Spray drying extract

Abstract

The present disclosure provides improved methods for stabilizing spray-dried bacterial extracts using compositions containing stabilizers so that the extracts can be used for cell-free protein synthesis. Also provided herein are formulations for stable spray-dried bacterial extracts that have increased protein synthesis activity compared to spray-dried bacterial extracts that do not contain stabilizers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 396,143, filed August 8, 2022, the entire disclosure of which is incorporated herein by reference for all purposes.

[0002] The present disclosure provides compositions and methods for producing spray-dried bacterial extracts with improved stability after long-term storage at different temperatures. The spray-dried extracts can be used in commercial-scale cell-free protein synthesis reactions. Summary of the Invention

[0003]

[0003] Provided herein are compositions and methods for producing a spray-dried bacterial extract for use in a cell-free protein synthesis reaction. In one aspect, the method includes: (i) combining a bacterial extract containing dissolved bacterial components with a composition containing trehalose, lactose, leucine, or raffinose to obtain a mixture, wherein the bacterial extract is capable of synthesizing a target protein from a template nucleic acid encoding the target protein in a cell-free protein synthesis reaction; and (ii) spray-drying the mixture to produce a stable spray-dried bacterial extract.

[0004] In another aspect, the present disclosure provides a method for expressing a target protein in a cell-free protein synthesis reaction, comprising: i) combining a bacterial extract comprising lysed bacterial components with a composition comprising trehalose, lactose, leucine, or raffinose to obtain a mixture, wherein the lysed bacterial components are capable of synthesizing a target protein from a template nucleic acid encoding the target protein in a cell-free protein synthesis reaction; ii) spray-drying the mixture to produce a stable spray-dried bacterial extract; iii) rehydrating the spray-dried bacterial extract; and iv) adding a template nucleic acid encoding the target protein to the rehydrated extract, wherein the template nucleic acid is translated in the rehydrated extract, thereby expressing the target protein.

[0005] In another aspect, the disclosure provides a method for expressing a target protein in a cell-free protein synthesis reaction, comprising rehydrating a spray-dried bacterial extract comprising lysed bacterial components and a composition comprising trehalose, lactose, leucine, or raffinose; adding a template nucleic acid encoding the target protein to the rehydrated extract, wherein the template nucleic acid is translated in the rehydrated extract, thereby expressing the target protein.

[0006] In some embodiments, the bacterial extract comprising lysed bacterial components is a liquid bacterial extract or a rehydrated bacterial extract.

[0007] In some embodiments, the composition comprises trehalose or lactose. In some embodiments, the mixture comprises about 25-200 g / kg trehalose. In some embodiments, the mixture comprises about 50-100 g / kg trehalose. In some embodiments, the trehalose is trehalose dihydrate (TDH).

[0008] In some embodiments, the mixture comprises about 25-200 g / kg lactose, in some embodiments, the mixture comprises about 50-100 g / kg lactose, in some embodiments, the lactose is lactose monohydrate (LMH).

[0009] In some embodiments, the mixture comprises about 5-10 g / L of leucine, hi some embodiments, the mixture comprises about 25-200 g / L of raffinose.

[0010] In some embodiments, the stable spray-dried bacterial extract comprises about 40-70 g / L of bacterial extract solids, hi some embodiments, the stable spray-dried bacterial extract comprises about 50-60 g / L of bacterial extract solids.

[0011] In some embodiments, the bacterial extract is further combined with one or more high glass transition temperature (Tg) non-polar, uncharged amino acids. In some embodiments, the one or more high Tg non-polar, uncharged amino acids are selected from valine, tryptophan, isoleucine, leucine, alanine, glycine, proline, and any combination thereof. In some embodiments, the one or more high Tg non-polar, uncharged amino acids are selected from L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, or L-proline, and any combination thereof.

[0012] In some embodiments, the bacterial extract is further combined with one or more amino acids selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, arginine, and any combination thereof.

[0013] In some embodiments, the mixture comprises 5-15 g / kg of amino acids.

[0014] In some embodiments, in step (i), the mixture further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), or 2-hydroxypropyl-β-cyclodextrin.

[0015] In some embodiments, the bacterial spray-dried extract contains about 15% (w / w) or less residual water. In some embodiments, the bacterial spray-dried extract contains about 10% (w / w) or less residual water. In some embodiments, the bacterial spray-dried extract contains about 5% (w / w) or less residual water.

[0016] In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% of the control extract after storage at 2°C-8°C for at least 6 months. In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% of the control extract after storage at 2°C-8°C for at least 12 months. In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% of the control extract after storage at 2°C-8°C for at least 18 months.

[0017] In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% of the control extract after storage for at least 6 months at about −20° C. In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% of the control extract after storage for at least 12 months at about −20° C. In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% of the control extract after storage for at least 18 months at about −20° C.

[0018] In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% greater than that of a control extract after storage at about room temperature (20° C.) for at least 6 months. In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% greater than that of a control extract after storage at about room temperature (20° C.) for at least 12 months. In some embodiments, the bacterial spray-dried extract is capable of synthesizing a target protein at a titer that is at least 80% greater than that of a control extract after storage at about room temperature (20° C.) for at least 18 months.

[0019] In some embodiments, the titer is determined by the Phytip® method.

[0020] In some embodiments, the spray-dried extract and the control extract comprise trehalose, and the spray-dried extract is stored at 2°C to 8°C for at least 12 months and, after reconstitution, is capable of synthesizing a target protein at a titer of at least 80% compared to the control extract reconstituted at time 0. In some embodiments, when the spray-dried extract is stored at 2°C to 8°C and then rehydrated, the protein synthesis activity of the spray-dried extract declines by less than about 5% per month compared to the control extract.

[0021] In some embodiments, the control extract does not contain trehalose, lactose, leucine, or raffinose. In some embodiments, the control extract contains trehalose, lactose, leucine, or raffinose.

[0022] In some embodiments, the spray-dried bacterial extract comprises an active oxidative phosphorylation system for cell-free protein synthesis. In some embodiments, the bacterial extract is derived from Escherichia species.

[0023] In some embodiments, prior to step (i), the bacterial extract is heated at about 20° C. to 45° C. for about 30 minutes to about 10 hours.

[0024] In some embodiments, spray drying in step (ii) comprises atomizing the mixture to form droplets, contacting the droplets with a gas to evaporate the liquid from the droplets, separating the dried extract from the gas and smaller particles, and collecting the spray-dried extract.

[0025] In some embodiments, 90% (w / w) or more of the liquid is removed from the mixture. In some embodiments, 95% (w / w) or more of the liquid is removed from the mixture.

[0026] In some embodiments, the method further includes (iii) rehydrating the spray-dried bacterial extract, and (iv) synthesizing the target protein under conditions that support a cell-free protein synthesis reaction. In some embodiments, the rehydrated bacterial extract comprises about 20% to 60% (by volume) of the cell-free protein synthesis reaction. In some embodiments, the rehydrated bacterial extract comprises about 30% to 40% (by volume) of the cell-free protein synthesis reaction.

[0027] In another aspect, the disclosure provides a spray-dried bacterial extract composition for cell-free protein synthesis, the extract comprising dried, lysed bacterial components and a composition comprising trehalose, lactose, leucine, or raffinose, wherein the extract, upon rehydration, is capable of synthesizing a target protein from a template nucleic acid encoding the target protein.

[0028] In some embodiments, the composition comprises trehalose or lactose. In some embodiments, the composition comprises about 25-200 g / kg trehalose. In some embodiments, the composition comprises about 50-100 g / kg trehalose. In some embodiments, the trehalose is trehalose dihydrate (TDH). In some embodiments, the composition comprises about 25-200 g / kg lactose. In some embodiments, the composition comprises about 50-100 g / kg lactose. In some embodiments, the lactose is lactose monohydrate (LMH). In some embodiments, the composition comprises about 5-10 g / L leucine. In some embodiments, the composition comprises about 25-200 g / L raffinose.

[0029] In some embodiments, the extract further comprises one or more high glass transition temperature (Tg) non-polar, uncharged amino acids. In some embodiments, the high Tg non-polar, uncharged amino acids are selected from the group consisting of valine, tryptophan, isoleucine, leucine, alanine, glycine, proline, and any combination thereof. In some embodiments, the high Tg non-polar, uncharged amino acids are selected from the group consisting of L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, L-glycine, L-proline, and any combination thereof. In some embodiments, the extract comprises 5-15 g / kg of amino acids.

[0030] In some embodiments, the extract further comprises one or more, any combination, or any subset of amino acids selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine, hi some embodiments, the extract comprises about 5-15 g / L of amino acids.

[0031] In some embodiments, the extract further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), or 2-hydroxypropyl-β-cyclodextrin.

[0032] In some embodiments, the spray-dried extract contains about 15% (w / w) or less residual water. In some embodiments, the spray-dried extract contains about 10% (w / w) or less residual water. In some embodiments, the spray-dried extract contains about 5% (w / w) or less residual water.

[0033] In some embodiments, the spray-dried extract is stored at 2°C-8°C for at least 6 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract. In some embodiments, the spray-dried extract is stored at 2°C-8°C for at least 12 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract. In some embodiments, the spray-dried extract is stored at 2°C-8°C for at least 18 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract.

[0034] In some embodiments, the spray-dried extract is stored at about -20°C for at least 6 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract. In some embodiments, the spray-dried extract is stored at about -20°C for at least 12 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract. In some embodiments, the spray-dried extract is stored at about -20°C for at least 18 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract.

[0035] In some embodiments, the spray-dried extract is stored at about room temperature (20° C.) for at least 6 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract. In some embodiments, the spray-dried extract is stored at about room temperature (20° C.) for at least 12 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract. In some embodiments, the spray-dried extract is stored at about room temperature (20° C.) for at least 18 months and is capable of synthesizing a target protein at a titer that is at least 80% of that of a control extract.

[0036] In some embodiments, the titer is determined by the Phytip® method. In some embodiments, the control extract does not contain trehalose, lactose, leucine, or raffinose. In some embodiments, the control extract contains trehalose, lactose, leucine, or raffinose.

[0037] In some embodiments, the spray-dried extract and the control extract contain trehalose, and the spray-dried extract is stored at 2°C to 8°C for at least 12 months and, after rehydration, is capable of synthesizing a target protein at a titer of at least 80% compared to the control extract rehydrated at time T=0.

[0038] In some embodiments, the protein synthesis activity of the rehydrated spray-dried extract declines by less than about 5% per month when the extract is stored at 2°C to 8°C before rehydration. In some embodiments, the protein synthesis activity of the rehydrated spray-dried extract is equal to or greater than the protein synthesis activity of the rehydrated control spray-dried extract when the extract is stored at 2°C to 8°C for 8 months or more before rehydration. In some embodiments, the spray-dried extract is stored at 2°C to 8°C for 13 months before rehydration. In some embodiments, the control spray-dried extract does not contain trehalose.

[0039] In some embodiments, the spray-dried extract contains trehalose or lactose and is stored at 2° C. to 8° C. for at least 4 months and is capable of synthesizing a target protein at a titer of at least 75% compared to a control extract that does not contain trehalose or lactose and is stored at −20° C. In some embodiments, the spray-dried extract contains about 75 g / kg to 105 g / kg of trehalose or about 100 g / kg of lactose.

[0040] In some embodiments, the rehydrated extract has 80% or more of the initial protein synthesis activity when stored at 2°C to 8°C for at least 18 months prior to rehydration, compared to the protein synthesis activity of the rehydrated extract at T=0.

[0041] In some embodiments, the spray-dried bacterial extract has an active oxidative phosphorylation system for cell-free protein synthesis. In some embodiments, the extract is derived from Escherichia species. In some embodiments, the extract is a powder. In some embodiments, the extract does not have a cake-like appearance or is not a dry cake.

[0042] In another aspect, the disclosure provides a method for preparing a spray-dried extract, comprising the steps of: (i) providing a liquid bacterial extract containing components for cell-free synthesis of a target protein from a template nucleic acid encoding the target protein; (ii) generating droplets of the liquid bacterial extract; (iii) contacting the droplets with a gas to evaporate the liquid from the droplets; (iv) separating the dried extract from the gas and smaller particles; and (v) collecting the spray-dried extract.

[0043] In some embodiments, prior to step (i), the liquid bacterial extract is sterile filtered. In some embodiments, the sterile filtered liquid bacterial extract is activated by heat.

[0044] In some embodiments, a composition comprising trehalose, lactose, leucine, or raffinose is added to the activated sterile-filtered liquid bacterial extract prior to step (ii), hi some embodiments, the composition comprises about 25-200 g / kg trehalose, about 25-200 g / kg lactose, about 5-10 g / L leucine, or about 25-200 g / L raffinose.

[0045] In some embodiments, step (i) further comprises adding one or more amino acids to the activated sterile-filtered liquid bacterial extract. In some embodiments, the one or more amino acids comprise high glass transition temperature (Tg) non-polar, uncharged amino acids selected from the group consisting of L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, L-proline, and any combination thereof. In some embodiments, the one or more amino acids are selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine.

[0046] In some embodiments, one or more of maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), 2-hydroxypropyl-β-cyclodextrin, or any combination thereof, are added to the activated sterile-filtered liquid bacterial extract.

[0047] In some embodiments, step (ii) comprises atomizing the liquid bacterial extract to generate droplets. In some embodiments, atomizing comprises passing the liquid bacterial extract through an atomization device selected from a nozzle or a rotary atomizer. In some embodiments, the median droplet size is about 20-100 microns (Dv50) at an atomization gas pressure of 10-50 psig.

[0048] In some embodiments, step (iii) comprises contacting the droplets with a drying gas through a drying chamber, the drying gas having an outlet temperature of about 60° C. to about 90° C. In some embodiments, step (iv) comprises separating the dried extract from the gas and smaller particles using centrifugal force. In some embodiments, step (v) comprises collecting the spray-dried extract in a vessel.

[0049] In some embodiments, the collected spray-dried extract contains about 15% (w / w) or less, about 10% (w / w) or less, or about 5% (w / w) or less residual water, hi some embodiments, 85% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more of the liquid is removed from the spray-dried extract.

[0050] In some embodiments, the protein synthesis activity of the rehydrated spray-dried extract decreases by less than about 5% per month when the extract is stored at 2°C to 8°C before rehydration. In some embodiments, the protein synthesis activity of the rehydrated spray-dried extract is equal to or greater than the protein synthesis activity of the rehydrated control extract when the extract is stored at 2°C to 8°C for 8 months or more before rehydration. In some embodiments, the spray-dried extract is stored at 2°C to 8°C for 13 months before rehydration.

[0051] In some embodiments, the spray-dried bacterial extract has an active oxidative phosphorylation system for cell-free protein synthesis. In some embodiments, the liquid extract is derived from Escherichia sp.

[0052] In another aspect, the disclosure provides a spray-dried extract for cell-free protein synthesis, the spray-dried extract comprising dried lysed bacterial components and one or more stabilizers, wherein the stabilizer has a glass transition temperature (Tg) of at least about 90°C, and the concentration of the stabilizer in the liquid extract before spray drying is between about 5 g / L and 200 g / L, or between about 25 g / kg and 200 g / kg.

[0053] In some embodiments, the stabilizer is selected from trehalose, lactose, and leucine. In some embodiments, the stabilizer comprises about 25-200 g / kg trehalose, about 25-200 g / kg lactose, or about 5-10 g / L leucine. In some embodiments, the trehalose is trehalose dihydrate (TDH) and the lactose is lactose monohydrate (LMH). In some embodiments, at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w) of the liquid is removed from the mixture.

[0054] In some embodiments, the extract further comprises one or more high glass transition temperature (Tg) non-polar, uncharged amino acids selected from L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, L-proline, and any combination thereof. In some embodiments, the extract further comprises one or more, all combinations, or a subset of amino acids selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine. In some embodiments, the extract further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), 2-hydroxypropyl-β-cyclodextrin, or any combination thereof.

