We have faced unprecedented growth in cell and gene therapy submissions in recent years. While the industry expects and anticipates approval of several products each year, there are signs that this pace of approval is not sustainable in view of deficiencies identified by the US Food and Drug Administration that are primarily focused on product manufacturing and consistency. This talk discusses the criticality of establishing phase-based chemistry, manufacturing, and controls (CMC) readiness, by developing a proactive plan to gain product knowledge iteratively while implementing a phase-appropriate approach to achieving full CGMPs.

We will demonstrate how you can achieve high single-cell seeding efficiencies and clonal outgrowth whilst conforming to the needs of regulatory bodies through proof of monoclonality. We will outline how to optimise and develop a robust GMP-compatible process by utilising a recombinant matrix, GMP media and dissociation reagents and outline some potential new GMP compatible consumables that will be available in combination with the Advanced Instruments technologies.

The characterization and testing of cellular therapeutic products (CTPs) is a critical aspect of product development, translation and release. Here I will describe recent efforts in the standardization of the characterization and testing of CTPs. I will also describe recently published standards on cell counting as well as NIST technical programs for increasing confidence in cell count and viability assays.

Flow cytometry data analysis is extremely time-consuming and the principal source of variation in the application of the technology. While many unsupervised approaches have been developed, poor performance has limited their application to discovery applications. As an alternative, we have developed the best performing approach for cell population identification. It consistently achieves up to 94% accuracy, and is in use by pharma for clinical studies and cell therapy manufacturing.

Qualification of raw materials used in the manufacturing of cell therapies requires the use of risk assessment strategies to categorize the critical components of a manufacturing process. In addition to cellular starting materials, cell culture supplements, excipients, and other components must meet the required quality criteria to ensure consistency in manufacturing, quality, and safety.

Diverse product lines require compliant, robust, and versatile quality systems. We present the quality requirements of cell-based products produced by Organogenesis and its partners. The gold standard quality systems developed for the commercial large-scale manufacturing of Apligraf, a cell-based product regulated as a Class III medical device to treat diabetic foot and venous leg ulcers, are adapted for the specific regulatory requirements of the clinical and commercial products in Organogenesis’s portfolio.

Manufacturing process changes are inevitable, but present risk to critical quality attributes (CQAs). Comparability mitigates this risk. Changes with greater risk to CQAs or later in development require more stringent comparability. Ideally, comparability uses starting material split between old and new processes, run in parallel, but other designs are possible. Limited understanding of CQAs, CPPs, and insufficient analytical methods present challenges. Assays are not limited to release tests, however, and an assay matrix, using orthogonal methods where possible, increases certainty. Acceptance criteria are narrower than for release and should be pre-defined, based on robust statistical analysis. 

Many advanced therapies rely on plasmid DNA to generate viral vectors and RNA vaccines; however, manufacturing pDNA poses an array of challenges. Learn how GenScript ProBio's multidisciplinary plasmid team tackles technical challenges around plasmid production and developed a well-established plasmid manufacturing process producing high quality and purity, with faster turn-around time and significant cost-savings compared to other CDMOs.

Considering that the first human-induced pluripotent stem cells were generated only 15 years ago, there is currently an impressive number of clinical trials to test their therapeutic value. However, tumorgenicity and heterogeneity remain major challenges to be addressed in manufacturing of clinical-grade iPS cells. Here, we discuss risk-based and process-stage-appropriate quality considerations for consistent manufacturing of uniform iPS cells to maximize patient safety in clinical trials.

Differentiation of human pluripotent stem cells (hPSCs) to cardiomyocytes (CMs) is a lengthy process prone to batch failures. Currently, in process monitoring of differentiation progression is rarely utilized. We performed a longitudinal multi-omics analysis of successful and failed batches of hPSC-derived CMs. I will describe gene, protein, and metabolite multivariate parameters that when measured early in differentiation will allow prediction of CM purity in the end product.

Growth-based mycoplasma testing is expensive, cumbersome, and represents a major bottleneck in QC testing. Consequently, manufacturers have traditionally outsourced testing to labs that specialize in the 28-day culture-based test method. For cell therapy manufactures or other low-dose, short shelf-life therapeutics, the 28-day turnaround time of the culture-based test is unsuitable. Real-time PCR based assays provide a viable alternative, with same day results. Here we describe a real-time PCR based mycoplasma assay and two case studies on the successful validation and approval by regulatory agencies to use the assay for lot-release testing.

Over the past decade, the development of cell therapies has gained significant momentum. Processes executed in a hospital setting such as the isolation of tumor infiltrating lymphocytes (TILs), transformed clones of hematopoietic stem cells (HSCs), or of other rare cells, is challenging to replicate in a cGMP environment. We will discuss the cell sorting process and analytical development considerations when implementing fluorescence-activated cell sorting (FACS) in a cGMP environment.

Rubius Therapeutics is developing a novel cell therapy platform, called the RED PLATFORM, by genetically engineering donor-derived precursor cells to express therapeutic proteins inside or on the cell surface and culturing them into erythroid cells, called Red Cell Therapeutics. Developing analytical tools to characterize the details of this maturation is key to understanding process robustness and Critical Quality Attributes. Approaches to understand in-process cellular phenotype, cellular impurities, and overall cell health will be discussed.

The business model for allogeneic cell therapies can appear similar to that of traditional biotech products, however with the exception of some stem cells, banked cells from a single donor are unlikely to last the whole product lifecycle. This means various aspects of banking will differ between products. This talk will consider some of these factors, their regulatory implications, and how to address them.

Lentiviral vectors are utilized in cell and gene therapy programs for both ex vivo and in vivo delivery. Tracking the quantity, size distribution, charge, and protein content of viral particles during production, purification, and formulation development can aid developers in making process decisions. In this presentation, we will discuss the use of DLS, NTA, Flow Virometry, and other methods to characterize lentiviral particles.

Data are presented demonstrating the utility of SV-AUC with case studies covering characterization of AAV vectors and DNA drug substance. For an AAV vector with increased HMW by SEC we show presents as a low molecular weight peak on SV-AUC, with a higher DNA content than the full capsid. Use of pseudo-absorbance and software tools to increase throughput and ultra-low loading concentrations to reduce sample requirements are also discussed.