence concerning high variability in titration assays indicates poor standardization of the methods undertaken to date. This review summarizes successes in the field of rAAV quality control and emphasizes ongoing challenges in PCR applications for rAAV characterization. General considerations regarding possible solutions are also provided.

Thermostability and in vivo performance of AAV9 in a film matrix

“…For AAV vectors, these have traditionally fallen into two classifications: those that characterize the number of physical particles or genomes present in a preparation and those that assess vector potency [38][39][40][41] . To date, PCR-based assays are the most widely used and accepted method for quantification of AAV vectors as they are simple and robust under ideal conditions [42][43][44] . However, the number of virus genomes present in a given production lot, cell, or tissue sample does not fully demonstrate that the virus is functional.…”

Methods and Analysis of Biological Contaminants in the Biomanufacturing Industry

“…For example, Polymerase chain reaction (PCR) can detect bacterial and virus contaminants, and cell analysis can evaluate if there is any aberrant cell development that could indicate bacterial or other contamination [54]. The technique employed is often decided by the unique needs of the process and the product being manufactured, with the ultimate goal of preventing and detecting biological contamination [11].…”

“…PCR is widely used in bioprocess monitoring to amplify specific DNA sequences of interest, such as those of bacterial or biological contaminants [61]. PCR can be used to detect and quantify the presence of specific microorganisms in a bioprocess by tracing the expression of specific genes linked to microbes [11]. The method entails repeatedly heating and cooling a nucleic acid sample to denature and re-anneal distinct DNA strands, followed by strand extension by a polymerase enzyme.…”

“…There are many current methods that have been continuously used for monitoring different biological contaminants that affect the yield of the bioprocess. These range from gold-standard microbial monitoring [10,11] to evolving reagent-based tests for protein and nucleic acid markers of biological contaminants [12], as well as the collection of sophisticated tools such as process analytical technologies [13]. Many emerging methods are also finding foot in the industrial process by offering facile operations, rapid results, and automation [14,15].…”

PCR-based analytics of gene therapies using adeno-associated virus vectors: Considerations for cGMP method development

“… 21 The use of recombinant AAV (rAAV) has increasingly and systematically become an important vector for gene therapy and will be used as the primary vector example in this review. 22 , 23 For disease treatment, AAVs have become popular for therapy design because of efficiency of transgene expression, safety, selective tropism, prolonged transgenic expression, and moderate packaging capacity. 24 They also have attributes favorable in manufacturing, such as rapid onset of replication in industrial cell culture, scalability, and storage stability.…”