Search

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research.

We describe a novel epitope discovery strategy for creating an affinity agent/peptide tag pair. A synthetic polymer nanoparticle (NP) was used as the "bait" to catch an affinity peptide tag. Biotinylated peptide tag candidates of varied sequence and length were attached to an avidin platform and screened for affinity against the polymer NP. NP affinity for the avidin/peptide tag complexes was used to provide insight into factors that contribute NP/tag binding. The identified epitope sequence with an optimized length (tMel-tag) was fused to two recombinant proteins. The tagged proteins exhibited higher NP affinity than proteins without tags. The results establish that a fusion peptide tag consisting of optimized 15 amino acid residues can provide strong affinity to an abiotic polymer NP. The affinity and selectivity of NP/tMel-tag interactions were exploited for protein purification in conjunction with immobilized metal ion/His6-tag interactions to prepare highly purified recombinant proteins. This strategy makes available inexpensive, abiotic synthetic polymers as affinity agents for peptide tags and provides alternatives for important applications where more costly affinity agents are used.

Enabling real‐time monitoring and control of the biomanufacturing processes through product quality insights continues to be an area of focus in the biopharmaceutical industry. The goal is to manufacture products with the desired quality attributes. To realize this rigorous attribute‐focused Quality by Design approach, it is critical to support the development of processes that consistently deliver high‐quality products and facilitate product commercialization. Time delays associated with offline analytical testing can limit the speed of process development. Thus, developing and deploying analytical technology is necessary to accelerate process development. In this study, we have developed the micro sequential injection process analyzer and the automatic assay preparation platform system. These innovations address the unmet need for an automatic, online, real‐time sample acquisition and preparation platform system for in‐process monitoring, control, and release of biopharmaceuticals. These systems can also be deployed in laboratory areas as an offline analytical system and on the manufacturing floor to enable rapid testing and release of products manufactured in a good manufacturing practice environment. [ABSTRACT FROM AUTHOR]

Research has been conducted on the amide-backbone substitution in the solid-phase synthesis of 'difficult' peptides. The advantages of this substitution are discussed.

Research is presented describing the development of a peptide cyclization strategy which proceeds through a ring closure/ring contraction process. A photolabile peptide cyclization auxiliary has been developed.

The p-cresol and p-thiocresol adducts produced from acylium ions during HF cleavage after contemporary Boc/benzyl solid-phase peptide synthesis were examined. Findings did not conform with previous reports because both p-cresol and p-thiocresol were observed to predominantly produce aryl esters under the usual cleavage conditions. The p-cresol esters were stable at 0 deg C but rearranged irreversibly at higher temperatures to afford aryl ketones. On the other hand, p-thiocresols did not undergo a Fries rearrangement but readily underwent further additions of p-thiocresol to produce ketenebisthioacetals and trithio ortho esters.

The study explains a new chemical approach for improving the multifaceted pharmaceutical profile of small proteins with an anti-HIV activity. These molecules can also help in the development of new anti-HIV therapeutics.

A new approach is applied to the site-specific attachment of poly(ethylene glycol) (PEG) to a synthetic CCL-5 (RANTES) analogue. The objective is to attach a PEG polymer in the GAG binding site at position 45 to form CCL-5--PEG, to reduce aggregation and to increase the circulating lifetime.

Two-dimensional nuclear magnetic resonance spectroscopy was used to describe the three-dimensional solution structure of the polypeptide alpha-conotoxin MII. A comparison was made for MII in three different solvent systems with that of an aqueous solution as well as to other alpha-conotoxins that target neuronal nicotinic acetylcholine receptors. Results showed that the presence of a hydrophobic solution environment enhances a helical region within MII and results in improved structural definition.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers. [ABSTRACT FROM AUTHOR]

The feasibility of developing a protecting group strategy primarily for keto groups that would be compatible with contemporary chemical protein synthesis strategies is explored. A reversible protection strategy that would allow for the complete protection of the keto group throughout peptide assembly, peptide side-chain deprotection, cleavage from resin, purification and native chemical ligation processes is implemented.