Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF.

Recombinant human IFNα2b (rhIFNα2b), as an important immune-related protein, has been widely used in clinic for decades. It is also at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Although with the same amino acid sequence, recombinant proteins are generally heterogeneous due to post-translational modification and chemical reactions during expression, purification, and long-term storage, which could have significant impact on the final product quality. So therapeutic rhIFNα2b must be closely monitored to ensure consistency, safety, and efficacy. In this study, we compared seven rhIFNα2b preparations from six manufacturers in China and one in America, as well as four batches of rhIFNα2b preparations from the same manufacturer, measuring IFNα2b variants and site-specific modifications using a developed LC/Q-TOF approach. Three main forms of N-terminus, cysteine, methionine, and acetylated cysteine were detected in five rhIFNα2b preparations produced in E. coli (1E~5E) and one in Pseudomonas (6P), but only the native form with N-terminal cysteine was found in rhIFNα2b preparation produced in Saccharomyces cerevisiae (7Y). Two samples with the lowest purity (4E and 6P), showed the highest level of acetylation at N-terminal cysteine and oxidation at methionine. The level of oxidation and deamidation varied not only between samples from different manufacturers but also between different batches of the same manufacturer. Although variable between samples from different manufacturers, the constitution of N-terminus and disulfide bonds was relatively stable between different batches, which may be a potential indicator for batch consistency. These findings provide a valid reference for the stability evaluation of the production process and final products.

Purification of protein therapeutics via high-affinity supramolecular host-guest interactions.

Efficient purification is crucial to providing large quantities of recombinant therapeutic proteins, such as monoclonal antibodies and cytokines. However, affinity techniques for manufacturing protein therapeutics that use biomolecule-conjugated agarose beads that harness specific biomolecular interactions suffer from issues related to protein denaturation, contamination and the need to maintain biomolecule-specific conditions for efficient protein capture. Here, we report a versatile and scalable method for the purification of recombinant protein therapeutics. The method exploits the high-affinity and controllable host-guest interactions between cucurbit[7]uril (CB[7]) and selected guests such as adamantylammonium. We show that the Herceptin (the brand name of trastuzumab, a monoclonal antibody drug used to treat breast cancer) and the much smaller cytokine interferon α-2a can be purified by site-specifically tagging them with adamantylammonium using the enzyme sortase A, followed by high-affinity binding with CB[7]-conjugated agarose beads and the recovery of the protein using a guest with a stronger affinity for CB[7]. The thermal and chemical stability of CB[7] beads and their scalability, recyclability and low cost may also make them advantageous for the manufacturing of biosimilars.

PASylation Enhances the Stability, Potency, and Plasma Half-Life of Interferon α-2a: A Molecular Dynamics Simulation.

In this study, the effectiveness of PASylation in enhancing the potency and plasma half-life of pharmaceutical proteins has been accredited as an alternative technique to the conventional methods such as PEGylation. Proline, alanine, and serine (PAS) chain has shown some advantages including biodegradability improvement and plasma half-life enhancement while lacking immunogenicity or toxicity. Although some experimental studies have been performed to find the mechanism behind PASylation, the detailed mechanism of PAS effects on the pharmaceutical proteins has remained obscure, especially at the molecular level. In this study, the interaction of interferon α-2a (IFN) and PAS chain is investigated using molecular dynamics simulation method. Several important parameters including secondary structure, root-mean-square distance, and solvent accessible surface area to investigate the stability, bioavailability, and bioactivity of the PASylated protein are studied. The results demonstrate that IFN conformation is not affected critically through PASylation while it results in improvement of the protein stability and bioactivity. Therefore, PASylation can be considered as a proper biological alternative technique to increase the plasma half-life of the biopharmaceutical proteins through enlarging apparent volume. The proposed simulation represents a computational approach that would provide a basis for the study of PASylated pharmaceutical proteins for different future applications.

New Combined Eye Gel with Alpha-2-Beta Interferon.

The results of the development of combined eye gel with interferon alpha-2-beta are presented. Experimental samples of the gel based on different gelling agents were prepared and their biotechnological and technological characteristics (the absence of the cytotoxic effect, aggregation stability, osmotic activity, bioadhesion, and rheological parameters) were evaluated. The composition with hydroxyethyl cellulose, Natrosol 250HHX, in a concentration of 1.5% as a gelling agent showed the best results and the best one-year stability.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

2D NMR Analysis of the Effect of Asparagine Deamidation Versus Methionine Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Protein.

Two of the most common forms of chemical modifications that compromise the efficacy of therapeutic proteins are the deamidation of asparagine residues and oxidation of methionine residues. We probed how deamidation affects the structure, stability, aggregation, and function of interferon alpha-2a (IFNA2a), and compared with our earlier results on methionine oxidation. Upon deamidation, no significant changes were observed in the global secondary structure of IFNA2a with minor changes in its tertiary structure. However, deamidation destabilized the protein, and increased its propensity to aggregate under accelerated stress conditions. Cytopathic inhibition and antiproliferation assays showed drastic decrease in the functionality of deamidated IFNA2a compared to the wild-type. 2D NMR measurements showed structural changes in local protein regions, with no effect on the overall global structure of IFNA2a. These local protein regions corresponded well with the aggregation hot-spots predicted by computational programs, and the functional hot-spots identified by site-directed mutagenesis. When compared to the effects of methionine oxidation, deamidation caused lesser aggregation, because of lesser structural unfolding observed in aggregation hot-spots by 2D NMR. In comparison to oxidation, deamidation showed larger decrease in function, because deamidation affected key amino acid residues in functional hot-spots as observed by 2D NMR and structural modeling. Such quantitative comparison between the effects of deamidation and oxidation on a pharmaceutical protein has not been done before, and the high-resolution structural information on local protein regions obtained by 2D NMR provided a better insight compared to low-resolution methods that probe global protein structure.

Novel glycosylated human interferon alpha 2b expressed in glycoengineered Pichia pastoris and its biological activity: N-linked glycoengineering approach.

Human interferon alpha 2b (IFN α2b) is a type I interferon exhibiting antiviral, anti-proliferative and immunomodulatory activities. The clinical outcome of the approved recombinant human IFN α2b drugs in the market suffers from short plasma half-life, rapid clearance and other side effects. Human IFN α2b expression in mammalian cell lines results in significant heterogeneity in glycan moieties, inconsistent product quality and high production cost. Potential scope exists for the design and development of a successful expression platform for enhanced human IFN α2b production with improved pharmacokinetic property. Glycoengineering strategy was employed to construct IFN α2b with potential N-glycosylation site to evade the drawbacks of approved recombinant human IFN α2b drugs. Heterogeneity of glycosylation and hypermannosylation in the wild-type strains of Pichia pastoris was circumvented by employing glycoengineered strain (SuperMan5) to produce glycosylated IFN α2b with human type N-glycans. Recombinant SuperMan5 strain expressed human type N-glycosylated IFN α2b with greater homogeneity elucidated by glycan analysis (MALDI-TOF/MS). The purified glycosylated IFN α2b was biologically active, inhibiting the viral replication of HCV and HEV at 85% and 66%, respectively. Pharmacokinetic studies in Wistar rats revealed 1.3 fold increase in plasma half-life for glycosylated IFN α2b compared to standard IFN α2b produced by E. coli.

Rapid sample-saving biophysical characterisation and long-term storage stability of liquid interferon alpha2a formulations: Is there a correlation?

The knowledge and tools to characterise proteins have comprehensively developed in the last two decades. Some of these tools are used in formulation development to select formulation conditions suitable for long-term storage. However, there is an ongoing debate whether the predictions obtained with these tools are in good agreement with the outcome from real-time long-term stability studies. In this work, we investigate whether some of the state-of-the-art microscale, microvolume and non destructive biophysical techniques can be applied to promptly select formulations that minimise the aggregation of interferon alpha2a during storage. Interferon alpha2a was used as a model protein as it is known to form aggregates at concentrations over an order of magnitude higher than used in the commercial product. We apply a systematic formulation approach in which we investigate the effect of pH and ionic strength on protein stability. The predictions from the sample-saving biophysical characterisation are validated by long-term stability studies at 4 °C and 25 °C for 12 months on selected formulations. Interferon alpha2a shows minimal aggregation in 10 mM sodium acetate buffer with pH 4 and low ionic strength. The latter is indicated by the rapid sample-saving biophysical characterisation and confirmed by the long-term stability data.

Molecular and Biological Characterization of a Prepared Recombinant Human Interferon Alpha 2b Isoform.

Recombinant human interferon alpha2b (rhIFN-α2b) protein is FDA approved for treatment of many tumors and viral diseases. A rhIFN-α2b isoform has been produced and purified from the refolding reaction using high-resolution anion ion exchange chromatography. This isoform has a proper MW (19 kDa) and high purity and homogeneity. The conservation of native linear and conformational epitopes in this isoform was immunologically confirmed by Western blot and ELISA. Mass spectrometry assessment of its intact mass showed average mass (19,337 Da) equivalent to that of the expressed rhIFN-α2b protein without any chemical modification and without the first methionine. Peptide mapping of rhIFN-α2b through tryptic digestion of reductive/alkylated protein using urea as a denaturing agent gave the best pattern. The rhIFN-α2b had a high specific antiviral activity (2.5 × 108 ± 1.1 × 108IU/mg protein). In vivo clearance study of rhIFN-α2b in female SD rats (500 µg/kg, intramuscularly) revealed rapid clearance (elimination half-life 0.54 h with a maximum plasma concentration of 33,792 pg/ml) compared with the commercial rhIFN-α2 (elimination half-life 0.75-0.96 h). In conclusion, the prepared rhIFN-α2b isoform has high purity, homogeneity, native like chemical and structural composition, high antiviral activity, and proper biological stability, which reduce its immunogenicity and raise its therapeutic efficiency.

Serum immunoglobulin fused interferon-alpha inhibited tumor growth in athymic mice bearing colon 26 adenocarcinoma cells

Interferon (IFN) has therapeutic potential for a wide range of infectious and proliferative disorders. However, the half-life of IFN is too short to have a stable therapeutic effect. To overcome this problem, serum immunoglobulin has been fused to IFN. In this study, the efficacy of serum immunoglobulin fused INFs (si-IFN1 and si-IFN2) was evaluated on athymic mice bearing colon 26 adenocarcinoma cells. Seven days after the implantation of tumor cells, each group of mice was injected once a week with si-IFN1 and si-IFN2 at two different concentrations (10 x : 30 microgram/kg and 50 x : 150 microgram/kg). A slight anti-tumoral effect was observed in all 10 x groups compared to the control. In the 50 x groups, however, si-IFN1 and si-IFN2 showed significant anti- tumoral effects compared to the control. To gain more information on the mechanisms associated with the decrease of tumor size, a Western blot assay of apoptosis-related molecules was performed. The protein expression of cytochrome c, caspase 9, 6, and 3 were increased by si-IFN1 and si-IFN2. These 2 IFNs also increased the expressions of p53, p21, Bax and Bad. Interestingly, si-IFN1 and si-IFN2 decreased the expression of VEGF-beta. Taken together, serum immunoglobulin fused IFNs increased therapeutic efficacy under current experimental condition.