Since several decades, PEGylation is known to be the clinical standard to enhance pharmacokinetics of biotherapeutics. In this study, we introduce polyglycerol (PG) of different lengths and architectures (linear and hyperbranched) as an alternative polymer platform to poly(ethylene glycol) (PEG) for half-life extension (HLE). We designed site-selective Nterminally modified PG-protein conjugates of the therapeutic protein anakinra (IL-1ra, Kineret) and compared them systematically with PEG analogues of similar molecular weights. Linear PG and PEG conjugates showed comparable hydrodynamic sizes and retained their secondary structure, whereas binding affinity to IL-1 receptor 1 decreased with increasing polymer length, yet remained in the low nanomolar range for all conjugates. The terminal half-life of a 40 kDa linear PG-modified anakinra was extended 4-fold compared to the unmodified protein, close to its PEG analogue. Our results demonstrate similar performances of PEG-and PG-anakinra conjugates and therefore highlight the outstanding potential of polyglycerol as a PEG alternative for half-life extension of biotherapeutics.
Poor water solubility and low bioavailability of active pharmaceutical ingredients (APIs) are major causes of friction in the pharmaceutical industry and represent a formidable hurdle for pharmaceutical drug development. Drug delivery remains the major challenge for the application of new small-molecule drugs as well as biopharmaceuticals. The three challenges for synthetic delivery systems are: (i) controlling drug distribution and clearance in the blood; (ii) solubilizing poorly water-soluble agents, and (iii) selectively targeting specific tissues. Although several polymerbased systems have addressed the first two demands and have been translated into clinical practice, no targeted synthetic drug delivery system has reached the market. This Review is designed to provide a background on the challenges and requirements for the design and translation of new polymer-based delivery systems. This report will focus on chemical approaches to drug delivery for systemic applications.
The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.
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