[0055] In another aspect, there is provided a method of producing a target protein from a spray-dried extract, comprising reconstituting the spray-dried extract of the present disclosure, providing a template nucleic acid encoding the target protein, and producing the target protein. [Brief explanation of the drawings]

[0056] [Figure 1]Figure 1 shows data from laboratory-scale spray drying. Drying was performed using a laboratory-scale Buchi B-290 spray dryer, with the inlet and outlet temperatures reported in parentheses for dried samples as (inlet temperature / outlet temperature). XpressCF® (XCF) testing used batch reactions in Micro-24 microbioreactors (m24) and Flower Plates (FP) with 30% (by volume) XtractCF® to express the product anti-CD74 antibody. The feedstock and dried XtractCF® were from lot DR 3c H4 B5.

[0057] [Figure 2A] Figure 2A shows data from the pilot-scale spray-dried formulation and drying process. Samples were prepared and dried at 100 g / L, 50 g / L, and 25 g / L trehalose. Drying was performed using a pilot-scale Mobile Minor PSD-1 spray dryer. XCF testing used a batch reaction in a Micro-24 microbioreactor with 30% (by volume) XtractCF® to express the product anti-CD74 antibody. The feedstock and dried XtractCF® were from lot ER 15-4.

[0058] [Figure 2B] Figure 2B shows data from the pilot-scale spray-dried formulation and drying process. Samples were prepared and dried at 100 g / L, 50 g / L, and 25 g / L of trehalose. Drying was performed using a pilot-scale Mobile Minor PSD-1 spray dryer. XCF testing used a batch reaction in a Micro-24 microbioreactor with 30% (by volume) XtractCF® to express the product anti-CD74 antibody. The feedstock and dried XtractCF® were from lot ER 15-4.

[0059] [Figure 3A] FIG. 3A shows the long-term stability data of spray-dried trehalose dihydrate formulated XtractCF® at 2-8° C., −20° C., and room temperature, respectively. [Figure 3B] FIG. 3B shows the long-term stability data of spray-dried trehalose dihydrate formulated XtractCF® at 2-8° C., −20° C., and room temperature, respectively. [Figure 3C] Figure 3C shows the long-term stability data for spray-dried trehalose dihydrate-formulated XtractCF® at 2-8°C, -20°C, and room temperature, respectively. Samples containing 25, 50, and 100 g / L amounts of trehalose dihydrate formulation and associated residual moisture levels (batch and % residual moisture listed in the legend) were spray-dried using a Mobile Minor (PSD-1). The titer loss rate (% / month) was calculated from a linear fit of the data from 6 months to 24.5 months. The titer of the antibody product, anti-CD74 antibody, was measured at the first time point (t0), while the trastuzumab antibody was used at all other time points in this study.

[0060] [Figure 4A] Figure 4A shows data from the 100 L pilot study from a 100 L spray drying study. Drying was performed using a modified PSD-2. The XCF study used a batch reaction in a Micro-24 microbioreactor using 30% extract (by volume) to express the product trastuzumab. The feedstock and dried XtractCF® were: (1) pilot study (DR) 1, extract lot ER11:ER17 80%:20% (2) DR 2, extract lot ER17 (3) DR 3, extract lot ER 18.

[0061] [Figure 4B]Figure 4B shows experimental data from a 100 L spray drying study. Drying was performed using a modified PSD-2. The XCF study used a batch reaction in a Micro-24 microbioreactor with 30% extract to express the product trastuzumab. The feedstock and dried XtractCF® were: (1) DR 1, ER11:ER17 80%:20% (2) DR 2, ER17 (3) DR 3, ER 18.

[0062] [Figure 5A] Figure 5A shows long-term stability data at 2-8 °C from a 100 L spray drying study. The XCF study used a batch reaction in a Micro-24 microbioreactor with 30% (by volume) extract to express the product trastuzumab.

[0063] [Figure 5B] Figure 5B shows long-term stability data at -20°C from a 100 L spray drying study. The XCF study used a batch reaction in a Micro-24 microbioreactor with 30% (by volume) extract to express the product trastuzumab.

[0064] [Figure 5C] Figure 5C shows long-term stability data at room temperature from a 100 L spray drying study. The XCF study used a batch reaction in a Micro-24 microbioreactor with 30% (by volume) extract to express the product trastuzumab.

[0065] [Figure 6A] Figure 6A shows long-term stability data from a 100 L spray drying study at -20°C, 2-8°C, and room temperature. The XCF study used a batch reaction in a DASbox stirred tank bioreactor using 37.5% (by volume) extract to express the product anti-folate receptor alpha antibody with a preformed light chain (PFLC).

[0066] [Figure 6B] Figure 6B shows long-term stability data from a 100 L spray drying study at -20°C, 2-8°C, and room temperature. The XCF study used a batch reaction in a DASbox stirred tank bioreactor with 37.5% (by volume) extract to express the product anti-folate receptor alpha antibody with PFLC.

[0067] [Figure 7A] Figure 7A shows comparative data for various trehalose spray-dried formulations from the February 2020 Mobile Minor® spray drying study. [Figure 7B] Figure 7B shows comparative data for various trehalose spray-dried formulations from a Mobile Minor® spray drying study in February 2020. Figure 7A shows a comparison of the initial activity of 100 g / L trehalose and 75 g / kg trehalose at the same drying process conditions (inlet T / outlet T; 168°C / 80°C). In the XCF study, a 37.5% (volume basis) extract was used to express an anti-folate receptor alpha antibody with a PFLC. Figure 7B shows a comparison of the initial activity of 100 g / L trehalose and 75 g / kg trehalose at the same drying process conditions (inlet T / outlet T; 168°C / 80°C). In the XCF study, a 37.5% (volume basis) extract was used to express an anti-CD74 antibody.

[0068] [Figure 8A] Figure 8A shows comparative data for various drying conditions for lactose spray-dried formulations from a Mobile Minor® spray drying study in February 2020. Figure 8A shows a comparison of the initial activity of 100 g / kg lactose for three different drying process conditions (inlet T / outlet T; 168°C / 80°C, 150°C / 70°C, 117°C / 60°C). The XCF study used a 37.5% (volume basis) extract to express anti-folate receptor alpha antibody with PFLC.

[0069] [Figure 8B]Figure 8B shows comparative data for various drying conditions for lactose spray-dried formulations from the Mobile Minor® spray drying study in February 2020. Figure 9B shows a comparison of the initial activity of 100 g / kg lactose for three different drying process conditions (inlet T / outlet T; 168°C / 80°C, 150°C / 70°C, 117°C / 60°C). In the XCF study, a 37.5% (volume basis) extract was used to express anti-CD74 antibodies.

[0070] [Figure 9] Figure 9 shows long-term stability data at 2-8°C for the trehalose dihydrate and lactose monohydrate single-component formulation XtractCF®. Figure 9 shows data for samples spray-dried using Mobile Minor® (PSD-1) containing trehalose dihydrate formulations in amounts of 75 g / kg and 100 g / L, and lactose monohydrate at 100 g / kg, and associated residual moisture levels. The percent titer loss (per month) was calculated from a linear fit of the data from 1 month to 5 months. In this study, the titer of trastuzumab antibody was measured.

[0071] [Figure 10] Figure 10 shows activity data for PSD-3. All XCF studies were performed using a 30% extract expressing trastuzumab in the flower plate. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present disclosure provides compositions and methods for producing spray-dried bacterial extracts for use in cell-free protein synthesis reactions. The spray-dried bacterial extracts contain additives (e.g., one or more additives) that increase the stability of the extract during long-term storage compared to spray-dried extracts that do not contain additives. The spray-dried extracts offer the unexpected advantage of retaining the ability to produce biomolecules, e.g., proteins, when stored for relatively long periods of time at various temperatures compared to spray-dried extracts that do not contain additives. For example, spray-dried extracts can unexpectedly be stored for 6 to 18 months (or longer) at about -20°C (minus 20°C), about 2°C to 8°C, or about room / ambient temperature (e.g., about 20°C) and still retain the ability to produce biomolecules, e.g., proteins. In contrast, liquid bacterial extracts stored at about -20°C, with or without additives, typically retain activity for only several months, while liquid bacterial extracts stored at about 2°C to 8°C without additives typically lose about 25 to 50% activity after one day.

[0073] Spray-drying bacterial extract also offers the advantages of reducing the volume of the liquid extract and increasing the stability of the bacterial extract for long-term storage for commercial-scale production.

[0074] I. Definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0075] It is understood that this disclosure is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which is limited only by the appended claims.

[0076] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, and a reference to a "protein" includes a reference to one or more proteins and equivalents thereof known to those of skill in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0077] In the claims. The transitional term "comprising" is a term of art and is intended to be inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The term "consisting essentially of" refers to specified materials or steps, and materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter. The terms "consists of" or "consisting of" exclude any element, step, or ingredient not specified in the claim.

[0078] As used herein, the term "about," when modifying any quantity, refers to variations in quantity typically encountered by one of skill in the art, for example, in protein synthesis experiments. For example, the term "about" refers to the normal variations encountered in measurements of a given analytical technique within a batch or sample, and between batches or samples. Thus, the term "about" can include a 1-10% variation in the measurement, e.g., a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% variation in the measurement. The amounts disclosed herein include equivalents of those amounts, including amounts modified or unmodified by the term "about."

[0079] The term "additive," also called a "stabilizer" or "excipient," refers to a compound or composition that is added to or combined with the liquid bacterial extract prior to spray drying.

[0080] The term "bacterial extract" refers to a bacterial cell lysate or a fraction thereof in which the cell extract is capable of synthesizing proteins from a nucleic acid template. In other words, a bacterial extract contains an energy source, such as ATP, GTP, etc. A bacterial extract can be a portion of a lysate from which other cellular components of the lysate have been separated by centrifugation, filtration, selective precipitation, selective immunoprecipitation, chromatography, or other methods. A bacterial extract also includes a lysate or a fraction thereof containing exogenous materials, such as preservatives, stabilizers, and reagents that enhance cell-free protein synthesis (CFPS). The term "bacterial extract" can refer to a preparation of an in vitro reaction mixture in which DNA can be transcribed into mRNA and / or mRNA can be translated into a polypeptide. The mixture can include ribosomes, an energy source, such as ATP, GTP, glucose, glutamate, or pyruvate, amino acids, and tRNA. The mixture can be derived directly from lysed bacteria, purified components, or a combination of both.

[0081] The term "extract capable of synthesizing a target protein from a template nucleic acid encoding the target protein in a cell-free protein synthesis reaction" refers to a lysed bacterial extract containing essential bacterial components necessary to synthesize a protein of interest in a cell-free protein synthesis reaction.

[0082] "Cell-free protein synthesis" or "CFPS" refers to the in vitro synthesis of nucleic acids, polypeptides, small molecules, and / or virus particles in a reaction mixture containing biological extracts and / or defined reagents. The reaction mixture includes macromolecules, such as DNA, mRNA, etc.; monomers for the macromolecules to be synthesized, such as amino acids, nucleotides, etc.; and cofactors, enzymes, and other reagents required for synthesis, such as templates for the production of ribosomes, uncharged tRNAs, charged tRNAs for natural and / or unnatural amino acids, polymerases, transcription factors, tRNA synthetases, etc.

[0083] The term "spray-dried bacterial extract" refers to a bacterial extract that has been spray-dried as described herein. "Spray-dried bacterial extract" differs from "freeze-dried bacterial extract," which refers to a bacterial extract that has been subjected to freeze-drying, lyophilization, in-situ evaporation, microwave radiation sublimation, or the like.

[0084] The term "stable spray-dried bacterial extract" refers to a spray-dried bacterial extract that essentially retains its physical and chemical stability and integrity when stored, for example, at -20°C, 2°C to 8°C, or room / ambient temperature (e.g., about 20°C) for six months or more. For example, a stable spray-dried bacterial extract refers to an extract that retains at least 75% of its initial ability to synthesize a protein of interest when stored at -20°C, 2°C to 8°C, or room / ambient temperature (e.g., about 20°C) for six months or more.

[0085] The term "control bacterial extract" refers to a bacterial extract that does not contain a formulation additive, such as those described herein, or an extract that contains an additive described herein but is tested at time T=0. Thus, the control bacterial extract can be an unformulated bacterial extract. The control extract can be an unformulated spray-dried bacterial extract. The control bacterial extract can be an unformulated frozen bacterial extract. The control extract can be spray-dried and / or stored at various temperatures, such as -80°C, -20°C, 4°C, 20°C, and 37°C. Alternatively, the control bacterial extract is not spray-dried. In some cases, the control bacterial extract is a fresh bacterial extract. In some cases, the control bacterial extract is a liquid bacterial extract. The control extract can be a spray-dried extract with or without an additive described herein that is reconstituted prior to testing. The control extract can be a formulated spray-dried extract with an additive described herein that is tested at T=0.

[0086] The term "lysed bacterial components" refers to the cellular components of lysed bacteria. For example, the term can include bacterial components necessary for synthesizing a protein of interest from a template nucleic acid encoding the protein of interest in a cell-free reaction, such as ribosomes, amino acids, polymerases, and tRNAs, as well as components of an active oxidative phosphorylation system. Additional components, such as exogenous ATP, GTP, glucose, glutamate, or pyruvate, can be added to the lysed bacterial components to provide an energy source.

[0087] The term "carbohydrate" refers to a carbohydrate consisting of carbon, hydrogen, and oxygen atoms and having the empirical formula C m (H2O) n where m and n can be different numbers. Carbohydrates include monosaccharides, disaccharides, oligosaccharides and polysaccharides.

[0088] The terms "rehydrate" or "reconstitute" in relation to spray-dried bacterial extract refer to suspending the spray-dried bacterial extract in a diluent such as water or a buffer to disperse the components of the bacterial extract.

[0089] The term "moisture content" refers to the amount of water contained in a material and can be expressed as a relative amount in weight percent or volume percent.

[0090] The term "residual water" or "residual moisture" refers to the amount of water contained in a material after it has been processed, e.g., spray dried, and includes residual water / moisture in the range of about 1% to 15%.

[0091] The term "protein synthesis activity" refers to the protein yield (eg, amount of protein) from a protein synthesis reaction to produce a target protein compared to a control protein synthesis reaction.

[0092] The term "lysate" refers to any cell-derived preparation containing components necessary for the protein synthesis machinery, such that the majority of the biological components are not reconstituted, but are capable of expressing nucleic acids encoding desired proteins present at concentrations resulting from lysis of the cells. Lysates can be further modified so that the lysate is supplemented with additional cellular components, e.g., amino acids, nucleic acids, enzymes, etc. Lysates can also be modified so that additional cellular components are removed or degraded after lysis.

[0093] The terms "polypeptide," "peptide," or "protein" are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to natural and unnatural amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds. The terms also encompass polymers containing L-amino acids, D-amino acids, or both L- and D-amino acids.

[0094] A "non-natural" or "non-native" amino acid refers to an amino acid that is not one of the 20 natural amino acids that are the building blocks of all proteins, but which can nevertheless be biologically engineered to be incorporated into a protein. An unnatural amino acid can include a D-peptide enantiomer of one of the 20 natural amino acids or any post-translational modification. A wide variety of unnatural amino acids can be used in the methods of the disclosure. Unnatural amino acids can be selected based on desired characteristics of the unnatural amino acid, such as the function of the unnatural amino acid, e.g., the biological properties of the protein, e.g., toxicity, biodistribution or half-life, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic properties, ability to react with other molecules (either covalently or non-covalently), or modifications thereof.Non-naturally occurring amino acids that can be used in the disclosed methods include, but are not limited to, non-naturally occurring analogs of tyrosine amino acids; non-naturally occurring analogs of glutamine amino acids; non-naturally occurring analogs of phenylalanine amino acids; non-naturally occurring analogs of serine amino acids; non-naturally occurring analogs of threonine amino acids; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, or any combination thereof; amino acids with photoactivatable crosslinkers; spin-labeled amino acids; fluorescent amino acids; amino acids with novel functional groups; amino acids that can be covalently or non-covalently bound to other molecules. amino acids that interact with; metal-binding amino acids; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; biotin or biotin analog containing amino acids; glycosylated or carbohydrate-modified amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyethers; heavy-atom substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar-substituted amino acids, such as sugar-substituted serine; carbon-linked sugar-containing amino acids, such as sugar-substituted serine; carbon-linked sugar-containing amino acids; redox-active amino acids; alpha-hydroxy-containing acids; aminothioacid containing amino acids; alpha,alpha-disubstituted amino acids; beta-amino acids; cyclic amino acids other than praline, and the like.

