Advanced Therapy Medicinal Products (ATMPs) represent a significant breakthrough in modern medicine, offering new hope for conditions once considered untreatable - from rare diseases to cancer and more prevalent disorders. These innovative therapies form a diverse and rapidly evolving class of medicines, driven by continuous scientific advancements. While each ATMP holds the potential to transform patient care, they also present unique opportunities and challenges.

The development of ATMPs in Europe is governed by a highly complex and multilayered regulatory framework that ensures product safety, efficacy, and quality while balancing scientific innovation and patient access. ATMPs - including gene therapies, somatic-cell therapies, and tissue-engineered products - are primarily regulated under Regulation (EC) No 1394/2007 [1], which supplements the broader Directive 2001/83/EC governing medicinal products. The European Medicines Agency (EMA) and its Committee for Advanced Therapies (CAT) play a central role in scientific evaluation and marketing authorization.

However, ATMP development often intersects with additional regulatory regimes. For cell- and tissue-based products, the Substances of Human Origin (SoHO) framework - including Directive 2004/23/EC [2] - sets requirements for donation, procurement, and testing, ensuring traceability and donor protection. This adds an additional compliance layer, especially during early manufacturing stages. For ATMPs administered via or in combination with medical devices (e.g., cell-seeded scaffolds or gene delivery systems), the Medical Device Regulation (MDR, 2017/745) [3] and the In Vitro Diagnostic Regulation (IVDR, 2017/746) [4] introduce further obligations, including conformity assessments and clinical evaluation requirements. Determining the primary mode of action becomes critical for classification and regulatory pathway selection. Additionally, ATMPs involving genetically modified organisms (GMOs) - such as CAR-T cells or gene-edited stem cells - must adhere to Directive 2001/18/EC (deliberate release of GMOs) [5] and Directive 2009/41/EC (contained use of GMOs) [6]. This dual oversight can complicate clinical trial applications, as GMO-specific environmental risk assessments are required alongside clinical safety data, with national-level variations adding complexity.

Navigating these interconnected regulatory layers demands a holistic approach, early regulatory engagement, and strategic planning. Understanding the interplay of medicinal product, SoHO, device, and GMO frameworks is essential for successful ATMP development, ensuring both regulatory compliance and patient-centric innovation.

References
[1] Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004.
[2] Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells.
[3] Medical Device Regulation (MDR, 2017/745) of the European Parliament and of the Council of 5 April 2017 on medical devices.
[4] In Vitro Diagnostic Regulation (IVDR, 2017/746) f the European Parliament and of the Council of 5 April 2017 on in vitro diagnostic medical.
[5] Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms.
[6] Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009 on the contained use of genetically modified micro-organisms

Real-Time Polymerase Chain Reaction, also called PCR, is a very powerful and sensitive bioanalysis technique. It is used for a broad range of applications such as quantitative gene expression analysis, biodistribution studies of a nucleic acid target sequence, genotyping, single nucleotide polymorphisms (SNP) analysis, pathogen detection, drug target validation and for measuring RNA interference. The quantitative PCR (qPCR) method is used for the detection and quantitation of an amplified PCR product as the reaction progresses and is based on the incorporation of a fluorescent dye. qPCR can be combined with reverse transcription (rt) assay in case of RNA as starting material in cells or tissues in two separated analysis or in one-step reaction (rt-qPCR). This presentation is focused on the experimental activities and procedures required for the validation of the bioanalytical methods used to quantify by PCR a specific nucleic acid in support of Biodistribution studies. Biodistribution studies are required for the development of Gene Therapies (GT) and Cell Therapies (CT) modalities, since based on nucleic acids (DNA or RNA). The aim of these studies is to characterize the distribution and persistence of a gene/cell therapy product from the site of administration to target and non-target tissues and biofluids by measuring the vector genome or expression of the transgene, as well as drug-derived gene products. They are applied, to an extent defined by a careful risk-assessment evaluation agreed with the project owner and with the regulatory agencies if needed, to non-clinical and clinical studies that are conducted in support of regulatory applications and subjected to GLP/GCP requirements. To date, there has not been a regulatory guidance document with a descriptive approach on the validation of molecular assays that are used in regulated bioanalysis to support these novel modalities. Therefore, an internal evaluation was performed starting from available references [1] [2], to define the strategy to adopt for qPCR Method Validation for Biodistribution and Viral Shedding Studies and Sample Analysis. The procedure described consists in three different steps: PCR assay set-up and preliminary assessments that is part of method development and shall not constitute part of the validation study, validation of the qPCR reaction with the specific primers and probe set and validation of the qPCR process starting from biological samples. The same process applies for RT-qPCR assay. Each of these phases will be further detailed in each of the critical parts.

