D trehalose

For this purpose, needles were obtained in batches from about 20 L total of bacteria growing in M9-type minimal media (Studier, 2005 (link)) supplemented with 2 g ml⁻¹¹⁵N labelled ammonium chloride and U–¹³C₆ labelled d-glucose (Cambridge Isotope Laboratories/CK Gas). Overnight cultures of 5 ml M9 minimal Media with labelled glucose and NH₄Cl, were inoculated with ΔmxiH (pACT3mxiH; (Shen et al., 2010 (link))), with kanamycin and chloramphenicol. The cultures were incubated at 37 °C with shaking at 180 rpm. After 16 hrs, the optical density at 600 nm (OD₆₀₀) was measured. If the OD₆₀₀ was >1.00, all of the 5 ml culture was used to inoculate 1 L of M9 minimal media, labelled as above, in a 5 L sterile conical flask, with 200 μM IPTG and incubated at 37 °C with shaking at 180 rpm. After 16 hrs, the OD₆₀₀ was measured again, if OD₆₀₀ was >1.10, growth was halted and needles purified as previously described (Cordes et al., 2005 (link), Fujii et al., 2012 (link)). The final pellet was resuspended in 0.01% of the initial culture volume in sterile 20 mM Tris–HCl pH 7.4, 100 mM NaCl, 10% w/v d-(+)-Trehalose (Sigma) and 0.02% w/v sodium azide, generally attaining 3–7 mg ml⁻¹ in protein concentration, and then flash frozen in liquid nitrogen and stored at −80 °C until use. From 20 L of bacterial culture about 7 mg of labelled needles were obtained.

All incubations were carried out in a PBS solution supplemented with: 50 mM d-Trehalose (Sigma T9531), 0.4 mM d-Glucose (Sigma G8270), 5 mM CaCl2, 15 mM MgSO₄, 12.3 mM Glutamine. Late third instar larvae were dissected and incubated for 15 min with 5 μM ThiolTracker Violet (Molecular Probes T10095) at room temperature. Dissected larvae were then washed three times for 1 min before being mounted in the same solution on a glass slide. Slides were imaged immediately on a confocal microscope.

Following cultivation, the cell suspension was centrifuged using a Heraeus™ Biofuge™ Stratos™ Centrifuge (Thermo Fisher Scientific, Waltham, USA; 15,000 rpm, 0.3 L/min, 30 min) to enable cell harvesting. Then, the cells were washed by adding peptone salt solution (1 g/L peptone from casein; 8.5 g/L NaCl; 0.3 g/L KH₂PO₄; 0.6 g/L Na₂HPO₄·2H₂O) and centrifuged again under the same conditions for another 15 min. After homogenizing the cell pellet, the protectants maltodextrin (dextrose equivalent 4.0–7.0; Sigma-Aldrich, Steinheim, Germany), D(+) trehalose (Sigma-Aldrich, Steinheim, Germany), and glucitol (D(-) sorbitol, Merck KGaA, Darmstadt, Germany) were added in several concentrations (0, 10, 25, 50, 75, and 100%) related to the bacterial dry matter (BDM, w/w). Afterwards, 1-mL aliquots of the suspensions were pipetted into freeze-drying glass vials and frozen in a − 80 °C freezer BF-U538 (Buchner Labortechnik, Pfaffenhofen an der Ilm, Germany). The process scheme is illustrated in Fig. 1.Fig. 1

Process scheme applied in this study

Monoclonal antibody IgG1 type (adalimumab, ADA) was kindly provided by AbbVie (Cork, Ireland). The qualitative composition of ADA formulation includes mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, and Polysorbate 80. The samples were in their commercial liquid form (100 mg/mL) and preserved at 4 °C until use. The osmolarity of the original formulation was measured by a Cryoscopic Osmometer (OSMOMAT^® 030, Gonotec^®, Germany). This equipment measures the freezing point depression, thus determining the total osmolality of aqueous solutions. The osmolarity of commercial ADA was 0.274 ± 0.0175 Osm/L and possessed a pH of 5.3 ± 0.007. D-mannitol, D-trehalose, and polyethylene glycol 6000 and 8000 (PEG) were purchased from Sigma-Aldrich (Burlington, USA) and Dextran 40 kDa was obtained from Pharmacosmos (Holbæk, Denmark). These ingredients were explored as potential excipients to improve the stability of the freeze-dried, concentrated IgG₁ samples.

The chemical composition, encompassing free sugars and organic acids was evaluated following procedures previously described by Barros et al. [20 (link)].
Free sugars were analyzed by HPLC coupled to a refraction index detector (Knauer, Smartline system 1000). The compounds were identified by chromatographic comparisons with authentic standards (D(−)-fructose, D(+)-sucrose, D(+)-glucose, D(+)-trehalose, and D(+)-raffinose pentahydrate) which were purchased at Sigma-Aldrich (St. Louis, MO, USA), as also melezitose (PanReac AppliChem ITW Reagents Co., Darmstadt, Germany) which was applied as the internal standard (IS) and used in the quantification method. Data was analyzed using Clarity 2.4 software (DataApex, Podohradska, Czech Republic), and the results were expressed in g/100 g fw.
Organic acids were evaluated using an Ultra-Fast Liquid Chromatography (UFLC, Shimadzu 20A series, Kyoto, Japan) coupled to a diode array detector. Organic acids standards (L(+)-ascorbic acid, citric acid, malic acid, oxalic acid, shikinic acid, succinic acid, fumaric acid, and quinic acid; Sigma-Aldrich, St. Louis, MO, USA) were used for identification by performing chromatographic comparisons with the peaks of the samples. These standards were also used for quantification relying on the external standard methodology. Results were expressed g/100 g fw.