[0095] In the context of bacterial extracts, the term "active oxidative phosphorylation system" refers to a bacterial extract that exhibits active oxidative phosphorylation during protein synthesis. For example, the bacterial extract can generate ATP using the enzyme ATP synthase and the reduction of oxygen. It is understood that other translation systems known in the art can also use active oxidative phosphorylation during protein synthesis. Activation of oxidative phosphorylation can be demonstrated by inhibiting the pathway using specific inhibitors, such as electron transport chain inhibitors.

[0096] As used herein, "increase" or "decrease" refers to a detectable positive or negative change in quantity from a comparative control, e.g., an established standard control (such as an extract containing no additives or stabilizers). An increase is a positive change that is typically at least 10%, or at least 20%, or 50%, or 100%, and can be at least 2-fold, or at least 5-fold, or even 10-fold greater than the control value. For example, when used with respect to the bacterial extracts described herein, the term "increased stability" refers to an extract that has greater or higher stability (i.e., greater or higher protein synthesis activity) when stored at a given temperature for a given period of time compared to the control extract. Similarly, a decrease is a negative change that is typically at least 10%, or at least 20%, at least 30%, or at least 50%, or even at least 80% or at least 90% greater than the control value. For example, when used with respect to the bacterial extracts described herein, the term "reduced stability" refers to an extract that has reduced stability (i.e., reduced protein synthesis activity) when stored at a given temperature for a given period of time, and is typically associated with extracts that do not contain the additives or stabilizers of the present disclosure. Other terms indicating quantitative changes or differences from a comparative standard, e.g., "more," "less," "higher," and "lower," as well as terms indicating the action that causes such changes or differences, e.g., "increase," "promote," "enhance," "reduce," "inhibit," and "suppress," are used in this application in the same manner as above. In contrast, the terms "substantially the same" or "substantially no change" indicate little or no change in quantity from a standard control value, typically within ±10% of the standard control, or within ±5%, 2%, or even less variation from the standard control.

[0097] The term gram per liter (g / L) refers to a unit of measure for mass concentration that indicates how many grams of a substance are present in one liter of a liquid mixture.

[0098] The term gram / kilogram refers to a unit of mass fraction expressed as grams of a substance per kilogram of a mixture.

[0099] II. Detailed Description of the Embodiments Standard methods in molecular biology are described in Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA. Standard methods are also found in Bindereif, Schon, & Westhof (2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany, which describes detailed methods for the manipulation and analysis of RNA, and in Walker, JM, (2009) The Protein Protocols Handbook, 3rd ed., Humana Press, New York, NY, which describes detailed methods for the manipulation and analysis of proteins.

[0100] A. Bacterial Culture Bacterial culture is well known to those skilled in the art. Bacterial lysate from any bacterial strain can be used in the methods of the present disclosure. Bacteria suitable for use in cell-free synthesis systems include Gram-negative and Gram-positive bacteria, such as Enterobacteriaceae, e.g., Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, Examples of bacteria that may be used in the formulations and methods provided herein include Escherichia species, such as Escherichia coli or its derivatives, as well as Bacilli, such as B. subtilis and B. licheniformis, and Pseudomonas, such as P. aeruginsa and Steptomyces. In preferred embodiments, the bacteria used in the formulations and methods provided herein are from the genus Escherichia, such as Escherichia coli or its derivatives.

[0101] The bacterial strain used to prepare the cell extract may have reduced nuclease and / or phosphatase activity, thereby increasing the efficiency of cell-free synthesis. For example, the bacterial strain used to prepare the cell-free extract may have mutations in the genes encoding the nucleases RNase E and RNase A. The strain may also have mutations that stabilize components of the cell synthesis reaction, such as deletions of genes such as tnaA, speA, sdaA, or gshA, which prevent the degradation of the amino acids tryptophan, arginine, serine, and cysteine, respectively, in the cell-free synthesis reaction. Furthermore, the strain may have mutations that stabilize the protein products of cell-free synthesis, such as knockouts in the proteases ompT or lonP.

[0102] Bacterial cultures can be obtained as follows: A bacterium of choice is grown overnight in any of several growth media under growth conditions that are well known in the art and easily optimized by the practitioner for the growth of a particular bacterium. Generally, bacterial isolates are grown in media until they reach a stable exponential or stationary phase. This can be achieved by growing the bacteria .... 6 ~10 9 In some embodiments, the culture is harvested when the pH of the culture rises above a set point, indicating depletion of glucose in the medium. Bacterial cultures are harvested at an OD of 10-60, depending on the bacterial strain used. 595 - 600 In some embodiments, the bacteria are cultured at a growth rate of about 0.06 to about 0.6 to about 0.8 doublings / hour.

[0103] Bacterial cells can be grown in a medium containing glucose and phosphate, where glucose is present at a concentration of at least about 0.25% (weight / volume), more usually at least about 1%, usually not more than about 4%, and more usually not more than about 2%. An example of such a medium is 2YTPG medium, but those skilled in the art will understand that many culture media can be adapted for this purpose, as there are many published media suitable for growing bacteria such as E. coli using defined and undefined nutrient sources. For specific species, optimal media and growth conditions are known. For example, E. coli is commonly grown in YT broth (yeast extract and tryptone) or its variants. Media can be defined (synthetic) or complex (undefined).

[0104] Bacterial cells may be transfected or transformed with expression or cloning vectors as described herein and cultured in conventional nutrient media modified appropriately for inducing promoters, selecting for transformants, and preparing bacterial extracts.

[0105] In some cases, bacteria are grown in aerobic conditions to induce protein expression, and then the culture is switched to anaerobic conditions, for example, by bubbling nitrogen, argon, etc. through the culture medium.

[0106] When large quantities of bacteria are required, continuous culture methods are used instead of closed batch systems. These continuous systems involve the continuous introduction of nutrients and the removal of waste products. Optimally, this allows cells to grow at a constant biomass concentration over an extended period of time. Two well-known systems are the chemostat and the turbidostat. In a chemostat system, sterile medium is supplied at a constant rate while the medium containing the bacteria is removed at the same rate. A turbidostat system uses a photocell to measure absorbance or turbidity and regulates the inflow of sterile medium and the outflow of bacteria according to a preset signal.

[0107] Methods for culturing bacteria are described, for example, in Zawada et al., Biotechnol. Bioeng., 108(7):1570-1578 (2011); Zawada, J. "Preparation and Testing of E. coli S30 In Vitro Transcription Translation Extracts", Douthwaite, J.A. and Jackson, R.H. (eds.), Ribosome Display and Related Technologies: Methods and Protocols, Methods in Molecular Biology, vol. 805, pp. 31-41 (Humana Press, 2012); Jewett et al., Molecular Systems Biology: 4, 1-10 (2008); Shin J. and Norieaux V., J.Biol.Eng., 4:8 (2010).

[0108] In some cases, engineered E. coli strains (e.g., engineered K-12-derived E. coli strain KGK10) have been shown to produce microbial growth in approximately 0.7 h. -1Using a fed-batch fermentation of glucose and amino acids at a maximum growth rate of 0.1% to 0.2% (OD of approximately 45 OD), the medium was grown to mid-log phase (OD of approximately 45 OD). 595 The culture is grown to a glucose concentration of about 1000 kJ / L (or about 140 g / L wet cell weight). Glucose can be increased during the culture so that excess glucose is present during harvest. See, e.g., Zawada et al., Biotechnol. Bioeng., 108(7):1570-1578 (2011).

[0109] B. Preparation of Bacterial Extracts When the bacterial culture is ready to be harvested, it can be cooled to 2-8°C, typically on ice or via a heat exchanger, if the culture is large-scale. The culture can be centrifuged to separate the spent medium from the cell paste (cell slurry). Preferred centrifuges include disc stack centrifuges, tubular bowl centrifuges, and other centrifuges for large-scale or small-scale bacterial culture. The cell paste is typically resuspended in S30 buffer, any equivalent buffer, or water. S30 buffer contains 10 mM Tris acetate, 14 mM magnesium acetate, and 60 mM potassium acetate. In some embodiments, a 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or greater dilution (liquid:solid; ml of buffer:gram weight of cells) is performed for washing. The cell paste can be washed again in S30 buffer or any equivalent buffer and centrifuged to remove any residual buffer. For small-scale cultures, a second wash step is typically performed. Upon washing, the cell paste (cell pellet) can be stored at -80°C for later use or can be further processed by homogenization to lyse the cells.

[0110] Cell extracts can be prepared from cultured bacteria as described above. Cells fermented overnight can be lysed by suspending the cell pellet in a suitable cell suspension buffer and disrupting the suspended cells by sonication, disrupting the suspended cells in a French press or with glass beads, continuous-flow high-pressure homogenization, or any other method known in the art useful for efficient cell lysis. The cell lysate is then centrifuged or filtered to remove large cellular debris, including DNA, and unlysed cells.

[0111] In some embodiments, the bacterial culture is pelleted by centrifugation at greater than 14,000 x g for about 45 minutes twice at about 8-20°C in a tubular bowl centrifuge in continuous or batch mode, or in a disc-stack continuous centrifuge with a maximum bowl speed of about 12,000 rpm and a feed flow rate of about 3.0-3.3 L / min. The pelleted cells are resuspended and repelleted using S30 buffer. In some embodiments, the cells are stored at -80°C for later use or processed by homogenization.

[0112] Prior to homogenization, the cell pellet may be resuspended in S30 buffer or equivalent to generate a cell suspension. In some embodiments, a 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or greater dilution (liquid:solid; ml of buffer:gram weight of cells) is performed. Preferably, a 2:1 dilution is performed, such that 2 ml of S30 buffer is used per gram weight of cell pellet.

[0113] The cell suspension can be homogenized or disrupted in a standard high-pressure homogenizer (e.g., Avestin Emulsiflex C-55a Homogenizer) and / or a microfluidizer (e.g., Microfluidics Microfluidizer) set at an appropriate pressure, such as 3,000 psi, to generate a lysate. The homogenization process lyses the bacteria, releasing essential components required for protein synthesis; in some embodiments, the formed everted membrane vesicles provide energy for protein synthesis via respiration.

[0114] In some embodiments, the homogenizer pressure is about 3,000 to 20,000 psi. In some embodiments, the homogenizer pressure is set at about 20,000 psi. In some embodiments, the homogenizer speed (frequency setting) is about 20 Hz to about 60 Hz to provide a flow rate of about 340 mL / min to 1.0 L / min. Generally, the flow rate is proportional to the frequency setting and can be varied independently of the homogenization pressure. Preferably, the minimum speed setting for the homogenization step is about 20 Hz at a flow rate of about 340 mL / min.

[0115] Bacterial lysates are also commercially available from manufacturers such as Promega Corp., Madison, WI; Agilent Technologies, Santa Clara, CA; GE Healthcare Biosciences, Pittsburgh, PA; Life Technologies, Carlsbad, CA; and Roche Diagnostics, Basel, Switzerland.

[0116] The lysate can then be clarified by centrifugation such that at least about 45% to about 85% or more, e.g., about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, of the cell solids are separated from the collected cell-free extract. In some embodiments, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cell solids are separated by centrifugation. In some embodiments, the centrifugation is by a continuous centrifuge, e.g., a disc stack centrifuge, a tubular bowl centrifuge, or a suitable centrifuge. In some embodiments, a 200 L fermentation produces greater than 1.1 L of clarified extract per kg wet cell weight at a total protein concentration of about 20-25 g / L.

[0117] The extract may be filtered through one or more sterilizing grade filtration membranes, for example, a 0.45 μm filtration membrane and / or a 0.22 μm filtration membrane, where a 0.45 μm filtration membrane can be used first, followed by a 0.22 μm filtration membrane.

[0118] In some embodiments, the filtered extract is activated or pre-incubated for about 2-5 hours, preferably about 2.5 hours, at 30° C. After pre-incubation, the particulates from the extract can be centrifuged, for example, by spinning at at least 14,000×g for about 35 minutes.

[0119] The lysed bacterial extract can be aliquoted, frozen in liquid nitrogen, and then stored at −80° C. Optionally, a cell-free synthesis reaction mixture as described herein can be added to the cell-free extract before freezing.

[0120] Methods for preparing lysed bacterial extracts are described, for example, in Zawada, J. "Preparation and Testing of E. coli S30 In Vitro Transcription Translation Extracts", Douthwaite, J. A. and Jackson, R. H. (eds.), Ribosome Display and Related Technologies: Methods and Protocols, Methods in Molecular Biology, vol. 805, pp. 31-41 (Humana Press, 2012); Jewett et al., Molecular Systems Biology, 4, 1-10 (2008); Shin J. and Norieaux V., J. Biol. Eng., 4:8 (2010).

[0121] C. Activation of bacterial extracts The lysed bacterial extract prepared as described above can be reconstituted in a buffer or other liquid to form a liquid bacterial extract. The liquid bacterial extract can be "activated" by heating the bacterial extract. In some embodiments, the liquid bacterial extract is heated to about 20°C to 45°C for about 30 minutes to about 10 hours. In some embodiments, the liquid bacterial extract is heated to about 40°C for about 40 minutes. Activation improves protein expression in cell-free protein synthesis reactions. In some embodiments, the liquid bacterial extract is sterile filtered prior to activation (heat treatment). Activation of bacterial extracts is described in Groff, D., et al. (Development of an E. coli strain for cell-free ADC manufacturing. Biotechnology and Bioengineering, 119, 162-175. doi.org / 10.1002 / bit.27961).

[0122] After activation, the liquid bacterial extract is formulated by adding one or more additives as described herein below.

[0123] D. Formulation of Bacterial Extracts Before Spray Drying The present disclosure is based, in part, on the unexpected finding that certain additives, excipients, or stabilizers, when added to bacterial extracts prior to spray drying, maintain the protein synthesis activity of the extracts in cell-free protein synthesis reactions. Without being bound by theory, the additives may prevent protein denaturation during spray drying and / or long-term storage. In some embodiments, certain formulations of stable spray-dried bacterial extracts described herein can be stored at −20° C., 2° C. to 8° C., or room temperature for at least 8 months and retain at least about 60% of the protein synthesis activity of control extracts with or without the additives, excipients, or stabilizers. Experiments described in the Examples (see below) demonstrate that formulations containing the additives described herein have long-term storage stability.

[0124] In some embodiments, the formulation comprises a single additive that is added to the bacterial extract prior to spray drying. Formulations that comprise a single additive are referred to as "single-component" spray-dried extracts.

[0125] In some embodiments, the formulation comprises a carbohydrate additive, hi some embodiments, the carbohydrate additive is selected from trehalose, lactose, raffinose, maltodextrin, or cyclodextrin, or combinations thereof.

