Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system due to its low cost, genetic tractability, rapid gene expression, and scalability. We developed and optimized a protocol that permits routine generation of recombinant metalloproteases that are pure and proteolytically active. By altering the feeding regime we increased biomass production and yielded several milligrams of recombinant protease, large enough quantities to facilitate conjugation of the protease to nanoparticles.

We present a robust method for expressing proteins in human embryonic kidney 293 (HEK293)-derived stable pools, leading to recombinant protein products with much less clipped species compared to those expressed in CHO cells. The protocol is also applicable to HEK293S GnTI- (N-acetylglucosaminyltransferase I–negative) and Expi293F GnTI- suspension cells, facilitating production of high yields of proteins with less complex glycans for use in structural biology projects.

Introduction of chimeric antigen receptor (CAR)-encoding DNA into primary human T cells via viral transduction is a critical step in CAR T manufacturing. Characterization of infective viral particles is important to ensure a robust process. Here, we describe the design and production of a recombinant viral envelope protein to generate and screen for sensitive and specific detection antibodies, allowing additional characterization of viral particles.

Fluorescent proteins (FPs) are essential in biological research, but their wide excitation range causes issues in traditional cytometry due to spectral overlap. Full-spectrum flow cytometers simplify analysis by removing the need for frequent filter changes. Producing FPs in Escherichia coli, purifying, and coupling them to microspheres offers a cost-effective, convenient method with long-term storage capability and easy multi-color analysis. This simplifies experiments involving multiple FPs or protein-coupled microspheres.

Glycoproteins take part in nearly every biological process and make up a large percentage of the proteome. We are specifically interested in the properties of membrane glycoproteins, which are key components in a number of different disease states. Our experiments demonstrate that recombinantly expressed full-length membrane proteins that contain an N-glycosylation consensus sequence can be glycosylated by N-glycosyltransferase, even in the presence of membrane mimetic environments.

Recombinant expression has revolutionised protein production. However, many proteins cannot be expressed in available systems. This is especially true for membrane proteins and complex enzymes. An alternative strategy is the purification of these proteins directly from their host organism by the genetic introduction of affinity tags. Yields from this purification strategy are compatible with modern structural and biochemical characterisation—and preservation of the host context provides new biological insights.

The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target of neutralizing antibodies. LASV vaccine design is complicated by the metastability of the GPC. We describe the development of prefusion-stabilized, trimeric GPC ectodomains. Further, we define the polyclonal antibody response post-vaccination, demonstrating the immunodominance of off-target epitopes and emphasizing the need for further GPC engineering.

Biochemists have long used enzyme immobilization as a route to robust and reusable biocatalysts. However, the scope of enzymatic activities successfully recapitulated in immobilized form has remained relatively narrow, limiting potential applications. The advent of orthogonal protein and peptide coupling methodologies presents a broadly applicable solution to this problem. We will describe our approach to recapitulating the activities of TEV protease and cAMP-dependent protein kinase (PKA) in immobilized form and their potential uses as benchtop reagents by biochemists.

Recombinant protein production is an integral part of drug discovery. As therapeutic targets become more challenging, we evolve our methods to triage protein variants more efficiently, while reducing cost and shortening timelines. We have developed a multi-host mid-scale expression and screening platform to supplement our existing small-scale high-throughput expression analysis. The semi-automated workflow is integrated with robotic liquid handlers and provides sufficient quantities of proteins for biochemical and structural screening.

Sensor-Integrated Proteome on Chip (SPOC) technology enables real-time, high-throughput kinetic analysis of biomolecular interactions. By automating in situ production and capture-purification of fully-folded functional protein arrays on biosensors, SPOC can simultaneously analyze up to 1000 unique proteins on a single SPR sensor, providing quantitative, qualitative, and kinetic information for all targets. SPOC finds application in (1) drug discovery for off-target binding screening, ensuring safer and more effective medications; (2) serology and antigen identification for vaccine development, facilitating the creation of targeted and robust vaccines; and (3) biomarker discovery for diagnostics development, enabling early disease detection and personalized treatments.