References
[1] A. Hays, et al., The AAPS Journal., 2024, 26:24.
[2] A. Lauren, et al., Bioanalysis , 2022, 14(16), 1085-1093.

ATMPs are Advanced Therapy Medicinal Products including gene therapy, somatic cell therapy, and tissue-engineered products. Often the development of non-conventional analytical methods is required, according to ICH guidelines [1, 2].

Developing analytical methods for ATMPs involves several key steps: 1. Understanding the Product: First, it is essential to have a thorough understanding of the ATMP, including its composition, mechanism of action, and the specific characteristics that need to be analyzed; 2. Defining Analytical Objectives: Determine what needs to be measured, such as potency, purity, identity, and safety. This will guide the selection of appropriate analytical techniques; 3. Defining the application: often the same analytical method can be applied from pre-clinical safety studies to DP characterization and also for the analysis of clinical trial samples; 4. Method Development and Optimization: This involves refining the chosen techniques to ensure they are sensitive, specific, reproducible, and robust; 5. Validation: Once developed, the methods must be validated according to regulatory guidelines to ensure they are reliable and can be consistently applied; 6. Continuous Improvement: As more data is gathered and technologies advance, analytical methods may need to be revisited and improved.

The design of the validation strategy should consider all the applications of the method, to save time and reduce cost and to optimize the use of precious materials required for validation.

References
[1] EMA/CHMP/ICH/172948/2019 ICH guideline M10 on bioanalytical method validation and study sample analysis, 2023
[2] ICH guideline Q2(R2) on validation of analytical procedures, 31 July2022

When manufacturing a drug and ensuring its adequate therapeutic efficacy, one of the most important aspects is the purity of the active substance. Impurities must first be determined qualitatively to develop a proper purification method. This step in drug development can be particularly challenging, especially for antisense oligonucleotides, which are becoming increasingly important in the treatment of various diseases. They are synthetic, chemically modified compounds used to treat different diseases, mainly genetic ones, e.g., spinal muscular atrophy (SMA). The Spinraza drug with antisense oligonucleotide (nusinersen) is used to treat this disease. The structure of nusinersen is modified with 2-O-2-methoxyethyl and phosphorothioate groups, which increases its stability in the body since the activity of antisense oligonucleotides includes binding to complementary sequences of the targeted ribonucleic acids in cells. The application of these compounds as therapeutic agents has enforced the need for their purification, as well as the separation of impurities and their identification. Impurities may be classified into several categories: products of modification of a single phosphodiester bond, sugar or base residues, products of single nucleotide deletion, or single nucleotide addition. The challenges in characterizing impurities of therapeutical oligonucleotides arise from their large molecular masses and complex structure. As a result, fully elucidating their structure is a challenge, and the development of effective analytical methods is crucial to enable their separation from the parent oligonucleotide and from other impurities.

The study aimed to develop a chromatographic method for the separation and identification of nusinersen impurities. To increase resolution, different chromatographic modes were tested: hydrophilic interaction liquid chromatography (HILIC), reversed-phase chromatography (RP LC), ion-pair chromatography (IP RP LC) and two-dimensional liquid chromatography (2D LC). Ultra high-performance liquid chromatography was tested in each case. The identification of impurities was done by quadrupole time-of-flight mass spectrometry (Q-TOF-MS) in negative ion mode. The developed method allows the analysis of nusinersen s impurities and degradation products in a comparatively short time. Moreover, the method is selective, although it compromises the MS detector response and the separation of impurities.

Advanced Therapy Medicinal Products (ATMPs) form a new class of biological drugs that can be used to treat unmet medical conditions and go one step further the personalized medicine. One of the main concerns about the use of ATMPs is the safety of patients because ATMPs can induce an immunological response with a consequent impact on treatment efficacy rather than patients impairment.

So far, to put the new therapy on the market, a regulated risk-based approach for ATMPs development is strongly suggested [1] and, as per guideline, should include the immunogenicity evaluation starting from the non-clinical studies as part of the overall toxicology assessment [2].

