Multispecific purification presents unique challenges due to the complexity of the molecules involved and the large number of potential binder combinations. During lead selection, it is common to test hundreds to thousands of multispecific antibodies, requiring rapid purification of high-quality material. To meet these material demands, we have developed one-step purification methods of multispecific antibodies using newer capture resins.

An innovative system was developed as a continuous manufacturing system for biopharmaceuticals. This system can continuously capture target molecules such as antibody proteins directly from cell culture broth without any clarification. Its principle is based on an in-line static mixer that accelerates the binding reaction between the antibody and Protein A resin, and a hydrocyclone that separates the antibody-bound resin from the culture medium. This technology does not even use a chromatography column, so it can eliminate centrifuges, depth filters, and chromatography columns for capture and purification. Therefore, it shortens the steps and reduces the footprint of biopharmaceuticals production.

Magnetic bead protein affinity purification provides a fast and efficient method for isolating proteins. Despite its advantages, recombinant tags may pose a significant challenge to some protein native structures and/or biological activity, potentially limiting the screening of promising therapeutic candidates. To address this, we have developed a magnetic bead platform that facilitates tagless affinity purification. This innovative technology aims to help accelerate and expedite protein research and drug discovery.

Our studies highlight the development of an adaptable purification system designed to optimize the balance among throughput, chromatographic flexibility, and total product yields. By integrating a 150 mL Superloop into an ÄKTA FPLC system for automated two-step tandem purifications, we managed to process various cell culture supernatant volumes ranging from 0.1 to 2 liters, achieving purification yields as high as 2 grams.

• Computational protein design
• Non-canonical amino acid incorporation via genetic code expansion
• Engineered cell lines and non-traditional expression hosts

• When you needed the protein yesterday - high pressure production
• How things change when this protein may be headed for the clinic
• Out of left field - strange things that proteins do, and what to do back

A generalizable, scalable, and efficient purification platform is critical for the success of mRNA therapeutics. Our goal in this work is to develop downstream bioseparation strategies to separate dsRNA impurities from ssmRNA. We designed peptide ligands that are selective towards dsRNA and coupled these peptides to filtration membranes to demonstrate the feasibility of purifying labile ss-mRNA from dsRNA at high yield and purity in a scalable fashion. dsRNA-selective peptides were successfully identified using two distinct strategies involving rational design and combinatorial library screening. These peptides demonstrated strong binding to dsRNA and showed selectivity for dsRNA over ssRNA.

Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. In this presentation, I will discuss our recent efforts taking advantage of split inteins and bacterial transpeptidases to simultaneously terminally functionalize and purify homogeneous protein populations in a single step and while maintaining native bioactivity.

We present an open-source chromatography platform for parallel protein purification at milligram scales. This device can purify up to four proteins and access eight buffers through a network of software-driven valves. It is controlled via Python scripting or a user-friendly graphical interface. We have released a detailed hardware build guide and open-sourced the control software, enabling others to create customized purification instruments.

In this work, we showed the development and utility of a split-intein-based expression and purification system. This technology works by utilizing self-splicing protein segments known as split-inteins to capture tagged proteins, while also removing the attached tag from column-bound proteins. Product quality studies confirmed proper tag cleavage and biological behavior, while process optimization was investigated using statistical DoE studies to enhance performance and yield. The findings in this work show that a novel split-intein-based affinity technology can be an effective capture strategy that can potentially provide a scalable platform for the purification of any expressed protein.

The vital role of protein QC is well established. By incorporating precise intact mass determination and MS2 analysis, a protein production lab can have valuable tools at their disposal. Case studies will be presented which illustrate how the detection of post-translational modifications have fundamentally altered projects.

Impurity isolation is a critical aspect of pharmaceutical development. Potential drug candidates are perpetually evolving, with optimizations in synthetic routes leading to new impurities that require characterization before downstream processing. Separations scientists are tasked with resolving these impurities at analytical-scale and finding conditions that are scalable and amenable to the stability of the impurity. Tools, tactics, and purification techniques that help overcome these challenges will be discussed.

Protein-biochemistry-related support of chimeric antigen receptor (CAR) T cell therapy programs from early development through commercialization requires effective project management, nimble business practices, and excellent cross-functional communication. This is facilitated by several tools, including a laboratory information management system (LIMS), SMART goal setting practices, and an environment that properly balances individual and teamwork-oriented tasks.

With the growing realization of clinically effective multispecific therapies, there has been a steady rise in the exploration of complex biologics specifically in immunology and oncology. This poses a unique challenge for drug development, with double or triple the number of antibody discovery campaigns needed per project, followed by the subsequent evaluation of libraries of multispecific antibodies with diverse formats, target clone properties, geometries, and valencies. In this study, we will showcase how robust functional assays using primary cells can be miniaturized and run in high throughput to provide early insights into what makes a potent multispecific drug.

Protein engineering is a highly iterative process, with multiple rounds of hypothesis-driven experimentation leading to better hypotheses in subsequent rounds on an overall trajectory toward a fitness optimum. Our Self-Driving Autonomous Machines for Protein Landscape Exploration (SAMPLE) platform automates the hypothesis, experiment, and data interpretation steps in a closed, autonomous loop, enabling researchers to focus on the overall experimental design rather than the lengthy iteration process, accelerating progress.