Modern biopharmaceutics pose a key challenge: how to produce large amounts of complex and labile biologics flexibly and affordably? Our team introduced allotropic chromatography using peptide ligands that respond to Ca2+/Mg2+ to purify antibodies, butyrylcholinesterase, Cas9, and a-1-antitrypsin: flow-through affinity chromatography wherein a feedstock is continuously fed to a peptide-functionalized adsorbent that selectively retains host cell proteins and DNA—and lets the product flow through—enabling continuous purification.

Single-step purification of proteins using affinity chromatography is often hampered by the lack of appropriate affinity ligands. We have developed a robust platform called the nanoCLAMP for producing custom affinity chromatography resins. nanoCLAMPs bind with mid- to low-nM Kd, have melting temperatures of >70°C, resist digestion by proteases, are compatible with multiple regeneration cycles with NaOH, and are elutable at neutral pH. Several single-step purification studies using custom nanoCLAMP resins will be described, highlighting the wide applicability of this platform.

The commercial availability of a practical self-removing affinity tag has provided a new platform for the rapid and inexpensive development of new therapeutic proteins.  These tags can be combined with additional expression- and solubility-enhancing domains, where the self-removal reaction is also largely insensitive to buffer excipients.  Hear case studies on the use of self-removing tags for expression and purification of difficult targets, including cytokines, membrane proteins, and multimeric proteins.

In this study, we explored various parameters for antibody production in the TGE cell host Expi293F and ExpiCHO-S with the transfection reagents ExpiFectamine and polyethylenimine. We discovered that there are significant differences between Expi293F and ExpiCHO-S cells with regards to DNA complex formation time and ratio, complex formation buffers, DNA complex uptake trafficking routes, responses to dimethyl sulfoxide and cell cycle inhibitors, as well as light-chain isotype expression preferences. This investigation mechanistically dissected the TGE processes and provided a new direction for future transient antibody production optimization.

Accessibility and affordability of proteins globally is poor. Disruptive innovations, such as highly automated small-footprint manufacturing, could enhance local biomanufacturing capacity and support global access. At Sunflower, we have developed integrated, continuous, and automated small-footprint manufacturing technologies for the production of pharmaceutical-quality proteins using eukaryotic microbes. Here, we describe the benefits of an alternative host (Pichia pastoris) for the production of high-quality proteins using Sunflower's small-footprint biomanufacturing equipment. 

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.

In order to obtain high-resolution NMR structural data, monoclonal antibody needs to be isotopically labeled. As labeling in mammalian cells is not practical, protein was expressed in E. coli and yeast. mAb expression in non-mammalian cell types was compared to mammalian-expressed mAb, and differences will be discussed, including a surprising result from E. coli codon optimization.

The drug discovery landscape is ever-evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have implemented several high-throughput small- and mid-scale screening strategies for challenging proteins to enable faster protein production for structural and biochemical studies. These strategies enable parallel testing of multiple parameters to improve protein yields and allow identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co-expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for aggregation-prone proteins. These platforms enable screening of intracellular, membrane and secreted proteins in E.coli, insect cells and mammalian expression systems. 

• Deciding the best expression strategy for new proteins: shotgun or informed guess?
• Balancing the best host: slow and reliable or quick and messy? 
• How pure is pure enough? Do we always need tags?
• What happens when you just can’t get what you need out of your strategy? What next? 
  

RaGene, an AI-driven codon optimization pipeline, integrates generative and evaluative components to address limitations of existing rule-based codon optimization tools, enhancing protein translation fidelity. Utilizing multi-omics data from the host and advanced protein features, RaGene consistently boosts yield by 2-10 fold and achieves 95-99% monomer percentages across monoclonal and multi-specific antibodies. By optimizing mRNA non-sequence features and controlling translation speed, the method ensures high yield and proper protein folding through precise ribosome regulation, significantly improving protein expression with significant implications on the efficacy and safety of cell and gene therapies. 

As biological engineering improves, the risk of release of a dangerous genetically modified organism becomes greater. It is important that authorities can carry out attribution, which can be achieved, in part, by discovering the design tools for the engineered genetic components. Previous methods focused on plasmid signatures. The next logical step is to address attribution using signatures from the tools that are used to create the engineered modifications. A classifier was developed that discriminates between codon optimization design tools, achieving more than 97% accuracy. The methods presented here lay the groundwork for the creation of effective organism engineering attribution techniques.

Research programs require high-quality recombinant proteins to enable proof-of-concept studies and structural biology. Some proteins present production and quality issues. To increase success, we developed a robust method for expressing proteins in HEK293-derived stable pools, producing recombinant proteins with less clipped species and high yields. This method also works with HEK293S GnTI- and Expi293F GnTI- suspension cells, facilitating production of proteins with less complex glycans for structural biology projects.

We identified a panel of GS mutants as potential selection markers in GS-knockout CHO cells. Our modified CMV promoter boosted antibody production over sixfold in stably transfected bulk pools compared to the leading commercial vector with a standard CMV promoter. Combing the GS mutant marker and modified CMV promoter in a single vector yielded a mini-pool antibody productivity of 4.98g/L. This was achieved in a simple fed-batch setup in 50 mL tubes without optimization.