ATMPs can elicit an immune response involving the innate or the adaptive immune systems. The difference of such activation is linked to the fact that ATMPs are delivered for the first time in the organism and are recognized as foreign or they are identified similar to a previously encountered substance.

Different bioanalytical methods can be used to monitor the immune response of ATMPs administered in vivo and the majority is represented by ligand binding assays (LBAs) such as ELISA. Each ATMP has unique properties which need to be considered when assessing its immunogenicity using LBAs.

A special focus on immunogenicity of gene therapy medicinal products (AAV, oligonucleotides) and cell-based products (CAR-T) will be provided with highlights to the LBA which can be used for the assessment and differences and similarities between non-clinical and clinical studies.

Oligonucleotide drugs have increasingly gained attention and the disease areas for which these therapeutic agents are evaluated have been rapidly expanding. Oligonucleotides encompass a wide array of chemistries and modes of action, e.g. from single stranded antisense oligonucleotides to double stranded conjugated siRNAs. To determine their PK/PD properties, sensitive and selective quantitative bioanalytical methods are required. LC-MS has emerged as a powerful tool and one of the most selective bioanalytical technologies to quantify a variety of oligonucleotide therapeutics in biological matrices. Compared with alternative assay types (e.g. hybridization-ELISA), LC-MS methods can selectively quantify the full-length oligonucleotides and their major metabolites, have a much wider dynamic range and do not require any specific reagents. However, significant challenges should be addressed while developing an LC-MS assay for oligonucleotides, including their very high plasma protein binding, nonspecific binding to containers, lack of chromatographic retention, conjugation (e.g. with lipids) affecting the extractability and chromatographic separation, multiple charge states and formation of cation adducts affecting the LC-MS method performance. The sensitivity of LC-MS methods for quantification of oligonucleotides has significantly improved since the last few years and reaching sub ng/mL lower limits of quantification is now feasible. This is mainly due to improved MS equipment, consolidated extraction techniques and the adoption of state-of-the-art chromatographic technologies. The LC-MS sensitivity is usually sufficient to support preclinical studies, particularly for analysis of tissues where these drugs exhibit their efficacy and are present at high concentrations. However, the sensitivity might not be sufficient to support clinical studies where the administered doses and the observed exposures are expected to be lower. Sensitivity will be also challenging in certain matrices, e.g. in plasma or serum when the administration of the oligonucleotide drug is not systemic. In all cases defined above, switching technology (e.g. LC-MS to hELISA) in order to meet a sensitivity target should be carefully evaluated because the selectivity of these assays can be different, and a direct comparison of the exposures obtained may not be possible.

According to the MIST guidance, the metabolites of oligonucleotide drugs should be quantified in case these significantly contribute to the pharmacological and toxicological effect. Again, the use of LC-MS offers significant advantages compared to other technologies due to the increased selectivity. However, additional complexity is because the metabolic profiles of oligonucleotides in plasma and in tissues are typically very different, resulting in different sensitivity requirements.

Considering all the above considerations and challenges, a proposed strategy is to generate preliminary metabolite i.d. information in plasma and tissues before a quantitative technology is selected and bioanalytical methods are fully validated to support regulatory toxicological studies. The pharmacological activities of predictable shortmers should also be evaluated to determine what analytes require quantification. The selectivity of LC-MS and hELISA assays vs. shortmers should also be assessed to select the technology to be fully validated for quantification.

All factors above will be discussed in this presentation, which includes an overview of recent advances in LC-MS bioanalysis of oligonucleotides, along with examples showing how different experimental conditions will affect the performance of the assay. Case studies will also be presented showing how the preliminary metabolite information and a comparison of selectivity/sensitivity of different technologies will help the bioanalytical team implement a proper analytical strategy in order to avoid complex bridging studies at later stages of drug development.

Cell and gene therapies represent a broad pipeline of bioengineered treatments for disease, encompassing gene editing and de novo expression, hematopoietic stem cell transplantation and immune reconstitution, targeted immunotherapy, and tissue regeneration. As cell and gene therapies are becoming increasingly important for the treatment of cancers and other diseases, the need for reliable characterization of such therapies is skyrocketing [1].

