Site-specific derivatization of human interferon ß-1a at lysine residues using microbial transglutaminase.

Microbial transglutaminase (TGase) has been successfully used to produce site-specific protein conjugates derivatized at the level of glutamine (Gln) or lysine (Lys) residues with diverse applications. Here, we study the drug human interferon ß-1a (IFN) as a substrate of TGase. The derivatization reaction was performed using carbobenzoxy-L-glutaminyl-glycine to modify Lys residues and dansylcadaverine for Gln residues. The 166 amino acids polypeptide chain of IFN ß-1a contains 11 Lys and 11 Gln residues potential sites of TGase derivatization. By means of mass spectrometry analyses, we demonstrate the highly selective derivatization of this protein by TGase at the level of Lys115 and as secondary site at the level of Lys33, while no reactive Gln residue was detected. Limited proteolysis experiments were performed on IFN to determine flexible regions of the protein under physiological conditions. Interestingly, primary and secondary sites of limited proteolysis and of TGase derivatization occur at the same regions of the polypeptide chain, indicating that the extraordinary selectivity of the TGase-mediated reaction is dictated by the conformational features of the protein substrate. We envisage that the TGase-mediated derivatization of IFN can be used to produce interesting derivatives of this important therapeutic protein.

Deciphering the Biophysical Effects of Oxidizing Sulfur-Containing Amino Acids in Interferon-beta-1a using MS and HDX-MS.

Introduction of a chemical change to one or more amino acids in a protein's polypeptide chain can result in various effects on its higher-order structure (HOS) and biophysical behavior (or properties). These effects range from no detectable change to significant structural or conformational alteration that can greatly affect the protein's biophysical properties and its resulting biological function. The ability to reliably detect the absence or presence of such changes is essential to understanding the structure-function relationship in a protein and in the successful commercial development of protein-based drugs (biopharmaceuticals). In this paper, we focus our attention on the latter by specifically elucidating the impact of oxidation on the HOS, structural dynamics, and biophysical properties of interferon beta-1a (IFNß-1a). Oxidation is a common biochemical modification that occurs in many biopharmaceuticals, specifically in two naturally-occurring sulfur-containing amino acids, methionine and cysteine. To carry out this work, we used combinations of hydrogen peroxide and pH to differentially oxidize IFNß-1a (to focus on only methionine oxidation versus methionine and cysteine oxidation). We then employed several analytical and biophysical techniques to acquire information about the differential impact of these two oxidation scenarios on IFNß-1a. In particular, the use of MS-based techniques, especially HDX-MS, play a dominant role in revealing the differential effects. Graphical Abstract ᅟ.

Biases affecting injected doses of an experimental drug during clinical trials.

BACKGROUND: During clinical trials, researchers rarely question nominal doses specified on labels of investigational products, overlooking the potential for inaccuracies that may result when calculating pharmacokinetic and pharmacodynamic parameters. This study evaluated the disparity between nominal doses and the doses actually administered in two Phase I trials of a biosimilar drug. METHODS: In Trial A, 12 healthy volunteers received various doses of an interferon ß-1a biosimilar via either subcutaneous or intravenous injection, prepared by partially emptying 0.53 ml syringes supplied by the manufacturer. In Trial B, 12 volunteers received three different formulations of the drug via intravenous injection (biosimilar with and without albumin and a comparator), followed by multiple subcutaneous injections. In both trials, the dose administered was calculated as D = C × V - losses, where C is the drug concentration assessed using ELISA, V is the volume administered calculated using syringe weighing and losses are deduced from in-vitro experiments. Interferon binding to added albumin and infusion lines was evaluated using a (125)I-interferon tracer with gel-filtration chromatography. RESULTS: In Trial A, measured concentrations were close to the nominal strength indicated by the manufacturer (median bias: -6 %), whereas in Trial B they differed significantly for all three formulations (median biases: +67 %, +73 % and +31 % for the biosimilar with albumin, the biosimilar without albumin and the comparator, respectively). In Trial A, the doses actually administered showed large variability and biases, especially at the lowest doses. Indeed, actually injected volumes differed by as much as 74 % from theoretical volumes - a phenomenon mainly attributed to unnoticed fluid re-aspiration through the syringe needle. This was corrected in Trial B. Interferon was not significantly adsorbed on the infusion lines used for intravenous administration. Its binding to albumin was slow, reaching 50 % after a 16 h incubation. CONCLUSIONS: These examples illustrate the importance of assessing the actual doses administered in clinical trials, to ensure accuracy in the determination of clearance, distribution volume, bioavailability and dose-response relationships. TRIAL REGISTRATION: Clinicaltrials.gov NCT02515695 (Trial A) and NCT02517788 (Trial B). Registered on 24 July and 5 August 2015, respectively.

Optimizing novel implant formulations for the prolonged release of biopharmaceuticals using in vitro and in vivo imaging techniques.

As a rapidly growing class of therapeutics, biopharmaceuticals have conquered the global market. Despite the great potential from a therapeutic perspective, such formulations often require frequent injections due to their short half-life. Aiming to establish a parenteral dosage form with prolonged release properties, a biodegradable implant was developed, based on a combination of nanoencapsulation of protein-heparin complexes, creation of a slow release matrix by freeze-drying, and compression using hyaluronan and methylcellulose. In order to investigate this novel delivery system, formulations containing IFN-ß-1a and trypsinogen as model proteins were developed. No degradation of the proteins was observed at any stage of the formulation processing. The potential of the delivery system was evaluated in vivo and in vitro after fluorescence-labeling of the biopharmaceuticals. An optimized agarose gel was utilized as in vitro release medium to simulate the subcutaneous environment in a biorelevant manner. In addition, the formulations were administered to female SJL mice and release was innovatively tracked by fluorescence imaging, setting up an in vitro-in vivo correlation. A prolonged time of residence of approximately 12days was observed for the selected formulation design.

Biological Functions of Interferon ß-1a Are Enhanced By Deamidation.

Human type I Interferons (IFN-ß, IFN-ɛ, IFN-κ, IFN-ω, and 12 subtypes of IFN-α) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory activities. They signal through the same cell surface receptors, IFNAR1 and IFNAR2, yet evoking markedly differential potency. One differentiating factor of IFN-ß from other type I interferons is the presence of a consensus sequence (NG) for deamidation. Comparing almost completely deamidated IFN-ß-1a with untreated IFN-ß-1a, this present study reports the increased activities in 3 in-vitro bioassays testing the antiviral, antiproliferative, and immunomodulatory properties, respectively, of the molecule. Deamidated IFN-ß-1a has the potential to improve current therapies in multiple sclerosis, and its ability to potentiate the MHC-Class I expression suggests a clinical benefit in diseases where the downmodulation of the MHC-class I expression plays a role (eg, in immuno-oncology combination therapies or antiviral agents). The present study on IFN-ß deamidation adds a new prospective on deamidation as part of a posttranslational modification code that allows the modulation of the biological properties of proteins. Moreover, it underlines the unique IFN-ß-1a properties that differentiate this molecule from other members of the type I interferon family.