Supplementary Table 1. Properties of bacterial IBs adjustable through process conditions (extended version of Table 1). Recombinant S vs. Parameter Culture conditions protein IS IBs structural features Ref. (E. coli strain) (%) β-lactamase IBs increased in median diameter from Bioreactor, fed-batch (37 °C), (Margreiter, (K12, HMS174- 5-95 325 nm to 410 nm, 2 and 6 h after IPTG induction et al., 2008) DE3) induction. IB size incremented from 200 up to 800 Culture time nm after 4 h of induction. IBs showed (Upadhyay, Batch (37 °C), IPTG induction hGH (M15) N.D. strong binding with CR and Th-T, and et al., 2012) were more resistant to proteolysis and denaturation. Sphingomyelin IBs Increased in a median diameter (Castellanos Bioreactor, batch (37 °C), 3– ase-D (BL21- from 450 nm (1 h post-induction) to 600 -Mendoza, IPTG induction 97 Gold-DE3) nm (3 h post-induction). et al., 2014) Bioreactor, fed-batch (37°C), 2– Similar aggregation, increasing ~25 % β-lactamase 1.0 µM IPTG / g dry biomass 98 RP content in IBs. (Margreiter, (K12, HMS174- Inductor concentration Bioreactor, fed-batch (37°C), 5– Similar aggregation, lower RP content in et al., 2008) DE3) 20 µM IPTG / g dry biomass 95 IBs. Shake flask, batch (37 °C) 13- Decreased aggregation. (Jhamb & 0.01 mM IPTG 87 XynB (BL21) Sahoo, Shake flask, batch (37 °C) 1.0 Increased IB formation. 4-96 2012) mM IPTG Batch (37°C), 0.1 mM IPTG N.D. Lower hydrodynamic diameter. (Luo, et al., GFP Hydrodynamic diameter increased from Batch (37°C), 2.0 mM IPTG N.D. 2006) 550 to 645 nm. EGFP (DH5- 5-10 times more RP are in IBs at the (Iafolla, et Growth rate Shake tubes, batch (37 °C) N.D. alfa) fastest growth rate. al., 2008) 73- Less RP aggregation Batch (30 °C), trp promoter IFN–α2 27 (HB101) Increase in temperature promoted RP (Schein & Batch (37 °C), trp promoter 5-95 aggregation Noteborn, Batch (30 °C), trp promoter 95-5 Less RP aggregation 1988) IFN–γ (C600/- 18- Increase in temperature promoted RP Temperature Batch (37 °C), trp promoter lys) 82 aggregation Bioreactor, batch (30°C in Lower IBs formation at initial post- growth phase), N.D. induction time. (Strandberg thermoinduction at 39 °C SpA-β gal (RR1 & Enfors, Bioreactor, batch (30°C in lacZAM15) The highest accumulation of IBs, 1991) growth phase), N.D. formed mainly during the first hours thermoinduction at 42°C after induction. Shake flask, batch (30°C), 0.1 99– Less RP aggregation. mM IPTG 1 Impurities were lower in IBs from Shake flask, batch (37 °C), 0.1 60- OmpA-β- cultures at 37 °C vs. 42 °C. The highest (Valax & mM IPTG 40 lactamase IBs concentration. Georgiou, Shake flask, batch (42°C), 0.1 (RB791) 83- Temperature increase promoted IB 1993) mM IPTG 17 formation. Batch (37 °C by 4 h), after 1 Less RP aggregation and high specific mM IPTG cultures were set N.D. fluorescence. to 18 °C The formed fluorescent IBs were Batch (37 °C by 4 h), after 1 solubilized and denatured faster by mM IPTG cultures were set N.D. proteases and chaotropic agents, than (de Groot & to 25°C A42(F19D)- those from cultures at 42 °C. Ventura, GFP Batch (37 °C by 4 h), after 1 Increase in temperature promoted IB 2006) mM IPTG cultures were set N.D. formation and decreased their to 37 °C fluorescence Batch (37 °C by 4 h), after 1 Increase in temperature promoted IB mM IPTG cultures were set N.D. formation and depleted their to 42 °C fluorescence. 2– Proteins inside IBs formed at 25°C G-CSF 98 presented similar structure as the (Jevsevar, et Shake flask, batch (25°C), 0.4 33- native versions, with increased al., 2005, GFP mM IPTG 77 extractability in mild detergents, Peternel, et 60- compared with those from 37°C and al., 2008) His7dN6TNF-α 40 42°C. Bioreactor, batch (30°C in When pH decreased to ~5.5, IB growth phase, N.D. accumulation was triggered, reaching (Strandberg thermoinduction at 39°C) SpA-βgal (RR1 up to 30 % of cell dry weight. & Enfors, Bioreactor, batch (30°C in lacZAM15) When pH decreases to ~5.5, IBs 1991) growth phase, N.D. formation increased vs. 39°C, reaching thermoinduction at 42°C) up to 75 % of cell dry weight. Bioreactor, batch (37 °C), 0.1 0- IBs were more resistant to proteolysis mM IPTG. pH 7.5 Sphingomyelin 100 and denaturation. (Castellanos pH Bioreactor, Batch (37 °C), 0.1 ase-D (BL21- IBs grown faster and were less resistant -Mendoza, 0- mM IPTG. pH uncontrolled Gold-DE3) to proteolysis and denaturation. et al., 2014) 100 (reach pH 8.5) Bioreactor, batch (37 °C), 0.1 IBs presented more -helices, were Phospholipase 0- mM IPTG. pH 7.5, and set to solubilized faster by proteinase-K and A2 (OrigamiTM) 100 (Calcines- 8.5 after induction bonded less Th-T. Cruz, et al., Bioreactor, batch (37 °C), 0.1 IBs presented less -helices, were less Phospholipase 0- 2018) mM IPTG. pH 7.5, and set to TM solubilized by proteinase-K and bonded A2 (Origami ) 100 6.5 after induction more Th-T. IBs showed sizes of ~400 nm with less Shake flask, batch (37 °C), 0.1 0- -helices fraction, compared with those mM IPTG, orbital (200 rpm) Phospholipase 100 (Valdez- formed under resonant acoustic. Agitation A2 (BL21-Gold- Cruz, et al., Shake flask, batch (37 °C), 0.1 Diffused protein clusters were seen DE3) 0- 2017) mM IPTG, resonant acoustic inside cells. IBs at 20 g were the most 100 (20 g) degraded after 120 min. 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