Flow cytometry has emerged as a key methodology for the analysis of cell and gene therapies during research, drug development, clinical manufacturing and follow-up in patients. Flow cytometry is a technology that rapidly analyzes single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution. Each particle is analyzed for visible light scatter and one or multiple fluorescence parameters. It allows for the simultaneous characterization of mixed populations of cells from blood and bone marrow as well as solid tissues that can be dissociated into single cells such as lymph nodes, spleen, mucosal tissues, solid tumors etc. [2]

Due to its potential applications and the impact on the results produced with flow cytometry assays, having robust and reliable validated assays is mandatory to allow the analysis of (pre)clinical samples. However, flow cytometry methods can be challenging to develop and validate due to several reasons: the inherent biological variability of cells, the appropriate selection and characterization of the reagents used and the lack of standardized cellular reference materials or quality controls, the limited stability of samples and the complexity of data output and interpretation of results [3,4]. Although there are no official regulatory guidance documents addressing the requirements for the validation of flow cytometric methods intended for use in drug development or for clinical testing, in the past few years, key stakeholders have published recommendation papers [4].

This lecture will present an overview on the use of flow cytometry and recommendations for method validation in accordance with regulatory requirements, based on the fit for purpose approach.

References
[1] Lazarski C.A. et al. Cytotherapy. 2024, 26(2),103-112
[2] McKinnon K.M. et al. Curr Protoc Immunol. 2018, 120, 5.1.1-5.1.11
[3] van der Strate B. et al. Bioanalysis, 2017, 9(16), 1253-1264.
[4] O Hara D.M. et al. Journal of Immunological Methods. 2011, 363 (2), 120-134

Adeno-associated viruses (AAVs) are icosahedral capsids comprised of 60 total copies of three viral proteins (VPs), VP1, VP2, and VP3, in an approximate 1:1:10 ratio, respectively. VP3 is the smallest of the three VPs, with VP2 containing the entire sequence of VP3 as the C-terminal sequence and VP1 containing the entirety of VP2 as its C-terminal sequence. These capsids contain single-strand (ss) DNA of approximately 4.7 kb that delivers the genetic payload to target cells. There is increasing interest in AAVs as gene therapy vectors because of their highly effective delivery mechanisms, low cytotoxicity, and minimal immunogenicity. Additionally, the variety of serotypes, each having different tropisms, provides the ability to target specific cell types and organs. Currently a handful of AAV therapy products are on the market, receiving conditional or full approval by either the US Food and Drug Administration (USFDA) or the European Medicines Agency (EMA). Monitoring AAV vector quality is crucial for ensuring product safety and efficacy. A key aspect of this is monitoring changes in post-translational modifications (PTMs) on the capsid VPs. PTMs on said VPs are known to occur during production and storage and can have an influence on product stability, infectivity, and transduction efficiency. They also are seen varying between production lots highlighting the importance of batch-to-batch monitoring. Additionally, final product yields for full AAV capsids are low due to the high levels of empty or partially filled capsids that are generated during production and need to be removed downstream. Therefore, having highly sensitive AAV characterization platforms is critical to minimize the amount of sample needed for quality control (QC) testing.

In this talk, several mass spectrometry based workflows for the analysis of different quality attributes will be explored. The talk will start from the monitoring of the full capsid filling states through advanced techniques such as charge detection mass spectrometry (CDMS) enabling characterization of complexes with a mass range between 2 and 10 MDa. We will then move to the viral protein level, to determine post-translational modifications at intact and peptide level. Final topic of the talk will cover the detection and quantitation through mass spectrometry techniques of the host cell contaminants derived by the process and how the purification strategies can impact their presence in the final product.

References
1. Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 2021;6(1):53.
2. Liu AP, Patel SK, Xing T, Yan Y, Wang S, Li N. Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry. Journal of pharmaceutical and biomedical analysis. 2020;189:113481.
3. Strasser L, Morgan TE, Guapo F, Füssl F, Forsey D, Anderson I, Bones J. A Native Mass Spectrometry-Based Assay for Rapid Assessment of the Empty:Full Capsid Ratio in Adeno-Associated Virus Gene Therapy Products. Anal Chem. 2021 Sep 28;93(38):12817-12821.
4. Smith J, Carillo S, Kulkarni A, Redman E, Yu K, Bones J. Rapid characterization of adeno-associated virus (AAV) capsid proteins using microchip ZipChip CE-MS. Anal Bioanal Chem. 2024 Feb;416(4):1069-1084.
5. Smith J, Strasser L, Guapo F, Milian SG, Snyder RO, Bones J. SP3-based host cell protein monitoring in AAV-based gene therapy products using LC-MS/MS. Eur J Pharm Biopharm. 2023 Aug;189:276-280.