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In this study, a microbioreactor\u2010based approach was employed to systematically design and evaluate mixed\u2010substrate fed\u2010batch strategies for enhanced HPV16 L1 VLP production. Four methanol\u2010based feeding compositions, including methanol\u2010only and combinations with glycerol and sorbitol, were investigated under pulse and linear feeding modes. Among the tested conditions, a linear feeding strategy using 70% methanol and 30% glycerol (70\u2009M\u2009+\u200930G) resulted in the highest biomass (DCW: 90.36\u2009g\/L) and VLP titer (42\u2009mg\/L), corresponding to a two\u2010fold improvement over methanol\u2010only feeding. Enhanced performance was attributed to reduced methanol\u2010induced metabolic stress and improved substrate utilization, leading to more stable dissolved oxygen (DO) profiles and sustained cellular activity. In contrast, pulse feeding resulted in significant DO fluctuations, indicating transient metabolic imbalances and lower overall productivity. The optimized feeding strategy was successfully translated to a 1\u2009L stirred\u2010tank bioreactor, achieving a DCW of 120\u2009g\/L and a VLP titer of 49\u2009mg\/L, with a\u2009~\u20092.3\u2010fold increase compared to the control. The consistency between microscale and bench\u2010scale results demonstrates the predictive capability of microbioreactor systems for fed\u2010batch process development. Overall, this study presents a rational and scalable feeding strategy that improves recombinant VLP production while reducing methanol usage, providing a practical framework for efficient bioprocess design in\n                    <jats:italic>K. phaffii<\/jats:italic>\n                    .\n                  <\/jats:p>","DOI":"10.1002\/btpr.88559","type":"journal-article","created":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T05:40:12Z","timestamp":1791610812000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Design and scale\u2010up of a mixed\u2010substrate fed\u2010batch strategy for enhanced\n                    <scp>HPV VLP<\/scp>\n                    production in\n                    <i>Komagataella phaffii<\/i>"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-4015-072X","authenticated-orcid":false,"given":"Abhilasha K.","family":"Rani","sequence":"first","affiliation":[{"name":"School of Interdisciplinary Research Indian Institute of Technology  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7821-5074","authenticated-orcid":false,"given":"Nida","family":"Khan","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering Indian Institute of Technology  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5913-4244","authenticated-orcid":false,"given":"Anurag S.","family":"Rathore","sequence":"additional","affiliation":[{"name":"School of Interdisciplinary Research Indian Institute of Technology  New Delhi India"},{"name":"Department of Chemical Engineering Indian Institute of Technology  New Delhi India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,9]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms23031818"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00705\u2010009\u20100484\u20108"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1590\/s0100\u2010879x2011007500141"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.3390\/vaccines8030391"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.4014\/jmb.2407.07033"},{"issue":"2","key":"e_1_2_10_7_1","first-page":"113","article-title":"Development of prophylactic HPV vaccines","volume":"31","author":"Gissmann L","year":"2007","journal-title":"Coll Antropol"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.3390\/pharmaceutics13111967"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1177\/0022034509333446"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978\u20100\u201012\u2010809633\u20108.09046\u20104"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20103\u2010031\u201028489\u20102_17"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1186\/s43141\u2010023\u201000514\u20109"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010020\u201061103\u20101"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.4161\/hv.18757"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.14218\/JCTH.2018.00037"},{"key":"e_1_2_10_16_1","doi-asserted-by":"crossref","DOI":"10.1016\/j.bej.2023.109188","article-title":"Bioprocessing of inclusion bodies from E. 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