Cell-free biology is the activation of complex biological processes without using intact living cells. While used for more than 50 years across the life sciences as a foundational research tool, a recent technical renaissance has made possible high-yielding cell-free gene expression systems (>g protein/L), including those for glycoprotein synthesis and on-demand biomanufacturing. Here we describe a rapid cell-free glycoprotein synthesis platform for characterizing SARS-CoV-2 neutralizing antibodies.

Developability and comparability assessment of current and next generation of biologics such as engineered mAbs, Fc-fusion proteins, BsAbs and 3G-ADCs requires state-of-the-art analytical and structural methods. Case studies will be presented based on native and ion mobility MS, Collision Induced Unfolding (CIU), multiplexed Top and Middle-Down MS, multiple fragmentation techniques, comprising high energy collisional-, electron-transfer and UV photo-dissociation (HCD, ETD and UVPD), CE-MS and quantification of trace-level HCPs by MS.

Since the introduction of the first recombinant DNA-derived insulin, biotherapeutics market has shown steady growth. Novel modalities such as ADCs and fusion proteins have received increasing attention in the pharmaceutical industry as targeted therapies. Integrated analytical strategies are required to address issues in attributes characterization. This presentation will discuss recent developments in protein analytics for elucidating key attributes, including phase-appropriate characterization strategy, integrated/orthogonal methodology and case studies.

In the summer of 2020, Bruker launched its next generation of SPR instruments, the “Pro series”. The “Pro series” instruments are available with either 32 sensor spots (8x4 array) or 24 sensor spots (8x3 array). The instruments are respectively named the SPR-32 Pro and the SPR-24 Pro. These instruments have outstanding sensitivity, fast cycle time with individual needle control capability and can run fully automated with an external robot. 

Many heavily engineered multispecific antibody formats have been generated and are analyzed in preclinical and clinical studies. As designed these molecules often encounter challenges during discovery, manufacturing and finally in patients. We employed the natural IgG format with minimal engineering to generate common light chain bi- and trispecific antibodies (Multiclonics®) with optimal biophysical and pharmacokinetic properties. The discovery, developability and in-patient behavior of our multispecific molecules will be discussed.

Early clinical data with CD3 bispecific antibodies has demonstrated transformational anti-tumor activity. Systemic cytokine release and on-target/off-tumor toxicity are the main adverse effects limiting the clinical utility of CD3 bispecific. Our data describe the cellular and molecular mechanism of CD3 bispecific-induced cytokine release and demonstrate that T cell killing activity is completely disconnected from the cytokine release providing a strong rationale for intervention by blocking various pyrogens without compromising efficacy.

The empty capsid are product-related impurities present in the rAAV products. It is important to quantify the full, empty and partial capsids in a rAAV product. This presentation will focus on different methods that are used for empty/full AAV characterization. In addition, orthogonal approaches will be discussed.

New biotherapeutic modalities, such as AAV gene therapy vectors, show significant promise in the clinic. As such, accelerated clinical development is common, requiring rapid analytical method implementation. Extensive protein therapeutic development experience can be leveraged to establish new modality product testing and characterization strategies. This talk will focus on crossing over from protein and antibody analytics to an informed and meaningful analytical test strategy for gene therapy vectors.

A common problem in drug discovery is a lack of clinical translation from recombinant screening systems. This is particularly true for chemokine receptors, where ligand/receptor redundancy provides an additional challenge; CXCR2 antagonist activity is not fully predicted by recombinant systems. We describe neutrophil dynamic mass redistribution assays which are sufficiently reliable for medium-throughput screening using endogenous receptors and demonstrate better correlation with the clinical target engagement assay, CD11b upregulation.

In mammals the members of the LRRC8 family of integral membrane proteins form heteromeric ion channels, which open an anion-selective pore in response to osmotic swelling. We have selected nanobodies from synthetic libraries to target the cytosolic domains of the obligatory channel subunit LRRC8A. These nanobodies are specific, recognize different epitopes and exhibit distinct functional properties with some acting as allosteric inhibitors and others as potentiators of ion channel function.

Ion channels constitute attractive drug targets for neurological diseases, pain and immunomodulation. However, achieving selectivity with small molecule drugs has been challenging and there currently is a growing trend to target ion channels with biologics such as peptides and antibodies. Using Nav1.7 targeting peptides and Kv1.3 targeting antibodies as examples, I will describe the challenges involved in screening and characterizing ion channel targeting biologics using binding assays and electrophysiology.

Protein glycosylation is a critical attribute of recombinantly produced proteins and can affect structure and function. Capturing site-specific glycan heterogeneity for highly glycosylated proteins can be challenging. Here we present a rapid peptide mapping approach that provides a glyco-fingerprint to each glycosylation site  thus allowing us to compare lots of the proteins produced under various conditions. Although semi-quantitative, this fingerprinting can be useful to monitor lot-to-lot variability.

At the beginning of 2020, SARS-CoV-2 presented a serious global threat and no therapies existed to slow the spread of infections around the world. As our part of this effort to develop effective therapies, the Marasco Lab used its 27-billion-member naïve phage library to perform parallel SARS-CoV-2 S1 and RBD panning campaigns and were able to rapidly isolate a large panel of anti-SARS-CoV-2 spike antibodies.  Utilizing the Octet RED96 platform, we performed high throughput kinetic screening to quickly eliminate sub-par antibodies. Subsequent competitive binding assays with CR3022 and soluble ACE2 allowed for broad epitope binning and identification of antibodies that blocked the ACE2-RBD interface. These ACE2 blocking antibodies were prioritized for further characterization, including fine resolution epitope binning within the group and viral neutralization assays. With the emergence of variant strains of SARS-CoV-2, further studies have investigated the effect of these spike mutations on antibody binding. The Octet RED96 system was critical in our efforts to identify potently neutralizing antibodies, allowing us to rapidly screen a large panel of unknown antibodies to identify lead candidates for characterization and development.