[0126] In some embodiments, the additive comprises trehalose (e.g., trehalose dihydrate (TDH); also known as D-(+)-trehalose dihydrate). In some embodiments, the formulation comprises about 25 g / L to about 200 g / L of trehalose, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / L of trehalose. In some embodiments, the formulation comprises about 50 g / L to about 100 g / L of trehalose. In some embodiments, the formulation comprises about 25 g / L to about 75 g / L of trehalose. In some embodiments, the formulation comprises about 75 g / L to about 125 g / L of trehalose. In some embodiments, the formulation comprises about 50 g / L of trehalose. In some embodiments, the formulation comprises about 75 g / L of trehalose. In some embodiments, the formulation comprises about 100 g / L of trehalose. In some embodiments, the formulation comprises about 125 g / L of trehalose. In some embodiments, the formulation contains about 25 g / kg to about 200 g / kg of trehalose, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / kg of trehalose. In some embodiments, the formulation contains about 50 g / kg to about 110 g / kg of trehalose. In some embodiments, the formulation contains about 25 g / kg to about 75 g / kg of trehalose. In some embodiments, the formulation contains about 75 g / kg to about 125 g / kg of trehalose. In some embodiments, the formulation comprises about 50 g / kg trehalose, in some embodiments, about 75 g / kg trehalose, in some embodiments, about 100 g / kg trehalose, in some embodiments, about 125 g / kg trehalose.

[0127] In some embodiments, the additive comprises lactose (e.g., lactose monohydrate (LMH)). In some embodiments, the formulation comprises about 25 to about 200 g / L of lactose, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / L of lactose. In some embodiments, the formulation comprises about 50 g / L to about 100 g / L of lactose. In some embodiments, the formulation comprises about 100 g / L of lactose. In some embodiments, the formulation contains about 25 to about 200 g / kg lactose, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / kg lactose. In some embodiments, the formulation contains about 50 g / L to about 100 g / kg lactose. In some embodiments, the formulation contains about 100 g / kg lactose.

[0128] In some embodiments, the additive comprises raffinose. In some embodiments, the formulation comprises about 25-200 g / L of raffinose, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / L of raffinose. In some embodiments, the formulation comprises about 25-200 g / kg raffinose, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / kg raffinose.

[0129] In some embodiments, the additive comprises maltodextrin. In some embodiments, the formulation comprises about 100 g / L of maltodextrin.

[0130] In some embodiments, the additive comprises a cyclodextrin. Cyclodextrins help improve the aqueous solubility and stability of hydrophobic compounds. In some embodiments, the cyclodextrin is alpha-, beta-, or gamma-cyclodextrin, or a combination thereof. In some embodiments, the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin (HP-β-CD; a cyclic oligosaccharide containing seven D-(+)-glucopyranose units). In some embodiments, the formulation comprises about 5 g / kg to about 50 g / kg of cyclodextrin (e.g., about 5 g / kg to about 50 g / kg of HP-β-CD).

[0131] In some embodiments, the formulation includes an additive selected from a sugar alcohol. In some embodiments, the sugar alcohol is selected from mannitol or sorbitol, or a combination thereof. In some embodiments, the formulation includes about 50 to about 100 g / L of mannitol. In some embodiments, the formulation includes about 15 g / L of sorbitol.

[0132] In some embodiments, the formulation includes an additive selected from an amino acid. In some embodiments, the amino acid is leucine. In some embodiments, the formulation includes about 5 g / L to about 15 g / L of leucine, e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 g / L of leucine.

[0133] In some embodiments, the formulation comprises an additive selected from a polymer. In some embodiments, the polymer is selected from polyethylene glycol (PEG) (e.g., PEG 200, or a PEG having a molecular mass less than 20,000 g / mol), polysorbate (e.g., polysorbate 20, 40, 60, or 80 (also known as Tween® 80) or polyvinylpyrrolidone (PVP or Kollidon 12 PF), or a combination thereof.

[0134] The additives may be mixed with the liquid lysed bacterial extract by combining concentrated stock solutions of one or more of the additives to achieve the desired formulation of the spray-dried extract. For example, a liquid extract containing an additive can have a total dry weight of about 5 g / L to 150 g / L of additive per volume of lysed bacterial extract, e.g., about 5 g / L, about 10 g / L, about 15 g / L, about 20 g / L, about 35 g / L, about 40 g / L, about 45 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, about 100 g / L, about 105 g / L, about 110 g / L, about 115 g / L, about 120 g / L, about 125 g / L, about 130 g / L, about 135 g / L, about 140 g / L, about 145 g / L, or about 150 g / L.

[0135] In some embodiments, the formulated bacterial extract does not comprise a composition, additive, or stabilizer selected from sucrose, mannitol, sorbitol, dextran, or combinations thereof, hi some embodiments, the formulated bacterial extract does not comprise sucrose.

[0136] E. Combination with other additives The bacterial extract formulations described herein can include other compounds, such as additives, excipients, stabilizers, chemicals, molecules, or reagents, that are added to the dissolved bacterial extract prior to spray drying. Thus, the bacterial extract can include a single additive described herein or a single additive combined with one or more other different additives. Examples of other additives include carbohydrates, sugar alcohols, polymers, and / or amino acids, or combinations thereof. Representative other additives include raffinose, maltodextrin, sucrose, cyclodextrins (e.g., 2-hydroxypropyl-β-cyclodextrin), mannitol, sorbitol, PEG (e.g., polyethylene glycol 200), polysorbates (e.g., polysorbate 80 (Tween® 80)), polyvinylpyrrolidone (PVP or Kollidon 12 PF), leucine, amino acid mixtures, and / or combinations thereof. Table 1 provides representative examples of single component and combination formulations that were tested for improved long-term stability, as described in the Examples. [Table 1]

[0137] The difference between g / L and g / kg is density, with a TDH of 100 g / L corresponding to approximately 104 g / kg, and a TDH of 75 g / kg corresponding to approximately 73 g / L.

[0138] In some embodiments, the bacterial extract formulation comprises one or more, any combination, or any subset of the naturally occurring amino acids leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine (referred to herein as a "PMA amino acid mix"). A representative PMA amino acid mix is ​​shown in Table 2. [Table 2]

[0139] In some embodiments, the amino acids tyrosine and / or phenylalanine are added to the formulation separately from the PMA mixed amino acids.

[0140] F. High glass transition component In some embodiments, the bacterial extract formulation includes one or more components with a high glass transition temperature (Tg). In some embodiments, the bacterial extract formulation includes one or more components with a Tg of about 70°C or higher. For example, the bacterial extract formulation includes one or more components with a Tg of about 80°C, about 90°C, or about 100°C or higher. In some embodiments, the bacterial extract formulation includes trehalose. In some embodiments, the bacterial extract formulation includes lactose. In some embodiments, the bacterial extract formulation includes raffinose. The Tg of various components tested in formulations of the present disclosure are shown in the table below. [Table 3] [Table 4]

[0141] In some embodiments, the bacterial extract formulations include one or more, or combinations or sub-combinations of high Tg non-polar, uncharged amino acids. Table 3 shows representative combinations of high Tg non-polar, uncharged amino acids (referred to herein as "selected amino acid mixtures") used in some formulations. [Table 5]

[0142] In some embodiments, the bacterial extract comprises trehalose and one or more additives selected from a carbohydrate, a sugar alcohol, an amino acid or a mixture of amino acids, a polymer, or a combination thereof. In some embodiments, the carbohydrate additive comprises a cyclodextrin. In some embodiments, the concentration of cyclodextrin present in the bacterial extract is about 5 g / kg to about 50 g / kg (e.g., about 5 g / kg to about 50 g / kg HP-β-CD).

[0143] In some embodiments, the sugar alcohol is selected from mannitol or sorbitol, or a combination thereof. In some embodiments, the bacterial extract comprises about 50 to about 100 g / L of mannitol. In some embodiments, the bacterial extract comprises about 15 g / L of sorbitol.

[0144] In some embodiments, the bacterial extract comprises trehalose and an amino acid selected from (i) leucine, (ii) one or more, any combination, or any subset of the PMA amino acid mixtures in Table 2, or (iii) one or more, any combination, or any subset of the selected amino acid mixtures in Table 3. In some embodiments, the bacterial extract comprises trehalose and about 5-10 g / L of leucine.

[0145] In some embodiments, the bacterial extract comprises trehalose and an additive selected from a polymer. In some embodiments, the polymer is selected from polyethylene glycol (PEG) (e.g., PEG 200, or a PEG having a molecular mass of less than 20,000 g / mol), polysorbate (e.g., polysorbate 20, 40, 60, or 80 (also known as Tween® 80) or polyvinylpyrrolidone (PVP or Kollidon 12 PF), or a combination thereof.

[0146] In any of the embodiments described herein, the trehalose can be trehalose dihydrate (TDH). In some embodiments, the concentration of trehalose or TDH present in the bacterial extract is about 25 to 200 g / L, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / L. In some embodiments, the bacterial extract comprises about 50 g / L to about 100 g / L of trehalose or TDH. In some embodiments, the bacterial extract comprises about 100 g / L of trehalose or TDH. In some embodiments, the bacterial extract comprises about 25 g / kg to about 200 g / kg of trehalose, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / kg of trehalose. In some embodiments, the bacterial extract comprises about 50 g / kg to about 110 g / kg of trehalose or TDH. In some embodiments, the bacterial extract comprises about 75 g / kg of trehalose or TDH.

[0147] In some embodiments, the bacterial extract comprises lactose and one or more additives selected from a carbohydrate, a sugar alcohol, an amino acid or a mixture of amino acids, a polymer, or a combination thereof. In some embodiments, the carbohydrate additive is selected from trehalose, raffinose, maltodextrin, sucrose, or cyclodextrin, or a combination thereof. In some embodiments, the trehalose present in the bacterial extract is present at a concentration of about 25 g / kg to about 200 g / kg, the raffinose present in the bacterial extract is present at a concentration of about 25-200 g / L, the maltodextrin present in the bacterial extract is present at a concentration of about 100 g / L, and the cyclodextrin present in the bacterial extract is present at a concentration of about 5 g / kg to about 50 g / kg (e.g., about 5 g / kg to about 50 g / kg HP-β-CD).

[0148] In some embodiments, the bacterial extract comprises lactose and a sugar alcohol selected from mannitol or sorbitol, or a combination thereof. In some embodiments, the bacterial extract comprises about 50 to about 100 g / L of mannitol. In some embodiments, the bacterial extract comprises about 15 g / L of sorbitol.

[0149] In some embodiments, the bacterial extract comprises lactose and an amino acid selected from (i) leucine, (ii) one or more, any combination, or any subset of the PMA amino acid mixtures in Table 2, or (iii) one or more, any combination, or any subset of the amino acid select mixtures in Table 3. In some embodiments, the bacterial extract comprises lactose and about 5-10 g / L of leucine.

[0150] In some embodiments, the bacterial extract comprises lactose and an additive selected from a polymer. In some embodiments, the polymer is selected from polyethylene glycol (PEG) (e.g., PEG 200, or a PEG having a molecular mass of less than 20,000 g / mol), polysorbate (e.g., polysorbate 20, 40, 60, or 80 (also known as Tween® 80) or polyvinylpyrrolidone (PVP or Kollidon 12 PF), or a combination thereof.

[0151] In any of the embodiments described herein, the lactose can be lactose monohydrate (LMH). In some embodiments, the bacterial extract comprises about 25 to about 200 g / L of lactose or LMH, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / L of lactose or LMH. In some embodiments, the bacterial extract comprises about 100 g / L of lactose or LMH. In some embodiments, the bacterial extract comprises about 25 to about 200 g / kg lactose, e.g., about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 g / kg lactose or LMH. In some embodiments, the bacterial extract comprises about 100 g / kg lactose or LMH.

[0152] In some embodiments, the bacterial extract comprises leucine and one or more additives selected from a carbohydrate, a sugar alcohol, an amino acid or a mixture of amino acids, a polymer, or a combination thereof. In some embodiments, the carbohydrate additive comprises trehalose. In some embodiments, the concentration of trehalose present in the bacterial extract is from about 25 g / kg to about 200 g / kg.

[0153] In some embodiments, the bacterial extract comprises leucine and a sugar alcohol selected from mannitol, sorbitol, or a combination thereof. In some embodiments, the bacterial extract comprises about 50 to about 100 g / L of mannitol. In some embodiments, the bacterial extract comprises about 15 g / L of sorbitol.

[0154] In some embodiments, the bacterial extract comprises leucine and an amino acid selected from (i) one or more, any combination, or any subset of the PMA amino acid mixtures of Table 2, or (ii) one or more, any combination, or any subset of the selected amino acid mixtures of Table 3.

[0155] In some embodiments, the bacterial extract comprises leucine and an additive selected from a polymer. In some embodiments, the polymer is selected from polyethylene glycol (PEG) (e.g., PEG 200, or a PEG having a molecular mass of less than 20,000 g / mol), polysorbate (e.g., polysorbate 20, 40, 60, or 80 (also known as Tween® 80) or polyvinylpyrrolidone (PVP or Kollidon 12 PF), or a combination thereof.

[0156] In some embodiments, the bacterial extract comprises raffinose and one or more additives selected from a sugar alcohol, an amino acid or a mixture of amino acids, a polymer, or a combination thereof.

[0157] In some embodiments, the bacterial extract comprises raffinose and a sugar alcohol selected from mannitol or sorbitol, or a combination thereof. In some embodiments, the bacterial extract comprises about 50 to about 100 g / L of mannitol. In some embodiments, the bacterial extract comprises about 15 g / L of sorbitol.

[0158] In some embodiments, the bacterial extract comprises raffinose and an amino acid selected from (i) one or more, any combination, or any subset of the PMA amino acid mixtures in Table 2, or (ii) one or more, any combination, or any subset of the selected amino acid mixtures in Table 3. In some embodiments, the bacterial extract comprises raffinose and about 5-10 g / L of leucine.

[0159] In some embodiments, the bacterial extract comprises raffinose and an additive selected from a polymer. In some embodiments, the polymer is selected from polyethylene glycol (PEG) (e.g., PEG 200, or a PEG having a molecular mass less than 20,000 g / mol), polysorbate (e.g., polysorbate 20, 40, 60, or 80 (also known as Tween® 80) or polyvinylpyrrolidone (PVP or Kollidon 12 PF), or a combination thereof.

[0160] In some embodiments, the formulated bacterial extract prior to spray drying does not comprise a composition, additive, or stabilizer selected from sucrose, mannitol, sorbitol, dextran, or combinations thereof, hi some embodiments, the formulated bacterial extract prior to spray drying does not comprise sucrose.

[0161] G. Methods for Producing Stable Spray-Dried Bacterial Extracts The bacterial extract containing the lysed bacterial components and one or more additive or stabilizer compositions described herein can then be spray-dried. In some embodiments, the bacterial extract is a liquid (rehydrated or reconstituted) bacterial extract containing the lysed bacterial components for cell-free synthesis of a target protein from a template nucleic acid encoding the target protein.

[0162] In some embodiments, the method includes atomizing a liquid bacterial extract to produce droplets, contacting the droplets with a gas to evaporate the liquid from the droplets, separating the dried extract from the gas and smaller particles, and collecting the spray-dried extract.

[0163] In some embodiments, the present disclosure provides a method of preparing a spray-dried bacterial extract, comprising providing a liquid bacterial extract containing components for cell-free synthesis of a target protein from a template nucleic acid encoding the target protein, generating droplets of the liquid bacterial extract, contacting the droplets with a gas to evaporate the liquid from the droplets, separating the dried extract from the gas and smaller particles, and collecting the spray-dried extract.

[0164] In some embodiments, a liquid bacterial extract containing dissolved bacterial components and one or more additive compositions is atomized by passing the liquid bacterial extract through an atomization device to generate droplets. In some embodiments, the atomization device is a tip, nozzle, or rotary atomizer. In some embodiments, the tip or nozzle has an opening suitable for atomizing the liquid bacterial extract, determined by commercially suitable means based on the type of drying device used and other factors. In some embodiments, the extract droplets are generated using a two-fluid nozzle system. The two-fluid nozzle system may include a first nozzle that provides the liquid bacterial extract and a second nozzle that provides pressurized gas that contacts the liquid bacterial extract as it exits the first nozzle, thereby generating droplets.

