en Pharmaceutical Biotechnology Pharmaceutical Biotechnology مقاله کامل Full Length/Original Article <span style="font-family:Times New Roman;"><span style="font-size:11pt"><span style="line-height:normal"><span class="Heading2Char"><span style="color:#4472c4"><span style="font-weight:bold"><span style="font-size:10.5pt"><span style="color:black">Background</span></span></span></span></span><span style="font-size:10.5pt"><span style="color:black">: Downstream processing of therapeutic recombinant proteins expressed as the </span></span><span style="font-size:10.5pt"><span style="color:black">inclusion bodies</span></span><span style="font-size:10.5pt"><span style="color:black"> (IBs) in <i>E. coli</i> is quite challenging. This study aimed to use the </span></span><span style="font-size:10.5pt"><span style="color:black">quality by design approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim.</span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span class="Heading2Char"><span style="color:#4472c4"><span style="font-weight:bold"><span style="font-size:10.5pt"><span style="color:black">Methods:</span></span></span></span></span><span style="font-size:10.5pt"><span style="color:black"> For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most </span></span><span style="font-size:10.5pt"><span style="color:black">critical quality attributes (CQAs) and effects of process parameters on these quality attributes. </span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span class="Heading2Char"><span style="color:#4472c4"><span style="font-weight:bold"><span style="font-size:10.5pt"><span style="color:black">Results:&nbsp;</span></span></span></span></span><span style="font-size:10.5pt"><span style="color:black">The solubilization of IBs was optimized by design of experiment</span></span><span style="font-size:10.5pt"><span style="color:black"> on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in </span></span><span style="font-size:10.5pt"><span style="color:black">anion exchange chromatography</span></span><span style="font-size:10.5pt"><span style="color:black"> that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on </span></span><span style="font-size:10.5pt"><span style="color:black">high molecular weight</span></span><span style="font-size:10.5pt"><span style="color:black"> impurities. After polishing by gel filtration, the final product&#39;s biological activity showed no statistically significant differences among the groups received romiplostim and Nplate^&reg;, as the reference product. </span><span style="color:black"></span></span></span></span><br> <span class="Heading2Char"><span style="color:#4472c4"><span style="font-weight:bold"><span style="font-size:10.5pt"><span style="line-height:115%"><span style="color:black">Conclusion</span></span></span></span></span></span><span style="font-size:10.5pt"><span style="line-height:115%"><span style="color:black">: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters</span></span></span><span style="font-size:10.5pt"><span style="line-height:115%">.</span></span> </span> Romiplostim, CQA, Design space, Quality by Design, risk assessment 414 425 http://ibj.pasteur.ac.ir/browse.php?a_code=A-10-1832-4&slc_lang=en&sid=1 Saeedeh Pouri saeedehpouri@yahoo.co 0000-0002-0093-1564 No Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Fatemeh Torkashvand fatemehtorkashvand4@gmail.com 0000-0003-3066-1144 No Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Hooman Aghamirza Moghim Aliabadi hooman.kntu2010@gmail.com 0000-0003-2150-5721 No Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Pezhman Fard-Esfahani pejman_fard@yahoo.com 0000-0001-5943-7846 No Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran Majid Golkar majid.golkar@gmail.com 0000-0002-0052-2410 No Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran Behrouz Vaziri behrouz-vaziri@pasteur.ac.ir 0000-0002-0295-5922 Yes Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran