Trends in Light and Temperature Sensitivity Recommendations among Licensed Biotechnology Drug Products.

PURPOSE: Inherent structural and functional properties of biotechnology-derived therapeutic biologics make them susceptible to light- and temperature-induced degradation and consequently can influence their quality. Photosensitivity of therapeutic proteins continues to be examined, but the commonalities and trends of storage conditions and information about light and temperature sensitivity among currently licensed therapeutic proteins has not been previously surveyed. METHODS: Using a comprehensive and relational database approach, we conducted a scientific survey of all licensed biotechnology-derived drug products with the goal of providing evidence-based information about recommended storage conditions of formulations sorted by light- and temperature-related attributes as described for each product at licensure. RESULTS: We report the prevalence of indications for light and temperature sensitivity in formulations categorized by their presentation type, number of doses, container type, dosage form and active molecule type. We also report the storage temperature range across formulations and diluents for reconstitution and dilution. Formulations with excipients that potentially facilitate light-induced and thermal degradation were also noted. CONCLUSIONS: The result of our analysis indicates that light and temperature sensitivity are prevalent across therapeutic protein formulations. However, when a formulation is reconstituted or diluted, both light and temperature sensitivity are less clear. In addition, light and temperature sensitivity are more well defined in liquid formulations than lyophilized powder formulations, and more well defined in products manufactured in autoinjectors, prefilled-syringes, and pens than products in vials. Overall, our report provides a data-driven summary of storage conditions among therapeutic protein formulations to support the development of future biologic drug products.

Understanding RNA Binding by the Nonclassical Zinc Finger Protein CPSF30, a Key Factor in Polyadenylation during Pre-mRNA Processing.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains five CCCH domains and one CCHC domain and recognizes two conserved 3' pre-mRNA sequences: an AU hexamer and a U-rich motif. AU hexamer motifs are common in pre-mRNAs and are typically defined as AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs and can affect polyadenylation efficiency or be linked to diseases. The effects of disease-related variations on CPSF30/pre-mRNA binding were determined using a construct of CPSF30 that contains just the five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify the variability within the AU hexamer sequence in pre-mRNAs. The effects of this sequence variability on CPSF30-5F/RNA binding affinities were measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer were found to be important for RNA binding. Bioinformatics revealed that the three bases flanking the AU hexamer at the 5' and 3' ends are twice as likely to be adenine or uracil as guanine and cytosine. The presence of A and U residues in these flanking regions was determined to promote higher-affinity CPSF30-5F/RNA binding than G and C residues. The addition of the zinc knuckle domain to CPSF30-5F (CPSF30-FL) restored binding to AU hexamer variants. This restoration of binding is connected to the presence of a U-rich sequence within the pre-mRNA to which the zinc knuckle binds. A mechanism of differential RNA binding by CPSF30, modulated by accessibility of the two RNA binding sites, is proposed.

Unraveling the RNA Binding Properties of the Iron-Sulfur Zinc Finger Protein CPSF30.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a "zinc finger" protein that plays a crucial role in the transition of pre-mRNA to RNA. CPSF30 contains five conserved CCCH domains and a CCHC "zinc knuckle" domain. CPSF30 activity is critical for pre-mRNA processing. A truncated form of the protein, in which only the CCCH domains are present, has been shown to specifically bind AU-rich pre-mRNA targets; however, the RNA binding and recognition properties of full-length CPSF30 are not known. Herein, we report the isolation and biochemical characterization of full-length CPSF30. We report that CPSF30 contains one 2Fe-2S cluster in addition to five zinc ions, as measured by inductively coupled plasma mass spectrometry, ultraviolet-visible spectroscopy, and X-ray absorption spectroscopy. Utilizing fluorescence anisotropy RNA binding assays, we show that full-length CPSF30 has high binding affinity for two types of pre-mRNA targets, AAUAAA and polyU, both of which are conserved sequence motifs present in the majority of pre-mRNAs. Binding to the AAUAAA motif requires that the five CCCH domains of CPSF30 be present, whereas binding to polyU sequences requires the entire, full-length CPSF30. These findings implicate the CCHC "zinc knuckle" present in the full-length protein as being critical for mediating polyU binding. We also report that truncated forms of the protein, containing either just two CCCH domains (ZF2 and ZF3) or the CCHC "zinc knuckle" domain, do not exhibit any RNA binding, indicating that CPSF30/RNA binding requires several ZF (and/or Fe-S cluster) domains working in concert to mediate RNA recognition.

Fe-S clusters masquerading as zinc finger proteins.

Metal ions are commonly found as protein co-factors in biology, and it is estimated that over a quarter of all proteins require a metal cofactor. The distribution and utilization of metals in biology has changed over time. As the earth evolved, the atmosphere became increasingly oxygen rich which affected the bioavailability of certain metals such as iron, which in the oxidized ferric form is significantly less soluble than its reduced ferrous counterpart. Additionally, proteins that utilize metal cofactors for structural purposes grew in abundance, necessitating the use of metal co-factors that are not redox active, such as zinc. One common class of Zn co-factored proteins are zinc finger proteins (ZFs). ZFs bind zinc utilizing cysteine and histidine ligands to promote structure and function. Bioinformatics has annotated 5% of the human genome as ZFs; however, many of these proteins have not been studied empirically. In recent years, examples of annotated ZFs that instead harbor Fe-S clusters have been reported. In this review we highlight four examples of mis-annotated ZFs: mitoNEET, CPSF30, nsp12, and Fep1 and describe methods that can be utilized to differentiate the metal-cofactor.

Iron-Sulfur Clusters in Zinc Finger Proteins.

Zinc finger (ZF) proteins are proteins that use zinc as a structural cofactor. The common feature among all ZFs is that they contain repeats of four cysteine and/or histidine residues within their primary amino acid sequence. With the explosion of genome sequencing in the early 2000s, a large number of proteins were annotated as ZFs based solely upon amino acid sequence. As these proteins began to be characterized experimentally, it was discovered that some of these proteins contain iron-sulfur sites either in place of or in addition to zinc. Here, we describe methods to isolate and characterize one such ZF protein, cleavage and polyadenylation specificity factor 30 (CPSF3O) with respect to its metal-loading and RNA-binding activity.