[0165] Those skilled in the art will appreciate that the droplet size produced by an atomization device depends on several factors, including the atomization gas pressure, the desired liquid feed rate, the nozzle or tip design, and the drying device used. The drying gas flow rate, as well as the inlet and outlet temperatures of the drying gas, can affect droplet size. These parameters can be adjusted based on the desired droplet size and residual moisture in the spray-dried extract. In some embodiments, the desired droplet size is about 20 to 100 microns (Dv50). In some embodiments, the atomization gas pressure is about 10 to 50 psig. In some embodiments, the drying gas outlet temperature is about 60°C to about 90°C. In some embodiments, the drying gas outlet temperature is about 65°C to about 80°C. For example, the drying gas outlet temperature can be about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80°C. In some embodiments, the outlet temperature of the drying gas is from about 65°C to about 76°C.

[0166] Those skilled in the art will understand that the temperature used to dry the extract can vary based on the size (volume) of the dryer used, since the activity of the extract is temperature-constrained. For example, a lower temperature may be required in a large, industrial-scale dryer compared to a smaller-volume dryer due to the longer residence time of the extract droplets in the large dryer. Those skilled in the art will understand that there are multiple ways to reduce heat exposure in a large dryer by shortening the residence time and / or special equipment design to cool the powder particles more quickly after they are dried. Exemplary laboratory-scale dryers include a Buchi B-290 spray dryer. Exemplary pilot-scale dryers include a Mobile Minor PSD-1 spray dryer. Exemplary industrial-scale dryers include modified PSD-2 and PSD-3 dryers and an IFN-Welko spray dryer.

[0167] In some embodiments, the gas is dehumidified air, hi some embodiments, the gas is nitrogen.

[0168] The spray-dried extract particles / droplets produced in contacting step (ii) above may then be separated from the gas and any smaller particles using centrifugal force. In some embodiments, the spray-dried extract particles / droplets are separated from the gas and smaller particles using a cyclone. In some embodiments, the smaller particles are about 1-10 microns and exit the cyclone in a separate stream from the desired spray-dried extract particles / droplets.

[0169] After separation step (iii) above, the spray-dried extract particles / droplets may then be collected. In some embodiments, the spray-dried extract particles / droplets are collected in a container.

[0170] As noted above, prior to spray drying, the liquid bacterial extract may be sterile filtered and heat activated. In some embodiments, the liquid bacterial extract is activated by heating the extract to about 20-45°C for about 30 minutes to about 10 hours.

[0171] In some embodiments, an additive or stabilizer composition comprising trehalose, lactose, leucine, or raffinose is added to the activated, sterile-filtered liquid bacterial extract prior to spray drying (e.g., prior to generating droplets of the liquid bacterial extract). In some embodiments, about 25-200 g / kg of trehalose, about 25-200 g / kg (or about 25-200 g / L) of lactose, about 5-10 g / L of leucine, and / or about 25-200 g / L of raffinose are added to the activated, sterile-filtered liquid bacterial extract prior to spray drying.

[0172] In some embodiments, one or more or a combination of amino acids are added to the activated, sterile-filtered liquid bacterial extract prior to spray drying. In some embodiments, the one or more amino acids comprise high glass transition temperature (Tg) non-polar, uncharged amino acids selected from the group consisting of L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, and L-proline, and combinations thereof. In some embodiments, the one or more or a combination of amino acids is selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and / or arginine.

[0173] In some embodiments, one or more of maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), and / or 2-hydroxypropyl-β-cyclodextrin, or combinations thereof, are added to the activated sterile-filtered liquid bacterial extract prior to spray drying.

[0174] In some embodiments, about 85% to 95% (w / w) or more (e.g., about 85% w / w or more, about 86% w / w or more, about 87% w / w or more, about 88% w / w or more, about 89% w / w or more, about 90% w / w or more, about 91% w / w or more, about 92% w / w or more, about 93% w / w or more, about 94% w / w or more, about 95% w / w or more, about 96% w / w or more, about 97% w / w or more, about 98% w / w or more, or about 99% w / w or more) of liquid is removed from the bacterial extract after spray drying. In some embodiments, the spray-dried extract contains about 15% (w / w) or less of residual water (e.g., about 15% w / w or less, about 14% w / w or less, about 13% w / w or less, about 12% w / w or less, about 11% w / w or less, about 10% w / w or less, about 9% w / w or less, about 8% w / w or less, about 7% w / w or less, about 6% w / w or less, about 5% w / w or less, about 4% w / w or less, about 3% w / w or less, about 2% w / w or less, or about 1% w / w or less).

[0175] H. Residual water content Residual moisture or the amount of residual water can affect the protein synthesis activity and / or stability of the spray-dried extract formulation. In some embodiments, the spray-dried extract contains less than about 15% by weight of residual moisture, e.g., about 15% (w / w) or less, about 14% (w / w) or less, about 13% (w / w) or less, about 12% (w / w) or less, about 11% (w / w) or less, about 10% (w / w) or less, about 9% (w / w) or less, about 8% (w / w) or less, about 7% (w / w) or less, about 6% (w / w) or less, about 5% (w / w) or less, about 4% (w / w) or less, about 3% (w / w) or less, about 2% (w / w) or less, or about 1% (w / w) or less.

[0176] Methods for determining the water content in dried extracts include proton nuclear magnetic resonance (NMR) spectroscopy and Karl Fisher coulometric titration.

[0177] NMR spectroscopy is based on the fact that hydrogen protons possess a magnetic moment and angular momentum. When hydrogen atoms are excited by an alternating magnetic field from a transmitter in the presence of the Earth's static magnetic field, they generate a magnetic field. A relaxation field is generated by the protons excited by the excitation field. The amplitude of the relaxation field, measured after the excitation is turned off, is directly related to the number of excited protons and, therefore, the water content. Time-domain nuclear magnetic resonance (TD-NMR) spectroscopy and "Spin Track" NMR spectroscopy are variations of this technique that have been applied to biological cultures and protein solutions.

[0178] For purposes of the disclosure provided herein, one method for determining the percentage of water in a dry extract is Karl Fischer coulometric titration.

[0179] Karl Fischer titration utilizes the quantitative reaction of water with iodine and sulfur dioxide based on the Bunsen reaction in the presence of a primary alcohol, such as methanol, ethanol, or ethylene glycol monoethyl ether, as the solvent and an organic base, such as pyridine, as the buffer. For pyridine-free systems, replacing the base with imidazole or a primary amine can be used. For protein or sugar solutions, a 2:1 methanol:formamide mixture can be used as the solvent. Two variations of this method, volumetric titration and coulometric titration, utilize different iodine sources. In volumetric titration, the iodine required for the reaction is predissolved, and the water content is determined by measuring the amount of iodine consumed as a result of the reaction with water in the sample. Automated volumetric titration systems are commercially available. In coulometric titration, iodine is first produced by electrolysis of a reagent containing iodide ions, and then the water content is determined by measuring the amount of electricity (coulombs) [= current (amperes) x time (seconds)] required for electrolysis to produce iodine, based on the quantitative reaction of the produced iodine with water.

[0180] Karl Fischer titration can be performed using a drying oven (e.g., Model D03080, Mettler Toledo, Columbus, OH) directly connected to a Karl Fischer coulometric titrator (e.g., Model C20, manufactured by Mettler Toledo). Typically, the oven setpoint is set to 100 °C. An aluminum insert is placed in the sample holder compartment of the oven, and the extract to be measured is loaded into the insert through a port at the top of the oven. A nitrogen flow set at 200 mL / min is run through the oven to facilitate the transfer of water vapor from the oven to the titration vessel. The mixing time, i.e., the time from introducing the sample into the oven to the start of the titration, is set to 120 seconds to allow complete transfer of water in each sample to the titration vessel. Iodine for the titration is electrochemically generated in incremental amounts based on the drift observed by the instrument. The starting drift criterion is less than approximately 25 μg / min. The drift criteria to be achieved to terminate the measurement is less than approximately 3.0 μg / min, and the maximum titration time is approximately 3600 seconds. A voltammetric sensor (e.g., model DM143-SC) with a polarization current of 5.0 μA is used for detection. To capture variability in measurements, each sample can be run in triplicate.

[0181] I. Spray-dried bacterial extract The spray-dried bacterial extract described herein can be used in cell-free protein synthesis reactions. In some embodiments, the spray-dried bacterial extract comprises dried, dissolved bacterial components. In some embodiments, the spray-dried extract comprises components for synthesizing a target protein from a template nucleic acid encoding the target protein. In some embodiments, the spray-dried bacterial extract has an active oxidative phosphorylation system for cell-free protein synthesis. Additional components present in the spray-dried extract that can be used for cell-free protein synthesis are described below.

[0182] In some embodiments, the spray-dried bacterial extract comprises one or more stabilizers, wherein the stabilizer has a glass transition temperature (Tg) of at least about 70°C. In some embodiments, the stabilizer is selected from trehalose, lactose, leucine, and / or raffinose. In some embodiments, the liquid bacterial extract comprises about 25-200 g / kg trehalose, about 25-200 g / kg (or about 25-200 g / L) lactose, about 5-10 g / L leucine, and / or about 25-200 g / L raffinose prior to spray drying. In some embodiments, the trehalose is trehalose dihydrate (TDH) and the lactose is lactose monohydrate (LMH).

[0183] In some embodiments, about 85% to 95% (w / w) or more (e.g., about 85% w / w or more, about 86% w / w or more, about 87% w / w or more, about 88% w / w or more, about 89% w / w or more, about 90% w / w or more, about 91% w / w or more, about 92% w / w or more, about 93% w / w or more, about 94% w / w or more, about 95% w / w or more, about 96% w / w or more, about 97% w / w or more, about 98% w / w or more, or about 99% w / w or more) of the liquid is removed from the spray-dried bacterial extract. In some embodiments, the spray-dried bacterial extract contains about 15% (w / w) or less of residual water (e.g., about 15% w / w or less, about 14% w / w or less, about 13% w / w or less, about 12% w / w or less, about 11% w / w or less, about 10% w / w or less, about 9% w / w or less, about 8% w / w or less, about 7% w / w or less, about 6% w / w or less, about 5% w / w or less, about 4% w / w or less, about 3% w / w or less, about 2% w / w or less, or about 1% w / w or less).

[0184] In some embodiments, the spray-dried bacterial extract further comprises one or more or a combination of high glass transition temperature (Tg) non-polar, uncharged amino acids selected from L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, and / or L-proline, and combinations thereof. In some embodiments, the spray-dried bacterial extract further comprises one or more, any combination, or any subset of amino acids selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine. In some embodiments, the spray-dried bacterial extract comprises about 5-15 g / L (or about 5-15 g / kg) of amino acids.

[0185] In some embodiments, the spray-dried bacterial extract further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), or 2-hydroxypropyl-β-cyclodextrin.

[0186] J. Stability of Spray-Dried Bacterial Extracts The spray-dried extracts of the present disclosure have increased stability when stored for extended periods of time (e.g., 6-18 months or longer) compared to extracts that do not contain the additives or stabilizers described herein. To determine stability, the spray-dried extract can be reconstituted by adding a liquid to the spray-dried extract powder, and the reconstituted extract can be used in a cell-free protein synthesis reaction. In some embodiments, the spray-dried extract is reconstituted (also referred to as rehydration) by combining the spray-dried extract with a liquid, such as a buffer or water. In some embodiments, a range of 5-10 g of liquid is added per gram of dried extract powder (e.g., 5, 6, 7, 8, 9, or 10 g of liquid per gram of spray-dried extract powder). In some embodiments, 5 g of water is added per gram of spray-dried extract. In some embodiments, 6 g of water is added per gram of spray-dried extract. In some embodiments, 7 g of water is added per gram of spray-dried extract. In some embodiments, 8 g of water is added per gram of spray-dried extract. In some embodiments, the reconstituted spray-dried extract is stored on ice or at about 0°C to 8°C before use in a CFPS reaction.

[0187] In some embodiments, the reconstituted or rehydrated spray-dried extract comprises about 20% to 60% by volume (e.g., about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%) of the cell-free protein synthesis reaction.

[0188] The stability of a spray-dried extract can be determined based on the yield (also expressed as titer) of a protein of interest produced by a cell-free protein synthesis reaction containing the spray-dried extract. In some embodiments, the yield of the protein of interest is determined as described below. In some embodiments, the yield of the protein of interest is determined by passing the cell-free protein synthesis reaction mixture through a Protein A resin or Protein A column. Protein A can bind to a protein, such as an antibody, and then the bound protein of interest can be eluted from the Protein A column. In some embodiments, a Protein A PhyTip® column (Biotage®) is used to purify and quantify the amount of protein of interest produced during a cell-free protein synthesis reaction.

[0189] In some embodiments, the spray-dried extract is stored at about −20° C. for at least 3, at least 6, at least 12, at least 18, at least 24, at least 30, at least 36 months, or more than 36 months before being rehydrated. In some embodiments, the spray-dried extract is stored at about −20° C. for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months before being rehydrated. In some embodiments, a rehydrated spray-dried extract stored at about -20°C prior to rehydration for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months, is capable of synthesizing a target protein of interest in at least 80% yield compared to a control extract.In some embodiments, a rehydrated spray-dried extract stored at about -20°C prior to rehydration for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months, is capable of synthesizing a target protein of interest in at least 85% yield compared to a control extract. In some embodiments, a rehydrated spray-dried extract stored at about -20°C prior to rehydration for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months, is capable of synthesizing a target protein of interest in at least 90% yield compared to a control extract.

[0190] In some embodiments, the spray-dried extract is stored at about 2° C. to 8° C. for at least 3, at least 6, at least 12, at least 18, at least 24, at least 30, at least 36 months, or more than 36 months before being rehydrated. In some embodiments, the spray-dried extract is stored at about 2° C. to 8° C. for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months before being rehydrated. In some embodiments, a rehydrated spray-dried extract stored at about 2°C to 8°C prior to rehydration for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months, is capable of synthesizing a target protein of interest in at least 80% yield compared to a control extract.In some embodiments, a rehydrated spray-dried extract stored at about 2°C to 8°C prior to rehydration for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months, is capable of synthesizing a target protein of interest in at least 85% yield compared to a control extract. In some embodiments, a rehydrated spray-dried extract stored at about 2°C to 8°C prior to rehydration for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months, is capable of synthesizing a target protein of interest in at least 90% yield compared to a control extract.

[0191] In some embodiments, the spray-dried extract is stored at room temperature (RT) (e.g., about 20°C) for at least 3, at least 6, at least 12, at least 18, at least 24, at least 30, at least 36 months, or more than 36 months before being rehydrated. In some embodiments, the spray-dried extract is stored at about 20°C for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months before being rehydrated. In some embodiments, a rehydrated spray-dried extract stored at about −20° C. prior to rehydration for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 months, or more than 36 months, is capable of synthesizing a target protein of interest in at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% yield compared to a control extract.

[0192] In some embodiments, the control extract is a reconstituted, unformulated extract, i.e., an extract that does not contain the additives or stabilizers described herein. In some embodiments, the control extract is a frozen, unformulated extract (stored at about -20°C to -80°C) that does not contain the additives or stabilizers described herein. In some embodiments, the frozen, unformulated control extract is a frozen liquid extract that has not been previously dried. In other cases, the frozen, unformulated control extract is a frozen liquid rehydrated from a spray-dried extract. In some embodiments, the control extract does not contain an additive or stabilizer selected from trehalose, lactose, leucine, and raffinose.

[0193] In some embodiments, the control extract is a reconstituted formulated spray-dried extract containing an additive or stabilizer described herein, hi some embodiments, the control extract is a reconstituted formulated spray-dried extract containing an additive or stabilizer selected from trehalose, lactose, leucine, and raffinose.