Sutro Biopharma’s XpressCF+ cell-free protein synthesis (CFPS) system is a powerful platform to produce antibodies containing non-natural amino acids that facilitates homogenous site-specific conjugation of ADCs. A novel hybrid IgG production process has been developed in which light chains, pre-fabricated in E. coli, are added to CFPS reactions to produce full-length antibodies. This integrated process increases CFPS titers and enables the production of high DAR and dually conjugated ADCs.

Mammalian cell-free protein synthesis systems hold promise as platforms for rapid generation of proteins with complex posttranslational modifications (PTMs). However, current approaches suffer from low protein yields, variable expression levels, and insufficient PTMs. We describe Leidos’ approach combining novel mRNA engineering and biochemical technologies for the development of CHO cell-free systems enabling rapid, on-demand, high-yield production of various protein classes using plug-and-play chassis systems with optimized molecular components.

In vitro "Cell-Free" Protein Synthesis systems hold disruptive potential within the protein pharmaceutical and medical diagnostics industries. Recent engineering advancements have paved the way for the on-demand production of biotherapeutics at the point-of-care.  This capability is vital in facilitating magistral and personalized treatments, disrupting the way we approach healthcare. Furthermore, notable progress has been made in the development of "cell-free" at-home colorimetric biosensors, opening up new possibilities for personalized cancer treatment.

The KIWI-biolab enables efficient recombinant bioprocess development and optimization on a robotic platform with fully automatic orchestration of parallel bioreactor systems of different scales, analytical instruments, and a mobile laboratory robot. Based on FAIR data principles it allows self-controlled parallel fed-batch cultivations, integrated sample analysis, and mathematical model-based parameter calibration and CQA optimisation. The power of the platform is demonstrated by industrially relevant recombinant processes including Fabs, elastins, and hydrogenase.

The Structural Biology and Protein Sciences group plays a key role within Pfizer’s oncology research organization, supporting and enabling early-stage small-molecule drug discovery efforts. Operating at the interface of discovery technologies and medicinal chemistry, the group generates recombinant proteins for hit identification, lead optimization, biochemical assay development, and structure-based drug design campaigns. Navigating the fast-paced competitive landscape and scientific challenges of discovering first-in-class medicines, the group utilizes optimized parallel protein production workflows to support its project portfolio within tight resource constraints and aggressive timelines. Successful process development and workflow optimization strategies will be highlighted in this presentation.

Protein production and biochemistry teams encounter different challenges associated with supporting chimeric antigen receptor (CAR) T cell therapy development depending on the program stage. Effective and concurrent support of discovery research through clinical development requires different activities, careful planning, and effective task prioritization. These activities offer career development opportunities for individual team members, which must be properly balanced with meeting ambitious productivity goals.

Targeted lipid nanoparticles (tLNP), which allows delivery of payloads to selected populations of cells through targeting moieties, is one of the most promising therapeutic concepts of the near future. Due to the complexity of tLNPs as a drug product, it is very challenging to manage multiple CDMOs/CROs to develop and manufacture multiple intermediates, drug substance, and the eventual drug product. This talk will share some experiences in management of CDMOs/CROs.

A common issue in high throughput expression labs is data management. Typical work streams require managing multiple types of data across systems incapable of crosstalk. Tracking all of these data streams usually requires heavy IT support, increases the burden on scientists, and severely limits the ability to mine the data to identify trends. An interesting question follows: Is higher throughput better if it leads to a data management crisis?

Join a Think Tank group to share and experience and hear what others have learned:

1) Workflow vs. technology development?

2) Scale-up: when and how to go from research to manufacturing?

3) Doing more with less: how do you test new methods and workflows without blowing up your annual budget?

4) Keeping staff motivated and engaged?

5) Tearing down silos: how do you foster cross-functional collaborations to innovate and improve?