Cell therapy products exhibit great therapeutic potential but come with a deterring price tag partly caused by their costly manufacturing processes. The development of strategies that lead to cost-effective cell production is key to expand the reach of cell therapies. Growth factors are critical culture media components required for the maintenance and differentiation of cells in culture and are widely employed in cell therapy manufacturing. However, they are expensive, and their common use in soluble form is often associated with decreased stability and bioactivity. Immobilization has emerged as a possible strategy to optimize growth factor use in cell culture. To date, several immobilization techniques have been reported for attaching growth factors onto a variety of biomaterials, but these have been focused on tissue engineering. This review briefly summarizes the current landscape of cell therapy manufacturing, before describing the types of chemistry that can be used to immobilize growth factors for cell culture. Emphasis is placed to identify strategies that could reduce growth factor usage and enhance bioactivity. Finally, we describe a case study for stem cell factor.

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Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic’s pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.

Snake venoms are rich sources of proteins with potential biotechnological and pharmaceutical applications. Among them, metalloproteases (MPs) and phospholipases A2 (PLA2) are the most abundant. Their isolation involves a multistep chromatographic approach, which has proven to be effective, however implies high operating costs and long processing times. In this study, a cost-effective and simple method based on aqueous two-phase systems (ATPS) was developed to recover MPs and PLA2 from Crotalus molossus nigrescens venom. A system with PEG 400 g mol−1, volume ratio (VR) 1, tie line length (TLL) 25% w/w and pH 7 showed the best performance for PLA2 recovery. In systems with PEG 400 g mol−1, VR 1, TLL 15% w/w, pH 7 and 1 and 3% w/w of NaCl, selective recovery of MP subtype P-III was achieved; whereas, in a system with PEG 400 g mol−1, VR 1, TLL 25% w/w and pH 8.5, MP subtypes P-I and P-III were recovered. Due to their low costs, ethanol–salt systems were also evaluated, however, failed to differentially partition PLA2 and MPs. The use of ATPS could contribute to the simplification and cost reduction of protein isolation processes from snake venoms and other toxin fluids, as well as potentially aid their biochemical, proteomic and biological analyses. Graphic Abstract

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