[0194] In some embodiments, the spray-dried bacterial extract and the spray-dried control extract comprise trehalose, and the spray-dried extract is stored at about -20°C, about 2°C to 8°C, or about 20°C (RT) for 3 to 18 months or more (e.g., 3 months or more, 4 months or more, 5 months or more, 6 months or more, 7 months or more, 8 months or more, 9 months or more, 10 months or more, 11 months or more, 12 months or more, 13 months or more, 14 months or more, 15 months or more, 16 months or more, 17 months or more, or 18 months or more). After reconstitution, the spray-dried bacterial extract is capable of synthesizing a target protein of interest at a titer that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the potency of the control extract reconstituted at time 0. In some embodiments, the spray-dried bacterial extract and the control extract contain about 100 g / L (about 105 g / kg) of trehalose before rehydration, and the control extract is reconstituted at time T=0.

[0195] In some embodiments, the spray-dried extract and the control extract comprise about 100 g / L (about 105 g / kg) trehalose, and the spray-dried extract is stored at about −20° C. for about 13.75 months and, after reconstitution, is capable of synthesizing a target protein of interest at a titer greater than about 90% compared to the control extract reconstituted at time T=0. In some embodiments, the spray-dried extract and the control extract comprise about 100 g / L (about 105 g / kg) trehalose, and the spray-dried extract is stored at about 2° C. to 8° C. for about 13.75 months and, after reconstitution, is capable of synthesizing a target protein of interest at a titer of about 80% compared to the control extract reconstituted at time T=0. In some embodiments, the spray-dried extract and the control extract contain about 100 g / L (about 105 g / kg) trehalose, and the spray-dried extract is stored at about -20°C for about 13.75 months and, after reconstitution, is capable of synthesizing a target protein of interest at a titer of about 60 greater than the control extract reconstituted at time T=0.

[0196] In some embodiments, the protein synthesis activity of the rehydrated spray-dried extract is equal to or greater than the protein synthesis activity of a rehydrated control spray-dried extract when the extract is stored at 2°C to 8°C for 8 months or more before rehydration. In some embodiments, the protein synthesis activity of the rehydrated spray-dried extract is equal to or greater than the protein synthesis activity of a rehydrated control spray-dried extract when the extract is stored at 2°C to 8°C for 13 months or more before rehydration. In some embodiments, the protein synthesis activity of the rehydrated spray-dried extract is equal to or greater than the protein synthesis activity of a rehydrated control spray-dried extract when the extract is stored at about 20°C (RT) for 8 months before rehydration. In some embodiments, the rehydrated spray-dried extract contains trehalose. In some embodiments, the spray-dried extract contains about 100 g / L (about 105 g / kg) trehalose before rehydration. In some embodiments, the control extract is an unformulated extract that does not contain trehalose.

[0197] In some embodiments, the spray-dried extract has a protein synthesis activity loss (as determined by rate of titer loss) of about 2% to about 6% per month when stored at about 2° C. to 8° C. prior to reconstitution. In some embodiments, the spray-dried extract has a protein synthesis activity loss (as determined by rate of titer loss) of about 2% to about 6% per month when stored at 2° C. to 8° C. and a residual moisture content of about 7% (w / w) or less, about 6% (w / w) or less, about 5% (w / w) or less, about 4% (w / w) or less, about 3% (w / w) or less, or about 2% (w / w) or less residual moisture.

[0198] In some embodiments, the spray-dried extract contains trehalose and about 2-6% residual moisture and has a protein synthesis activity loss of about 2% to about 6% per month when stored at about 2°C to 8°C before reconstitution. In some embodiments, the spray-dried extract contains 100 g / L trehalose and about 2-3% residual moisture and has a protein synthesis activity loss of about 2% to about 6% per month when stored at about 2°C to 8°C before reconstitution. In some embodiments, the spray-dried extract contains 75 g / L trehalose and about 2-3% residual moisture and has a protein synthesis activity loss of about 4% to 5% per month when stored at 2°C to 8°C before reconstitution.

[0199] In some embodiments, the spray-dried extract contains lactose and about 3-4% residual moisture and has a protein synthesis activity loss of about 3% to about 4% per month when stored at about 2° C. to 8° C. before reconstitution. In some embodiments, the spray-dried extract contains about 100 g / L or about 100 g / kg lactose and about 3-4% residual moisture and has a protein synthesis activity loss of about 3% to 4% per month when stored at 2° C. to 8° C. before reconstitution.

[0200] In some embodiments, the spray-dried extract contains trehalose and is stored at 2°C to 8°C for about 4 months or at least 4 months before reconstitution, and is capable of synthesizing a target protein of interest at a titer of at least 75% compared to a frozen, unformulated control extract that does not contain trehalose and is stored at -20°C to -80°C. In some embodiments, the spray-dried extract contains about 70 to 100 g / L of trehalose (e.g., 70, 75, 80, 85, 90, 95, or 100 g / L of trehalose) before reconstitution. In some embodiments, the spray-dried extract contains about 75 to 105 g / kg of trehalose (e.g., 75, 80, 85, 90, 95, 100, or 105 g / kg of trehalose) before reconstitution.

[0201] In some embodiments, the spray-dried extract contains lactose and is stored at 2°C to 8°C for about 4 months or at least 4 months before reconstitution, and is capable of synthesizing a target protein at a titer of at least 75% compared to a frozen, unformulated control extract that does not contain lactose and is stored at -20°C to -80°C. In some embodiments, the spray-dried extract contains about 70 to 100 g / L lactose (e.g., 70, 75, 80, 85, 90, 95, or 100 g / L lactose) before reconstitution. In some embodiments, the spray-dried extract contains about 75 to 105 g / kg lactose (e.g., 75, 80, 85, 90, 95, 100, or 105 g / kg lactose) before reconstitution.

[0202] K. Use of Spray-Dried Bacterial Extracts in Cell-Free Protein Synthesis A biologically active protein of interest can be synthesized, properly folded, and / or assembled using a cell-free protein synthesis system, such as an E. coli-based cell-free (OCFS) system. In such a system, a cell extract from E. coli cells contains template DNA (such as a plasmid or linear DNA fragment), amino acids (including natural or unnatural amino acids), nucleotides, T7 RNA polymerase, and an energy source. Optionally, a disulfide isomerase chaperone is also added to aid in disulfide bond formation. The CFPS system has been used to produce a variety of proteins, including growth factors (Zawada et al., Biotechnol Bioeng, 108:1570-1578 (2011)), full-length antibodies and antibody fragments (Yin et al., mAbs, 4(2):217-225 (2012)), and antibody-drug conjugates (Zimmerman et al., Bioconjug Chem, 25(2):351-61 (2014)).

[0203] The bacterial strain used to prepare the cell extract may have reduced nuclease and / or phosphatase activity, thereby increasing the efficiency of cell-free synthesis. For example, the bacterial strain used to prepare the cell-free extract may have mutations in the genes encoding the nucleases RNase E and RNase A. The strain may also have mutations that stabilize components of the cell synthesis reaction, such as deletions of genes such as tnaA, speA, sdaA, or gshA, which prevent the degradation of the amino acids tryptophan, arginine, serine, and cysteine, respectively, in the cell-free synthesis reaction. Furthermore, the strain may have mutations that stabilize the protein products of cell-free synthesis, such as knockouts in the proteases ompT or lonP.

[0204] In a typical CFPS reaction, a gene encoding a protein of interest is expressed in a transcription buffer, resulting in mRNA that is translated into the protein of interest in a CFPS extract and translation buffer. The transcription buffer, cell-free extract, and translation buffer can be added separately, or two or more of these solutions can be combined before their addition or added simultaneously.

[0205] To synthesize a protein of interest in vitro, a bacterial extract initially contains mRNA molecules encoding the protein of interest. In some systems, mRNA is either purified from a natural source and then added exogenously, or synthetically prepared in vitro from cloned DNA using an RNA polymerase such as RNA polymerase II, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, RNA polymerase III, and / or a phage-derived RNA polymerase. In other systems, mRNA is produced in vitro from a template DNA; in this type of reaction, both transcription and translation occur. In some embodiments, the transcription and translation systems are combined with or include complementary transcription and translation systems that synthesize both RNA and protein in the same reaction. In such in vitro transcription and translation systems, the bacterial extract contains all components (exogenous or endogenous) necessary for both transcription (to produce mRNA) and translation (to synthesize protein) in a single system.

[0206] A CFPS reaction mixture can contain the following components: a template nucleic acid, such as DNA, comprising a gene of interest operably linked to at least one promoter and, optionally, one or more other regulatory sequences (e.g., a cloning or expression vector containing the gene of interest) or a PCR fragment; a promoter (e.g., T7 ribosomes; transfer RNA (tRNA); other or optionally present translation factors (e.g., translation initiation factors, elongation factors, and termination factors) and therefore cofactors; one or more energy sources (e.g., ATP, GTP); optionally, one or more energy regenerating components (e.g., PEP / pyruvate kinase, AP / acetate kinase, or creatine phosphate / creatine kinase); optionally, yield and / or efficiency enhancing factors (e.g., nucleases, nuclease inhibitors, protein stabilizers, chaperones) and therefore cofactors; and; optionally, a solubilizing agent. The reaction mixture may also contain amino acids and other materials specifically required for protein synthesis, including salts (e.g., potassium, magnesium, ammonium, and manganese salts of acetate, glutamate, or sulfate), polymeric compounds (e.g., polyethylene glycol, dextran, diethylaminoethyl dextran, quaternary aminoethyl and aminoethyl dextran, etc.), cyclic AMP, inhibitors of proteases or nucleases, inhibitors or modulators of protein synthesis, oxidation / reduction modulators (e.g., DTT, ascorbic acid, glutathione, and / or their oxides), non-denaturing detergents (e.g., Triton X-100), buffer components, spermine, spermidine, putrescine, etc. Components of such reactions are described in further detail in U.S. Pat. Nos. 7,338,789, 7,351,563, 8,715,958, and 8,778,631, the disclosures of each of which are incorporated by reference in their entirety for all purposes.

[0207] Depending on the particular enzymes present in the extract, for example, one or more of the many known nuclease inhibitors, polymerase inhibitors, or phosphatase inhibitors can be selected and used advantageously to improve the efficiency of synthesis.

[0208] Protein and nucleic acid synthesis typically requires an energy source. Energy is required for the initiation of transcription to produce mRNA (e.g., when a DNA template is used, a high-energy phosphate in the form of GTP is used to initiate translation). Each subsequent step of a codon by the ribosome (3 nucleotides; 1 amino acid) requires the hydrolysis of an additional GTP to GDP. ATP is also typically required. In order for an amino acid to be polymerized during protein synthesis, it must first be activated. Therefore, a significant amount of energy from high-energy phosphate bonds is required for protein and / or nucleic acid synthesis to proceed.

[0209] An energy source is a chemical substrate that can be enzymatically processed to provide energy for achieving a desired chemical reaction. Energy sources that allow the release of energy for synthesis by cleavage of high-energy phosphate bonds, such as those found in nucleoside triphosphates, such as ATP, are commonly used. Any source that can be converted into high-energy phosphate bonds is particularly suitable. ATP, GTP, and other triphosphates can generally be considered equivalent energy sources for supporting protein synthesis.

[0210] To provide energy for the synthesis reactions, the system can include additional energy sources such as glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, and glucose-6-phosphate, which can generate or regenerate high-energy triphosphate compounds such as ATP, GTP, other NTPs, etc.

[0211] If sufficient energy is not initially present in the synthesis system, it is preferred that an additional energy source be added. Energy sources can also be added or supplemented during the in vitro synthesis reaction.

[0212] In some embodiments, the cell-free protein synthesis reaction comprises NTP, E. coli tRNA, amino acids, Mg 2+ Acetate, Mg 2+ Glutamate, K + Acetate, K + Glutamate, folinic acid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfide isomerase, phosphoenolpyruvate (PEP), NAD, CoA, Na + This is performed using the PANOx-SP system, which contains oxalate, putrescine, spermidine and S30 extract.

[0213] In some embodiments, proteins containing unnatural amino acids (nnAAs) can be synthesized. In such embodiments, a reaction mixture can include the unnatural amino acid, a tRNA orthogonal to one of the 20 natural amino acids, and a tRNA synthetase capable of ligating the nnAA to the orthogonal tRNA. See, e.g., U.S. Pat. No. 8,715,958. Alternatively, the reaction mixture can contain the nnAA conjugated to a tRNA that has been depleted of natural tRNA synthetases. See, e.g., U.S. Pat. No. 8,778,631 and U.S. Patent Application Publication No. 2010 / 0184134. A variety of unnatural amino acids, including, but not limited to, detectably labeled amino acids, can be added to cell-free protein synthesis reactions and efficiently incorporated into proteins for specific purposes. See, for example, Albayrak, C. and Swartz, JR., Biochem. Biophys Res. Commun., 431(2):291-5; Yang WC et al., Biotechnol. Prog., (2012), 28(2):413-20; Kuechenreuther et al., PLoS One, (2012), 7(9):e45850; and Swartz JR., AIChE Journal, 58(1):5-13.

[0214] In some cases, cell-free synthesis reactions generally do not require the addition of a secondary energy source and utilize the coactivation of oxidative phosphorylation and protein synthesis.In some cases, CFPS is carried out in reactions such as the Cytomim (cytoplasmic mimic) system.The Cytomim system is defined as a reaction condition carried out in the absence of polyethylene glycol with an optimized magnesium concentration.This system does not accumulate phosphate, which is known to inhibit protein synthesis.A detailed description of the Cytomim system can be found, for example, in U.S. Patent No. 7,338,789; Jewett et al., Mol Syst Biol, (2008), 4:220; Spirin, AS and Swartz, JR (2008) Cell-free Protein Synthesis; Methods and Protocols, New Jersey: John Wiley & Sons, the entire contents of which are incorporated herein for all purposes.

[0215] The presence of an active oxidative phosphorylation pathway can be tested using inhibitors that specifically inhibit steps in the pathway, such as electron transport chain inhibitors. Examples of inhibitors of the oxidative phosphorylation pathway include toxins such as cyanide, carbon monoxide, azide, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and 2,4-dinitrophenol, antibiotics such as oligomycin, pesticides such as rotenone, and competitive inhibitors of succinate dehydrogenase such as malonate and oxaloacetate.

[0216] In some embodiments, the cell-free protein synthesis reaction comprises NTP, E. coli tRNA, amino acids, Mg 2+ Acetate, Mg 2+ Glutamate, K + Acetate, K + Glutamate, folinic acid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfide isomerase, sodium pyruvate, NAD, CoA, Na +This is carried out using a Cytomim system containing oxalate, putrescine, spermidine, and S30 extract. In some embodiments, the energy substrate of the Cytomim system is pyruvate, glutamate, and / or glucose. In some embodiments of the system, nucleoside triphosphates (NTPs) are replaced by nucleoside monophosphates (NMPs).

[0217] Cell extracts can be treated with iodoacetamide to inactivate enzymes that can reduce disulfide bonds and impair proper protein folding. As further described herein, cell extracts can also be treated with prokaryotic disulfide bond isomerases, such as, but not limited to, E. coli DsbC and PDI. Cell extracts can be treated with DsbC, FkpA, and peptidyl peryl isomerase. Exogenous chaperone proteins can be expressed by the bacterial strain of the cell extract. Glutathione disulfide (GSSG) and glutathione (GSH) can also be added to the extract in ratios that promote proper protein folding and prevent the formation of aberrant protein disulfides.

[0218] In some embodiments, the CFS reaction includes inverted membrane vesicles for carrying out oxidative phosphorylation. These vesicles may be formed during the high-pressure homogenization step of the preparation of cell extract process, as described herein, and may remain in the extract used in the reaction mixture.

[0219] Cell-free synthesis reaction conditions can be performed as batch, continuous flow, or semi-continuous flow, as known in the art. Reaction conditions are linearly scalable, for example, at a 0.3 L scale in a 0.5 L stirred tank reactor, a 4 L scale in a 10 L fermentor, and a 100 L scale in a 200 L fermentor.

