We report a novel methodology to accurately quantify charge variants of monoclonal antibody mixtures that span 40-fold in ratio. With the wide concentration ranges of combination products, one component may fall within the linear range while the other does not. Imaged capillary isoelectric focusing conjugated with the multiple detection techniques allowed us to overcome this challenge.

Alternative-format biopharmaceuticals which feature domains linked by (Gly4Ser)n linkers provide new therapeutic opportunities through potentially enhanced binding properties. However, characterization of the intermolecular domain interactions that give rise to oligomeric species remains challenging, due to the variety of possible interaction interfaces at the subdomain level. In this study, we present an online, middle-up LC-MS-based method to characterize covalent and non-covalent subdomain interactions in alternative format drug substances.

Gene therapy products have demonstrated great potential for treating devastating diseases and are being extensively evaluated in clinical trials for many disease indications. The structural and biological properties of these products are complex and yet to be fully understood. Analytics are key to developing safe and efficacious products. Strategies to overcome analytical challenges to ensure the highest product quality and consistency in gene therapy development will be presented.

Adeno-associated virus (AAV) has emerged as one of the most used vectors for gene therapy. Many structural features of AAV particles such as empty/full ratio, stoichiometry of capsid viral proteins, post-translational modifications of the viral proteins, as well as the residual host cell proteins and viral proteins are considered potential critical quality attributes (CQA). Characterization and quantitation of these CQAs using mass spectrometry techniques will be discussed in this presentation.

CE-SDS is a widely recognized platform technology for purity determination and impurity characterization in protein and gene therapy products. One of the major limitations of this method is its low throughput. Method development and optimization, and application of CE-SDS to support process development decision-making, where high throughput is essential, are made inefficient because of this limitation. A new instrument capable of running eight capillaries in parallel, thus enabling an 8-fold increase in throughput and decrease in turnaround time, has become available. This talk focuses on a thorough evaluation of this instrument. Details and results of experiments performed will be described.

This presentation will focus on the necessary steps to standardize data collection and automation practices in an established analytical development laboratory with 30 analysts running 40 different assays. We will detail our challenges and progress to standardize data storage practices, automate routine assay setup, and make our data searchable to our broader organization. We will discuss the advantages of this approach and present our roadmap for future development.

Cell cultures expressing biotherapeutic molecules are routinely monitored using cell counters to differentiate healthy and dead cells to measure overall viability. Process development scientists can infer additional cell morphology insights by inspecting cell counter images, but this is a time-intensive process that does not scale. We applied transfer learning and fine-tuning principles to the YOLOv5 algorithm to develop a computer vision model for monitoring cell morphology.

Manual execution of Vg titer assays (qPCR and dPCR) leads to sample testing bottlenecks, resulting in a long turnaround time for Vg titer results. This talk will present case studies highlighting how Spark Therapeutics is using automation to increase sample testing capacity (4X over manual). It will describe strategies to expand throughput using integrated work cells, which will culminate in faster delivery of life -saving gene therapies to patients.

Native MS is a technique enabling the study of intact and non-covalent protein species that has become increasingly popular as a biotherapeutic characterization tool during analytical development. Coupling nanospray MS with native chromatographic separation methods allows us to directly characterize variants without time-consuming offline fractionation. We have used native MS to investigate format-specific variants for multiple biotherapeutics, including co-formulations and novel bispecifics, which will be discussed in this presentation.

• Major challenges in CQA evaluation for novel modalities (e.g., AAV, FDC, Alternative-Format Molecules)
• Limitations of conventional analytical tools for the analysis of novel modalities
• Pros and Cons of new analytical technologies in the development of novel modalities
• Health Authority’s perspective ― Is it possible to achieve both high speed and high quality?​

• Opportunities and challenges of high-resolution and high-throughput analytical methods (e.g., MAM, NMR etc.)  ​
• Automation: successful stories and lessons learned
• Digital transformation: successful stories and lessons learned.
• Future perspective of Machine learning/Artificial Intelligence in biotherapeutics development
  

Affinity chromatography with online mass spectrometry hyphenation (AC-MS) on relevant binding partners of therapeutic proteins allows streamlining of the assessment of critical quality attributes. We are establishing an affinity column toolbox, including important receptors and targets, which are of high interest for the biopharmaceutical field. AC-MS as novel technology provides unprecedented resolution and sensitivity of functionally distinct proteoforms and, hence, is expected to overcome current analytical challenges of biopharmaceutical analysis.

MAM for analytical testing of biotherapeutics is gaining traction due to its potential to improve efficiency and provide more detailed information on PQAs as compared to conventional methods. This presentation will focus on the development of USP standards and tools to support MAM for product characterization and quality control. Considerations that impact the quality and consistency of MAM, approaches to enhance the robustness, and case studies will be discussed.

Biologics hold incredible promise in alleviating or curing life-altering illnesses and continue to be one of the fastest growing classes of therapeutics. In both discovery and development, characterization by mass-spectrometry and other analytical assays is often a bottleneck and necessitates high-throughput methods to optimize sample preparation, data analysis and interpretation. We have developed an end-to-end analytical mass-spectrometry pipeline using robotic automation and cloud-based databases that ensure correct processing and analysis of each sample. The system represents a lab-of-the-future concept eliminating the need for manual sample preparation and instrument operation, thereby improving consistency, bandwidth, and accelerating early molecule development.

Bio-capacitance has become a standard method to estimate biomass in cell-based bio-manufacturing processes. There is now a good understanding behind the apparent discrepancies between bio-capacitance and conventional trypan blue-based cell counts in the later stage of culture. Furthermore, capacitance measurements can determine the metabolic status of cells from changes in cytoplasmic conductivity that are indicative of the first stages of nutrient deprivation.

The use of MAM for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. We developed and qualified a MAM workflow for therapeutic mAbs and their coformulations with Lys-C digestion. The developed platform MAM workflow and assay performance evaluation paved the way for its GMP qualification and enabled clinical GMP release of mAb products.

Development and production of innovative biotherapeutics demands bioprocesses that consistently yield a high-quality product. However, current methods to determine product quality do not necessarily capture the actual mix of product and related impurities in cell culture supernatant, but rather what can be captured after purification. We developed a highly-sensitive method that can be applied to the detailed characterization of cell culture supernatants from bioreactors without a falsifying pre-purification step.

Glycosylation, a post-translational modification (PTMs), is a well-defined critical quality attribute with significant impact on Fc-receptor binding affinity which in turn impacts the Fc effector mediated antibody-dependent cellular cytotoxicity. Established methods to assess glycan structures and Fc effector activity are costly and time-consuming. Recently, the FcR-IIIA-NPR HPLC affinity column emerged which could provide a rapid monoclonal antibody (mAb) glycosylation assessment. The feasibility of the FcR-IIIA-NPR HPLC affinity column as a rapid assay to identify glycosylation PTMs of therapeutic mAbs is being ascertained.

Identification tests in the pharmaceutical industry should demonstrate that the intended components in a product are present at lot release. These tests should “be able to discriminate between compounds of closely related structure” (ICH Q6A), and so limited specificity in methods such as FTIR can make identification challenging. A case study on formulations with common pharmaceutical excipients and surfactants demonstrates how NMR can serve as a powerful identification method.