Hydrogels are of growing interest for the delivery of therapeutics to specific sites in the body. For use as a delivery vehicle, hydrophilic precursors are usually laden with bioactive moieties and then directly injected to the site of interest for in situ gel formation and controlled release dictated by precursor design. Hydrogels formed by thiol–ene click reactions are attractive for local controlled release of therapeutics owing to their rapid reaction rate and efficiency under mild aqueous conditions, enabling in situ formation of gels with tunable properties often responsive to environmental cues. Herein, we will review the wide range of applications for thiol–ene hydrogels, from the prolonged release of anti-inflammatory drugs in the spine to the release of protein-based therapeutics in response to cell-secreted enzymes, with a focus on their clinical relevance. We will also provide a brief overview of thiol–ene click chemistry and discuss the available alkene chemistries pertinent to macromolecule functionalization and hydrogel formation. These chemistries include functional groups susceptible to Michael type reactions relevant for injection and radically-mediated reactions for greater temporal control of formation at sites of interest using light. Additionally, mechanisms for the encapsulation and controlled release of therapeutic cargoes are reviewed, including i) tuning the mesh size of the hydrogel initially and temporally for cargo entrapment and release and ii) covalent tethering of the cargo with degradable linkers or affinity binding sequences to mediate release. Finally, myriad thiol–ene hydrogels and their specific applications also are discussed to give a sampling of the current and future utilization of this chemistry for delivery of therapeutics, such as small molecule drugs, peptides, and biologics.
Hydrogel-based depots are of growing interest for release of biopharmaceuticals; however, a priori selection of hydrogel compositions that will retain proteins of interest and provide desired release profiles remains elusive. Toward addressing this, in this work, we have established a new tool for the facile assessment of protein release from hydrogels and applied it to evaluate the effectiveness of mesh size estimations on predicting protein retention or release. Poly(ethylene glycol) (PEG)-based hydrogel depots were formed by photoinitiated step growth polymerization of four-arm PEG functionalized with norbornene (PEG-norbornene, 4% w/w to 20% w/w, M ∼ 5 to 20 kDa) and different dithiol cross-linkers (PEG M ∼ 1.5 kDa or enzymatically degradable peptide), creating well-defined, robust materials with a range of mesh sizes estimated with Flory-Rehner or rubber elasticity theory (∼5 to 15 nm). A cocktail of different model proteins was released from compositions of interest, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to facilely and quantitatively analyze temporal release profiles. Mesh size was predictive of retention of relatively large proteins and release of relatively small proteins. Proteins with diameters comparable to the mesh size, which is often the case for growth factors, were released by hindered diffusion and required experimental assessment of retention and release. With this knowledge, hydrogels were designed for the controlled release of a therapeutically relevant growth factor, PDGF-BB.
Real‐time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real‐time release (RTR) of biopharmaceuticals. A well‐defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real‐time data. We believe a comprehensive PAT roadmap should entail not only integration of analytical tools into the bioprocess but also should address automated‐data piping, analysis, aggregation, visualization, and smart utility of data for advanced‐data analytics such as machine and deep learning for holistic process understanding. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated‐sampling technologies. We also provide insights, based on our experience in clinical and commercial manufacturing, into data automation, data visualization, and smart utility of data for advanced‐analytics in PAT. This review is catered for a broad audience, including those new to the field to those well versed in applying these technologies. The article is also intended to give some insight into the strategies we have undertaken to implement PAT tools in biologics process development with the vision of realizing RTR testing in biomanufacturing and to meet regulatory expectations.
Mesenchymal stem cells (MSCs) are promising for the regeneration of tendon and ligament tissues. Toward realizing this potential, microenvironment conditions are needed for promoting robust lineage-specific differentiation into tenocytes/ligament fibroblasts. Here, we utilized a statistical design of experiments approach to examine combinations of matrix modulus, composition, and soluble factors in human MSC tenogenic/ligamentogenic differentiation. Specifically, well-defined poly(ethylene glycol)-based hydrogels were synthesized using thiol–ene chemistry providing a bioinert base for probing cell response to extracellular matrix cues. Monomer concentrations were varied to achieve a range of matrix moduli (E ∼ 10 – 90 kPa), and different ratios of integrin-binding peptides were incorporated (GFOGER and RGDS for collagen and fibronectin, respectively), mimicking aspects of developing tendon/ligament tissue. A face-centered central composite response surface design was utilized to understand the contributions of these cues to human MSC differentiation in the presence of soluble factors identified to promote tenogenesis/ligamentogenesis (BMP-13 and ascorbic acid). Increasing modulus and collagen mimetic peptide content increased relevant gene expression and protein production or retention (scleraxis, collagen I, tenascin-C). These findings could inform the design of materials for tendon/ligament regeneration. More broadly, the design of experiments enabled efficient data acquisition and analysis, requiring fewer replicates than if each factor had been varied one at a time. This approach can be combined with other stimuli (e.g., mechanical stimulation) toward a better mechanistic understanding of differentiation down these challenging lineages.
During biopharmaceutical process development, it is important to improve titer to reduce drug manufacturing costs and to deliver comparable quality attributes of therapeutic proteins, which helps to ensure patient safety and efficacy. We previously reported that relative high-iron concentrations in media increased titer, but caused unacceptable coloration of a fusion protein during early-phase process development. Ultimately, the fusion protein with acceptable color was manufactured using low-iron media, but the titer decreased significantly in the low-iron process. Here, long-term passaging in low-iron media is shown to significantly improve titer while maintaining acceptable coloration during late-phase process development. However, the long-term passaging also caused a change in the protein charge variant profile by significantly increasing basic variants. Thus, we systematically studied the effect of media components, seed culture conditions, and downstream processing on productivity and quality attributes. We found that removing β-glycerol phosphate (BGP) from basal media reduced basic variants without affecting titer. Our goals for late-phase process development, improving titer and matching quality attributes to the early-phase process, were thus achieved by prolonging seed culture age and removing BGP. This process was also successfully scaled up in 500-L bioreactors. In addition, we demonstrated that higher concentrations of reactive oxygen species were present in the high-iron Chinese hamster ovary cell cultures compared to that in the low-iron cultures, suggesting a possible mechanism for the drug substance coloration caused by high-iron media. Finally, hypotheses for the mechanisms of titer improvement by both high-iron and long-term culture are discussed.
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