Analysis of Biologics Molecular Descriptors towards Predictive Modelling for Protein Drug Development Using Time-Gated Raman Spectroscopy.

Pharmaceutical proteins, compared to small molecular weight drugs, are relatively fragile molecules, thus necessitating monitoring protein unfolding and aggregation during production and post-marketing. Currently, many analytical techniques take offline measurements, which cannot directly assess protein folding during production and unfolding during processing and storage. In addition, several orthogonal techniques are needed during production and market surveillance. In this study, we introduce the use of time-gated Raman spectroscopy to identify molecular descriptors of protein unfolding. Raman spectroscopy can measure the unfolding of proteins in-line and in real-time without labels. Using K-means clustering and PCA analysis, we could correlate local unfolding events with traditional analytical methods. This is the first step toward predictive modeling of unfolding events of proteins during production and storage.

Characterization, Stability, and in Vivo Efficacy Studies of Recombinant Human CNTF and Its Permeation into the Neural Retina in ex Vivo Organotypic Retinal Explant Culture Models.

Ciliary neurotrophic factor (CNTF) is one of the most studied neuroprotective agents with acknowledged potential in treating diseases of the posterior eye segment. Although its efficacy and mechanisms of action in the retina have been studied extensively, it is still not comprehensively understood which retinal cells mediate the therapeutic effects of CNTF. As with therapeutic proteins in general, it is poorly elucidated whether exogenous CNTF administered into the vitreous can enter and distribute into the retina and hence reach potentially responsive target cells. Here, we have characterized our purified recombinant human CNTF (rhCNTF), studied the protein's in vitro bioactivity in a cell-based assay, and evaluated the thermodynamic and oligomeric status of the protein during storage. Biological activity of rhCNTF was further evaluated in vivo in an animal model of retinal degeneration. The retinal penetration and distribution of rhCNTF after 24 h was studied utilizing two ex vivo retina models. Based on our characterization findings, our rhCNTF is correctly folded and biologically active. Moreover, based on initial screening and subsequent follow-up, we identified two buffers in which rhCNTF retains its stability during storage. Whereas rhCNTF did not show photoreceptor preservative effect or improve the function of photoreceptors in vivo, this could possibly be due to the used disease model or the short duration of action with a single intravitreal injection of rhCNTF. On the other hand, the lack of in vivo efficacy was shown to not be due to distribution limitations; permeation into the retina was observed in both retinal explant models as in 24 h rhCNTF penetrated the inner limiting membrane, and being mostly observed in the ganglion cell layer, distributed to different layers of the neural retina. As rhCNTF can reach deeper retinal layers, in general, having direct effects on resident CNTF-responsive target cells is plausible.

Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS).

Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.

Electron dispersive X-ray spectroscopy and degradation properties of hyaluronic acid decorated microparticles.

The purpose of this study was to produce poly(DL-lactic-co-glycolic acid) (PLGA) - based microparticles (MPs), externally decorated with hyaluronic acid (HA). The MPs are intended for intravitreal injections in the treatment of posterior eye segment and have been designed to prolong the release of growth factors into the vitreous body, therefore aiming to increase the time interval between two consecutive injections. The MPs, prepared by a modified double emulsion-solvent evaporation technique and loaded with bovine serum albumins (BSA) and ciliary neurotrophic factor (CNTF), were spherical, with a diameter around 70 µm and a >90% encapsulation efficiency. Energy Dispersive Spectroscopy (EDS) outcomes indicated that HA presence in the external aqueous phase of the emulsion did affect the surface properties of MPs. Moreover, poloxamers drastically slowed down MP degradation properties which are, in turn, closely related to their ability to prolong drug release. This is promising for the envisaged application of the produced MPs. Further work will be devoted to optimizing MP formulation with respect to the envisaged intravitreal route of administration.

Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation.

The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may affect the protein's stability and activity. We aimed to integrate protein expression, purification, and bioconjugation in small volumes coupled with cell free protein synthesis for the target protein, ciliary neurotrophic factor. Split-intein mediated capture by use of capture peptides onto a solid surface was efficient at 89-93%. Proof-of-principle of light triggered release was compared to affinity chromatography (His6 fusion tag coupled with Ni-NTA). The latter was more efficient, but more time consuming. Light triggered release was clearly demonstrated. Moreover, we transferred biotin from the capture peptide to the target protein without further purification steps. Finally, the target protein was released in a buffer-volume and composition of our choice, omitting the need for protein concentration or changing the buffer. Split-intein mediated capture, protein trans splicing followed by light triggered release, and bioconjugation for proteins synthesized in cell free systems might be performed in an integrated workflow resulting in the fast production of the target protein.

Pharmacokinetic aspects of retinal drug delivery.

Drug delivery to the posterior eye segment is an important challenge in ophthalmology, because many diseases affect the retina and choroid leading to impaired vision or blindness. Currently, intravitreal injections are the method of choice to administer drugs to the retina, but this approach is applicable only in selected cases (e.g. anti-VEGF antibodies and soluble receptors). There are two basic approaches that can be adopted to improve retinal drug delivery: prolonged and/or retina targeted delivery of intravitreal drugs and use of other routes of drug administration, such as periocular, suprachoroidal, sub-retinal, systemic, or topical. Properties of the administration route, drug and delivery system determine the efficacy and safety of these approaches. Pharmacokinetic and pharmacodynamic factors determine the required dosing rates and doses that are needed for drug action. In addition, tolerability factors limit the use of many materials in ocular drug delivery. This review article provides a critical discussion of retinal drug delivery, particularly from the pharmacokinetic point of view. This article does not include an extensive review of drug delivery technologies, because they have already been reviewed several times recently. Instead, we aim to provide a systematic and quantitative view on the pharmacokinetic factors in drug delivery to the posterior eye segment. This review is based on the literature and unpublished data from the authors' laboratory.

Codon optimization and factorial screening for enhanced soluble expression of human ciliary neurotrophic factor in Escherichia coli.

BACKGROUND: Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. Human ciliary neurotrophic factor (hCNTF) has neuroprotective properties and is also known to influence energy balance. Consequently, hCNTF has potential therapeutic applications in neurodegenerative, obesity and diabetes related disorders. Clinical and biological applications of hCNTF necessitate a recombinant expression system to produce large amounts of functional protein in soluble form. Earlier attempts to express hCNTF in Escherichia coli (E. coli) were limited by low amounts and the need to refold from inclusion bodies. RESULTS: In this report, we describe a strategy to effectively identify constructs and conditions for soluble expression of hCNTF in E. coli. Small-scale expression screening with soluble fusion tags identified many conditions that yielded soluble expression. Codon optimized 6-His-hCNTF construct showed soluble expression in all the conditions tested. Large-scale culture of the 6-His-hCNTF construct yielded high (10 - 20 fold) soluble expression (8 - 9 fold) as compared to earlier published reports. Functional activity of recombinant 6-His-hCNTF produced was confirmed by its binding to hCNTF receptor (hCNTFRα) with an EC50 = 36 nM. CONCLUSION: Our results highlight the combination of codon optimization and screening soluble fusion tags as a successful strategy for high yielding soluble expression of hCNTF in E. coli. Codon optimization of the hCNTF sequence seems to be sufficient for soluble expression of hCNTF. The combined approach of codon optimization and soluble fusion tag screen can be an effective strategy for soluble expression of pharmaceutical proteins in E. coli.