[0220] The protein synthesis reactions described herein can utilize large-scale reactors, small-scale, or multiplexed to perform multiple simultaneous syntheses. Continuous reactions can introduce reagent flows using a feeding mechanism and can isolate the final product as part of the process. Batch systems are also important, in which additional reagents can be introduced to extend the period of active synthesis. Reactors can be operated in any mode, including batch, extended batch, semi-batch, semi-continuous, fed-batch, and continuous, depending on the application.

[0221] L. How to Compare Cell-Free Protein Synthesis Yields The activity of the dried extract (e.g., the yield of a particular protein in a cell-free protein synthesis system) can be determined using assays such as performing cell-free protein synthesis to produce a model protein (test protein) that can be measured. Methods for cell-free protein synthesis are described, for example, in Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng. 66:180-8 (1999); Kim, D. M. and Swartz, J. R. Biotechnol. Prog. 16:385-90 (2000); Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng. 74:309-16 (2001); Swartz et al., Methods Mol. Biol. 267:169-82 (2004); Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng. 85:122-29 (2004); Jewett, M. C. and Swartz, J. R., Biotechnol. Bioeng. 86:19-26 (2004); Yin, G. and Swartz, JR, Biotechnol. Bioeng. 86:188-95 (2004); Jewett, MC and Swartz, JR, Biotechnol. Bioeng. 87:465-72 (2004); Voloshin, AM and Swartz, JR, Biotechnol. Bioeng. 91:516-21 (2005).

[0222] The amount of protein produced in a CFPS reaction can be measured using any method known to those of skill in the art. In some embodiments, the yield of the protein of interest is determined by dual flow chromatography (DFC) using Protein A resin. Protein A resin can be packed between two thin fritted screens at the end of a disposable pipette tip. In some embodiments, the yield of the protein of interest is determined by passing the product of the cell-free synthesis reaction through a Protein A column, such as a PhyTip® column (Biotage®). The yield of the protein of interest can be expressed as weight / volume of the CFPS reaction (e.g., mg / L or g / L) or as a percentage of the yield of a control extract.

[0223] In some embodiments, the yield of the protein of interest is determined by performing high performance liquid chromatography (HPLC) of the protein product from the CFPS reaction along with a protein standard.

[0224] In some embodiments, the yield of a protein of interest is determined by an assay that measures the activity of the specific protein being translated. Examples of assays for measuring protein activity include the luciferase assay system or the chloramphenicol acetyltransferase assay system for the production of related proteins. These assays measure the amount of functionally active protein produced from a translation reaction. Assays for measuring protein levels include, but are not limited to, Coomassie-stained polyacrylamide gels, silver-stained polyacrylamide gels, ELISA, immunoblotting, Western blotting, size-exclusion chromatography, affinity chromatography, and mass spectrometry. The activity of a specific protein being translated can be measured using any method known to those skilled in the art for measuring the activity (e.g., function) of a specific protein of interest. For example, the amount of a specific kinase produced in a translation reaction can be measured by a kinase assay, in which the activity of the specific kinase is determined by quantifying the kinase reaction.

[0225] Another method for measuring the amount of protein produced in a coupled in vitro transcription and translation reaction is to 35 S-methionine, 3 H-leucine, or 14 The incorporation assay involves carrying out a reaction using a known amount of radiolabeled amino acid, such as C-leucine, followed by measuring the amount of radiolabeled amino acid incorporated into the newly translated protein. The incorporation assay measures the amount of radiolabeled amino acid in all proteins produced in an in vitro translation reaction, including truncated protein products. The radiolabeled proteins may be further separated on a protein gel, and autoradiography may be used to confirm that the products are of the correct size and that no secondary protein products are produced.

[0226] Methods for measuring the ability of an expression system to express a protein include: 14 In some embodiments, the method for measuring the protein synthesis activity of the spray-dried extract includes a C Leu incorporation assay. 14 C Leu incorporation assay.

[0227] In some embodiments, the yield of soluble protein is determined by the amount of soluble protein incorporated into the soluble protein produced in the cell-free protein synthesis reaction. 14 The C is calculated from the amount of Leu. The extract may be treated with 50 μM iodoacetimide (IAM) for approximately 30 minutes at room temperature. IAM is added to allow for the formation of disulfide bonds within the protein of interest. Other thiol-capping reagents, such as iodoacetic acid (IAA) and N-ethylmaleimide (NEM), can be substituted for IAM.

[0228] Typically, then, to initiate the reaction, 14Add the extract to a microfuge tube containing the protein synthesis reaction mixture with C-Leu. Transfer approximately 60 μl of the reaction mixture to a 24-well plate and spread it evenly around the wells. Incubate the mixture at 30°C for 5 hours. At the end of the 5 hours, transfer the mixture to a new microfuge tube. Transfer two 10 μl aliquots to two slips of chromatography paper labeled "A" and "B." "A" represents the total count, and "B" represents the total protein count. Centrifuge the remaining mixture in the tube at approximately 13,000 rpm for approximately 15 minutes in a microfuge. Transfer two 10 μl aliquots of the supernatant to two slips of chromatography paper labeled "C" and "D." "C" and "D" represent the soluble protein. Dry all paper strips approximately 2 inches from a heat lamp for approximately 15 minutes. Transfer "A" to a microfuge tube. "B," "C," and "D" are washed three times on ice with 5% TCA for approximately 15 minutes, then with 100% ethanol. They are then dried under a heat lamp for approximately 15 minutes. "B," "C," and "D" slips are transferred to individual microfuge tubes. Scintillation cocktail (Optiphase Supermix, PerkinElmer, Waltham, MA) is added to each microfuge, and the slips are counted in a scintillation counter for 5 minutes.

[0229] The total protein yield can be determined by the following formula: (Countslip B / Countslip A) (Leucine concentration in cell-free medium / Number of leucines in the protein of interest) (MW of the protein of interest)

[0230] The yield of soluble protein for "C" and "D" can be determined by the following formula: (Countslip C or D / Countslip A) (Concentration of leucine in CF / Number of leu residues in the protein of interest) (MW of the protein of interest)

[0231] The average yield of soluble protein can be determined by averaging the "C" and "D" yields.

[0232] Alternatively, protein yields can be determined on polyacrylamide gels using conventional techniques. 14 The degree of denaturation can be determined by electrophoresis of proteins labeled with C Leu. The gel can be denaturing or non-denaturing depending on the polypeptide to be detected. When detecting proteins containing multiple subunits, non-denaturing gels are preferred.

[0233] Alternatively, protein yield can be determined by specific binding assays such as enzyme-linked immunosorbent assay (ELISA) or surface binding resonance (eg, Biacore).

[0234] Alternatively, protein yield can be determined by total or partial purification, such as using chromatography in combination with protein quantitation, such as UV absorbance or BCA analysis.

[0235] Although the foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present disclosure that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0236] Example Example 1. Preparation of bacterial cell-free extracts This example provides a representative method for preparing bacterial cell-free extracts. Exemplary methods for preparing bacterial cell-free synthetic extracts are described in Zawada, JF, et al. (Microscale to manufacturing scale-up of cell-free cytokine production—a new approach for shortening protein production development timelines. Biotechnol Bioeng. 2011 Jul; 108(7): 1570-8. doi:10.1002 / bit.23103) and Groff, D., et al. (Development of an E. coli strain for cell-free ADC manufacturing. Biotechnology and Bioengineering, 119, 162-175. https: / / doi.org / 10.1002 / bit.27961).

[0237] For small-scale bacterial extract preparation, 10 μl of a thawed glycerol stock of E. coli was used to inoculate 50 mL of 2YT medium in a 250 mL baffled flask. The culture was incubated overnight at 37°C with vigorous shaking. 50 mL of the culture was then transferred to 1 L of 2YTPG medium in a 2.5 L flask with a filter lining in the cap. The culture was incubated at 30°C with vigorous shaking, and growth rate was monitored. Cells were harvested and cooled during the exponential phase and before the growth rate decreased during the transition to stationary phase. After cooling the culture, cells were collected by centrifugation at 8,000 × g for 20–30 minutes. Approximately 8 g of wet cells were harvested from 1 L at an OD of 3. The cell pellet was resuspended in at least 5 mL of S30 buffer for every 1 gram of wet cell weight. The cell suspension was centrifuged at 8,000 × g for 20–30 minutes. The supernatant was discarded, and the washed cell pellet was frozen at -80°C.

[0238] For large-scale bacterial extract preparation, the culture was harvested after 16–20 hours of fermentation. The fermenter (200 L fermenter) was then pressurized to 20 psi, and the culture (approximately 200 L) was transferred via pressure to a chilled 200 L jacketed holding tank through two heat exchangers connected in parallel. The culture temperature in the holding tank (approximately 200 L) was cooled to 2–8°C by recirculating glycol through the jacket. After transfer, the culture was ready for the first centrifugation step via a disc-stack centrifuge. During the first centrifugation step, the cells were separated from the spent medium (supernatant). The discharged paste (cell slurry) was collected in a container, weighed, and then dispensed into a chilled 200 L holding tank containing 100 L of S30 buffer, mixed, and resuspended. The resuspended cells were then centrifuged to pellet the cells. The cell pellet was stored at -80°C.

[0239] One liter of 2YTPG medium contains 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 22 mM monobasic sodium phosphate, 40 mM dibasic sodium phosphate, 100 mM glucose, and, optionally, 100 μl of antifoam agent 204. 2YT medium contains 16 g / L tryptone, 10 g / L yeast extract, and 5 g / L sodium chloride. S30 buffer contains 10 mM Tris acetate, 14 mM magnesium acetate, and 60 mM potassium acetate.

[0240] Frozen S30 cell paste was broken into small pieces and thawed in 1 mL of room-temperature S30 buffer per gram of cell paste. After thawing, the cell suspension was kept on ice. Before processing the cell extract, the cell homogenizer was rinsed with S30 buffer. Cells were lysed by passing the homogenizer once at 17,500 psi. The lysate was then rapidly cooled through a cooling coil or heat exchanger. The lysate was kept on ice until the entire cell paste was lysed, as described herein. The lysate was centrifuged at 30,000 × g for 30 minutes at 4°C. The supernatant was collected in a clean tube, and the centrifugation step was repeated once more to collect the entire supernatant in the lysate.

[0241] To the collected supernatant, 0.2 mL of preincubation mixture was added for every mL of supernatant. The mixture was then incubated at about 37° C. for about 80 minutes. The preincubation mixture contained 370 mM Tris acetate pH 8.2, 11.1 mM magnesium acetate, 16.5 mM ATP, 50 μM each of the 20 amino acids or unnatural amino acids, 105 mM phosphoenolpyruvate (PEP), and 8.4 U / mL pyruvate kinase.

[0242] Example 2. Formulation of cell-free extracts with additives This example provides a representative method for formulating a bacterial cell-free extract with additives useful for stabilizing the spray-dried extract.

[0243] Liquid bacterial extracts were formulated by directly mixing in powdered excipients or by adding concentrated stock solutions of excipients to achieve various levels and combinations of excipients in the formulated extract prior to spray drying. For bioburden control, concentrated stock solutions can be sterile filtered before being added to the liquid extract. Some excipients demonstrated ready aqueous solubility at concentrations up to several hundred grams per liter under ambient and refrigerated conditions, while others had limited solubility by comparison. Some components demonstrated solubility in the extract up to at least 100 g / L or at least 100 g / kg and were tested at these levels, but the lower limit of extract solubility dictated the maximum concentration that could be evaluated for some other excipients. The excipients used included trehalose dihydrate (TDH), lactose monohydrate (LMH), raffinose, maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), and 2-hydroxypropyl-β-cyclodextrin. Over 33 formulations were made, dried, and evaluated for extract formulations. Representative single-component and combination formulations are shown in Table 1.

[0244] Additionally, some mixtures included one or more, all combinations, or subsets of the naturally occurring amino acids leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine, referred to herein as the "PMA amino acid mixture." The PMA amino acid mixture optionally does not include tyrosine or phenylalanine. In some embodiments, tyrosine and / or phenylalanine are added separately from the PMA mixture in the XCF reaction mixture. A representative PMA amino acid mixture is shown in Table 2.

[0245] Example 3. Spray Drying Process This example describes a typical spray drying process.

[0246] Spray drying is a scalable, continuous process that converts liquid feedstock into a fine powder while reducing material volume and bulk density. Spray drying of extracts such as XtractCF® has been achieved by (1) atomizing the liquid feedstock material into a fine mist by pumping it through an atomizing device, such as a nozzle or rotary atomizer; (2) interfacing the fine mist with convective flow of hot, dry gas in a chamber, which evaporates the liquid to dryness; (3) separating the dried product material from the wet, dry gas and smaller particulates using a cyclone or other similar technology designed for powder fractionation; and (4) collecting the dried material. Spray drying XtractCF® from liquid to powder form allows for a reduction in the volume of material required for commercial-scale production and increased storage capacity.

[0247] The liquid feedstock material was formulated prior to spray drying and consisted of extract solids, formulation solids, and water. The formulated feedstock was continuously pumped into the dryer at a liquid feed rate determined by the dryer process control scheme. The material was fed through piping to a two-fluid nozzle. The nozzle combined the liquid with an atomizing gas stream, with the atomizing gas pressure and flow rate affecting the droplet size formed based on the pressure and relative flow of the two streams. After atomization into a fine mist, the droplets were contacted with a drying gas at the inlet temperature. However, evaporative cooling occurred, and the solids did not directly experience the high inlet drying gas temperature. During the residence time in the drying chamber, the mist was exposed to the drying gas, during which the drying gas cooled to the outlet temperature. Initially, the solids in the droplets experienced the wet-bulb temperature, but as the water evaporated, there was a transition to where the solids experienced the dry-bulb temperature. Interdependencies exist between several of the above process parameters and output in the spray drying process, and the balance of momentum, heat, and mass transfer in the process, defined by these parameters, determined the particle size or particle size distribution, which affected the yield. Water evaporation during drying was significant. The dried material contained residual moisture or a portion of the moisture content, which negatively correlated with the observed shelf-life stability. Spray-dried extracts typically had a residual moisture content of less than 7% and could have a residual moisture content of less than 5%.

[0248] In XtractCF® spray drying, inlet and outlet temperatures significantly affected the performance of XpressCF®. Residual moisture or water content of spray-dried XtractCF® was observed to significantly affect shelf-life stability when stored at 2-8°C and ambient temperatures. Development of XtractCF® spray drying included laboratory-scale (Buchi B-290), pilot-scale (Mobile Minor PSD-1), and industrial-scale (modified PSD-2, PSD-3, and IFC Welko PSD-4) studies. These drying scales ranged from approximately 0.1 L to 1000 L batch sizes and throughputs of approximately 1 kg / h to 100 kg / h. Larger dryers typically have longer material residence times, which can result in greater heat exposure to XtractCF® during the drying process. As a result, downward temperature setpoint adjustments, particularly for outlet temperature, were made to maintain optimal XpressCF® activity during larger-scale drying. The outlet temperature of the large dryer was adjusted to approximately 65 to 76°C.

[0249] The process control scheme can take multiple configurations whereby some parameters are controlled near set points while other parameters are allowed to free float or adjust to accommodate control of the set parameters. These are not necessarily scale specific. Due to the thermal constraints of XtractCF®, the typical approach to drying has been to control the inlet temperature, outlet temperature, drying gas flow rate, and atomizing gas pressure / flow rate at set points and float and adjust the liquid feed rate to compensate.

[0250] Example 4. Testing various extract formulations and drying conditions This example describes various extract formulations and drying conditions that were tested using different spray dryers.

