In use for over four decades now, insect cell protein expression has produced thousands of high-quality proteins for drug discovery, X-ray crystallography, and vaccine development. Technological developments in the last decade have increased the value of this system and broadened the scope of its use to numerous new areas, including the production of multiprotein complexes and cryo-EM. We will discuss the numerous advantages of insect cells for producing high-value recombinant proteins.

Protein production in bacteria is limited by downstream processing (DSP), as purification accounts for most of the time and cost of protein manufacturing. Protein secretion streamlines DSP by enabling purification of the target protein out of the culture media, bypassing cell lysis and reducing unit operations. Opera Bioscience has developed the type 3 secretion system (T3SS) in a novel bacterial expression platform to achieve single-step secretion of heterologous proteins, achieving up to 90% purity. This enables efficient fermentation to manufacture proteins for reagents, enzymes, and therapeutics. We will present progress on the industrial development of Opera’s bacterial secretion host.

Heterologous expression of mammalian proteins can often be a significant challenge due to membrane integration, post-translational modifications, and prosthetic cofactor integration. This is particularly true for the human cytochrome (CYP) P450 enzymes, a large family of xenobiotic-detoxifying oxidoreductases, which suffer from all three limitations. In this presentation, we will describe our efforts to develop a CYP P450 expression system using the unicellular kinetoplast protozoan Leishmania tarentolae as a novel and highly advantageous host for CYP P450 expression.

Trichoderma reesei is a filamentous fungus known for its high productivity and extensive use in the enzyme and food industries. Our research shows it is also an attractive production platform for pharmaceutical proteins. We generated strains with high-expression levels of an IgA-Fc fragment and a model monoclonal antibody, which are secreted into the extracellular medium, facilitating downstream processing. We have also engineered fungal glycosylation pathways to append the proteins with mammalian type N-glycans. Our technoeconomic assessment indicates that using T. reesei as a production host for pharmaceutical proteins is significantly more cost-effective compared to mammalian cells.

Prolific Machines’ photomolecular platform uses molecular optogenetics to dynamically control gene expression in mammalian cell lines using light. The unique features of Prolific’s platform enable novel solutions for the production of next-generation, complex biologics. This presentation will describe the platform, demonstrate its application in the expression of therapeutic proteins in CHO cells, and explain how it enables a new level of performance and control while leveraging a proven mammalian production architecture.

We present a systems engineering approach to modifying E. coli for scalable, efficient protein production. By decoupling growth from expression, we implement strain modifications that are incompatible with growth but enable high-titer production of challenging proteins. This includes strain engineering to ease scale-up and dynamic modulation of protease and reductase activity to express nanobodies and degradation-prone proteins. We also demonstrate genetically programmed in-Bioreactor purification, greatly simplifying downstream processing.

Targeted integration (TI) of therapeutic protein-encoding transgenes into predetermined high and stably expressing transcriptional hotspots in CHO cells can simplify the cell line development processes for biologics production. We used TRIP (Thousands of Reporters Integrated in Parallel) technology to identify transcriptional hotspots in CHO cells through randomly integrated barcoded reporters tracked by sequencing. TRIP-identified hotspots achieved 9.4-fold higher mRNA levels and 5.6-fold increased protein titers compared to controls, providing a powerful functional screening method.

To overcome challenges in multi-chain and bispecific antibody expression, we created a new stable expression system using only the auxotrophic glutamine synthetase (GS) selection marker. Up to four different antibody chains can be stably expressed in GS-KO CHO-K1 cells using two plasmids in our split GS vector system through mRNA trans-splicing, a post-transcriptional event whereby exons from two separate RNAs join to produce a chimeric RNA.

As a protein expression core, we focus on hard-to-express targets for structure–function studies. Our current targets include multi-protein transmembrane complexes. These target proteins require co-expression of chaperone proteins as well. I will focus my talk on two targets: the Alpha 7 nicotinic receptor (which requires co-expression of a chaperone) and the ghrelin GPCR complex (four proteins, a chaperone, and a nanobody to help stabilize the complex).

Cell-free protein synthesis unlocks high-throughput biologics discovery and machine learning–driven protein engineering. By decoupling cell growth from protein production, it enables rapid expression in just 5–8 hours using preprepared, stable extracts. Unlike cell-based systems, it offers precise control over reaction conditions, efficient incorporation of non-natural amino acids, and production of complex molecules from diverse scaffolds—all with a single extract. This flexibility accelerates design–build–test cycles and supports parallel screening at scale. This cutting-edge platform is fully scalable from benchtop to commercial manufacturing, offering a transformative alternative to traditional expression systems.