[0251] Formulation testing was performed using a Buchi-290 laboratory-scale spray dryer to evaluate several common spray-dried excipients in single-component formulations. Spray-dried powder samples were reconstituted into liquid form before testing for XpressCF® performance. Figure 1 shows the initial titer and residual moisture results from the XpressCF® (XCF) testing. In Figure 1, the inlet and outlet temperatures for the dried samples are listed in parentheses as (inlet temperature / outlet temperature). Samples 1A-1E are unformulated spray-dried samples, while Samples 2A-2E are formulated spray-dried samples, with varying inlet / outlet temperatures as indicated. Protein A resin in a pipette tip, also referred to herein as "Phytip," is used in a high-throughput format with laboratory automation for rapid purification and titer determination using A280 absorbance. Flower plates, also referred to herein as "FP," are small multiwell plates (approximately 1 ml reactions) without pH or DO control. Trehalose dihydrate (100 g / L, Sample 2B) was the promising excipient, followed by leucine (10 g / L, Sample 2D) and then Tween 80 (0.1%, Sample 2A). PVP (100 g / L, Sample 2C) and maltodextrin (100 g / L, Sample 2E) resulted in low initial potency. Residual moisture was in the range of approximately 3-6% for the dried samples tested. Unformulated dried XtractCF® showed good recovery of initial activity. The control sample was an unformulated liquid extract. Some effect of freeze-thaw on potency relative to the control was observed.

[0252] Based on the results in Figure 1, the stability of dried XtractCF® formulated with 100 g / L trehalose dihydrate and unformulated dried XtractCF® was evaluated at 2-8° C. and room temperature. Testing included the use of multiple extraction lots and drying conditions, as shown in Table 4. [Table 6]

[0253] The samples in Table 4 were prepared and dried for both unformulated and 100 g / L trehalose formulations. Drying was performed using a laboratory-scale Buchi B-290 spray dryer. XCF studies used batch reactions in Micro-24 microbioreactors with 30% XtractCF® to express the product anti-CD74 antibody for the 8- and 13-month samples and trastuzumab for the 18-month sample. The feedstock and dried XtractCF® were derived from either DR 6-2, DR 7-6, or DR 8-7. The sample extraction lot, residual moisture, and dryer outlet temperature are listed for each sample. As shown in Table 4, promising stability was observed for up to 18 months at storage conditions of 2–8°C. Unformulated XtractCF®, by comparison, exhibited limited shelf-life stability, highlighting the importance of having stabilizing formulation components present. Even when stored at 2-8°C, enhanced performance is observed for the 100 g / L trehalose formulation compared to the unformulated extract upon room temperature storage.

[0254] The drying process was scaled up to pilot-scale drying using a Mobile Minor PSD-1. Several trehalose formulations, including 100 g / L, 50 g / L, and 25 g / L, were tested, including a preliminary investigation of several drying process conditions. As shown in Figures 2A and 2B, comparable initial activity was obtained at the three tested trehalose formulation levels. The moisture levels of the 50 g / L and 25 g / L formulations were observed to be higher than the 100 g / L formulation, due to the relatively lower total solids content of the feedstock, which resulted in lower drying efficiency and reduced overall moisture evaporation. A source unformulated liquid extract was used as a control.

[0255] A subset of samples from Figures 2A and 2B with promising initial activity were tested in long-term stability studies at storage conditions of -20°C, 2-8°C, and room temperature. Figure 3A and Table 5 show the stability results obtained from the 2-8°C storage condition. The 2-8°C storage condition was evaluated for up to 24.5 months. The calculated percent potency loss is listed in Table 5. [Table 7]

[0256] As shown in Table 5, the rates for total TDH concentration ranged from 0.3 to 1.6% / month. However, the 50 g / L and 25 g / L formulations have higher moisture content than the 100 g / L formulation, which confounds the ability to make direct comparisons based purely on formulation concentration, as moisture levels may affect stability. Storage conditions at -20°C showed the least potency loss evaluated over the 12-month period compared to conditions at 2-8°C, as shown in Figure 3B. Room temperature conditions showed significant activity loss over the 8-month period, with clear distinctions between formulation levels, with higher levels of TDH being more stable, as shown in Figure 3C. The control used in this study was a t=0 reconstituted dried extract stored frozen (≤-65°C) and thawed when needed for testing.

[0257] The above results suggest that trehalose is useful as a single component stabilizer, and therefore 100 g / L of trehalose was selected for further testing.

[0258] The minimum target drying feed rate was 10 kg / hr. Therefore, the experiment was repeated using a modified PSD-2 dryer to achieve a higher than typically designed gas flow rate, which was assessed as having the potential to achieve the desired throughput. The dimensions of the drying chamber compared to the gas flow rate of the modified PSD-2 stem from a different technical perspective than the PSD-3, namely, the PSD-2 chamber is narrower than the PSD-3 chamber. However, the drying capacity can be described similarly, since both chamber size / dimensions and gas flow rate contribute to drying performance and efficiency.

[0259] The study involved an initial process range of four conditions, each 25 L, from which a single process condition was selected. This condition was run in three consecutive 100 L experiments, each using a different extract lot, to demonstrate process robustness. The results are shown in Figures 4A and 4B. As shown in Figures 4A and 4B, in each demonstration, the samples tested included the unformulated extract pool (UEP), the formulated extract pool (FEP), a blend of formulated liquid samples taken throughout each 100 L demonstration (FEP dried feed blend), and a blend of reconstituted dried XtractCF® samples taken throughout each 100 L demonstration (spray-dried blend). The formulated samples included 100 g / L trehalose. All three experiments demonstrated approximately 100% activity recovery in each dried blend compared to the formulated blend.

[0260] Materials from the demonstration experiment were tested for long-term stability. As shown in Figures 5A, 5B, and 5C, the results indicate minimal, if any, loss of activity over approximately 19.5 months when the extract was stored at -20°C and 2-8°C, and a slight loss to approximately 70-90% recovery of activity over approximately 19.5 months when the extract was stored at room temperature (approximately 20°C). The control used in the test was the t=0 reconstituted dried extract.

[0261] Select stability sample time points were tested in an expanded stirred tank reactor configuration (DASbox, 200 ml XCF reaction volume), which is considered more representative of large-scale bioreactors compared to the Micro-24. The DASbox, also referred to herein as "DB," is a miniature stirred tank bioreactor system with pH and DO control (approximately 100-200 ml reactions). Figures 6A and 6B show stability data at approximately 14 months, demonstrating activity recovery of >90% when the extract was stored at -20°C, approximately 80% when the extract was stored at 2-8°C, and approximately 60-65% when the extract was stored at room temperature. This study utilized expression of the product anti-folate receptor alpha antibody using a preformed light chain (PFLC). The control used in the study was the t=0 reconstituted dried extract.

[0262] Additional formulation screening was performed. The scope of testing included screening of numerous single and multi-component formulations, including (1) sugar / polyol, (2) amino acid, (3) detergent / polyethylene glycol, and (4) macromolecular crowding agents. Motivated by the above data suggesting acceptable stability at 50 g / L trehalose compared to 100 g / L trehalose, an intermediate 75 g / kg trehalose formulation was evaluated here.

[0263] Table 6 lists the formulations tested, the corresponding XpressCF® initial activity results, the reaction setup (batch or fed-batch) tested, and the expression product used. As shown in Table 6, sample codes 1119, 0120, and 0220 represent November 2019, January 2020, and February 2020, respectively. For each formulation tested, the XpressCF® reaction setup (batch or fed-batch) and expression product are listed. All tests were performed using 37.5% extract by volume. The extract lots used were 19006-10 (November 2019), 19013-01 (January 2020), and 19011-04 (February 2020). The m24, also known as the micro-24, is a miniature multiwell microbioreactor system with pH and DO control (approximately 5 ml reactions). 19013-01 was a high bioburden lot suspected to be at least partially responsible for the lower than expected titers from the January 2020 testing, which prompted additional testing in February 2020. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

[0264] The data in Table 6 are based on the glass transition temperatures (T g ) and recovered activity after drying. The top candidate formulations were the single components (A) trehalose dihydrate 100 g / L, (B) trehalose dihydrate 75 g / kg, and (C) lactose monohydrate 100 g / kg, as shown in Figures 7A, 7B, 8A, and 8B, which showed promising results for the expression of two different antibody products. Because the initial activity with 100 g / kg lactose was better at outlet temperatures of 60°C and 70°C compared to 80°C, only these two conditions were pursued further. These single component formulations were tested for long-term stability and show similar rates of potency loss, as shown in Figure 9 and Table 7. High T g Several combination formulations containing either a polar / non-charged amino acid mixture or 2-hydroxypropyl-β-cyclodextrin showed promising initial activity but did not offer any improvement over the TDH or LMH single component options. [Table 9]

[0265] PSD-3 (Sutro) A series of range experiments were conducted during the start-up of the PSD-3 dryer. A water run was first conducted to test the operating range of the drying process. Several replicate tests with extracts were then conducted to gain a better understanding of the process parameter range and output. Process and analytical data from these experiments were used to select GMP manufacturing run conditions. As shown in Figure 10, a 75 g / kg TDH formulation was used in the GEA PSD-3 drying experiment, producing an SDE with high activity recovery.

[0266] In summary, the spray-dried bacterial extracts described in this example exhibit high percent activity and improved stability during storage in various formulations. Such extracts have a longer shelf life at room temperature, 2-8°C, and -20°C compared to control extracts, such as additive-free (unformulated) extracts. The results of the above studies demonstrate that additive-containing spray-dried bacterial extracts increase the stability of the bacterial extracts in CFPS reactions.

[0267] All publications, issued patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. i. combining a bacterial extract containing lysed bacterial components with a composition containing trehalose, lactose, leucine, or raffinose to obtain a mixture, wherein the bacterial extract is capable of synthesizing a target protein from a template nucleic acid encoding the target protein in a cell-free protein synthesis reaction; and ii. spray drying the mixture to produce a stable spray-dried bacterial extract; 1. A method for producing a stable spray-dried bacterial extract for cell-free protein synthesis, comprising:

2. 10. The method of claim 1, wherein the bacterial extract containing lysed bacterial components is a liquid bacterial extract or a rehydrated bacterial extract.

3. 3. The method of claim 1 or claim 2, wherein the composition comprises trehalose or lactose.

4. The method according to any one of claims 1 to 3, wherein the mixture comprises about 25 to 200 g / kg of trehalose.

5. 5. The method of claim 4, wherein the mixture comprises about 50 to 100 g / kg of trehalose.

6. 6. The method of claim 4 or claim 5, wherein the trehalose is trehalose dihydrate (TDH).

7. 10. The method of claim 1, wherein the mixture comprises about 25 to 200 g / kg of lactose.

8. 8. The method of claim 7, wherein the mixture comprises about 50 to 100 g / kg of lactose.

9. 9. The method of claim 7 or claim 8, wherein the lactose is lactose monohydrate (LMH).

10. 10. The method of claim 1, wherein the mixture comprises about 5 to 10 g / L of leucine.

11. 10. The method of claim 1, wherein the mixture comprises about 25 to 200 g / L of raffinose.

12. 12. The method of any one of claims 1 to 11, wherein the stable spray-dried bacterial extract comprises about 40-70 g / L bacterial extract solids.

13. 12. The method of any one of claims 1 to 11, wherein the stable spray-dried bacterial extract comprises about 50-60 g / L bacterial extract solids.

14. In step (i), the bacterial extract is selected from the group consisting of one or more high glass transition temperatures (T ｇ 14. The method of any one of claims 1 to 13, further comprising combining the amino acid with a non-polar, uncharged amino acid.

15. the one or more high T ｇ 15. The method of claim 14, wherein the non-polar, uncharged amino acids are selected from valine, tryptophan, isoleucine, leucine, alanine, glycine, proline, and any combination thereof.

16. the one or more high T ｇ 15. The method of claim 14, wherein the non-polar, uncharged amino acid is selected from L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, L-proline, and any combination thereof.

17. The mixture contains 5 to 15 g / kg of the high T ｇ 17. The method of any one of claims 14 to 16, comprising a non-polar, uncharged amino acid.

18. 18. The method of any one of claims 1 to 17, wherein in step (i) the bacterial extract is further combined with one or more amino acids selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, arginine, and any combination thereof.

19. 19. The method of claim 18, wherein the mixture comprises about 5 to 15 g / kg of the one or more amino acids.

20. 18. The method of any one of claims 1 to 17, wherein in step (i), the mixture further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), or 2-hydroxypropyl-β-cyclodextrin.

21. 21. The method of any one of claims 1 to 20, wherein the spray-dried bacterial extract contains no more than about 15% (w / w) residual water.

22. 21. The method of any one of claims 1 to 20, wherein the spray-dried bacterial extract contains no more than about 10% (w / w) residual water.

23. 21. The method of any one of claims 1 to 20, wherein the spray-dried bacterial extract contains no more than about 5% (w / w) residual water.

24. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at 2°C to 8°C for at least 6 months.

25. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at 2°C to 8°C for at least 12 months.

26. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at 2°C to 8°C for at least 18 months.

27. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at about -20°C for at least 6 months.

28. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at about -20°C for at least 12 months.

29. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at about -20°C for at least 18 months.

30. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at about room temperature (20°C) for at least 6 months.

31. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at about room temperature (20°C) for at least 12 months.

32. 24. The method of any one of claims 1 to 23, wherein the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract after storage at about room temperature (20°C) for at least 18 months.

33. The method according to any one of claims 24 to 32, wherein the titer is determined by the Phytip® method.

34. 34. The method of any one of claims 24 to 33, wherein the control extract does not contain trehalose, lactose, leucine or raffinose.

35. 34. The method of any one of claims 24 to 33, wherein the control extract comprises trehalose, lactose, leucine or raffinose.

36. 24. The method of any one of claims 1 to 23, wherein the spray-dried extract and the control extract comprise trehalose, and the spray-dried extract is stored at 2°C to 8°C for at least 12 months and, after reconstitution, is capable of synthesizing the target protein at a titer of at least 80% compared to the control extract reconstituted at time 0.

37. 24. The method of any one of claims 1 to 23, wherein the protein synthesis activity of the spray-dried extract declines by less than about 5% per month compared to a control extract when the spray-dried extract is stored at 2°C to 8°C and then rehydrated.

38. 38. The method of claim 37, wherein the control extract does not contain trehalose, lactose, leucine, or raffinose.

39. 38. The method of claim 37, wherein the control extract comprises trehalose, lactose, leucine, or raffinose.

40. 39. The method of any one of claims 1 to 38, wherein the spray-dried bacterial extract comprises an active oxidative phosphorylation system for cell-free protein synthesis.

41. 41. The method of any one of claims 1 to 40, wherein the bacterial extract is derived from an Escherichia species.

42. 42. The method of any one of claims 1 to 41, wherein prior to step (i), the bacterial extract is heated at about 20°C to 45°C for about 30 minutes to about 10 hours.

43. In step (ii), the spray drying comprises: atomizing the mixture to form droplets; contacting the droplets with a gas to evaporate liquid from the droplets; separating the dried extract from the gases and smaller particles; and 42. The method of any one of claims 1 to 41, comprising collecting the spray-dried extract.

44. 44. The method of claim 43, wherein 90% (w / w) or more of the liquid is removed from the mixture.

45. 44. The method of claim 43, wherein 95% (w / w) or more of the liquid is removed from the mixture.

46. 46. ​​The method of any one of claims 1 to 45, further comprising: (iii) rehydrating the spray-dried bacterial extract; and (iv) synthesizing the target protein under conditions that support a cell-free protein synthesis reaction.

47. 47. The method of claim 46, wherein the rehydrated bacterial extract comprises about 20% to 60% (by volume) of the cell-free protein synthesis reaction.

48. 48. The method of claim 47, wherein the rehydrated bacterial extract comprises about 30% to 40% (by volume) of the cell-free protein synthesis reaction.

49. dried lysed bacterial components; a composition comprising trehalose, lactose, leucine or raffinose; 1. A spray-dried bacterial extract for cell-free protein synthesis comprising: A spray-dried bacterial extract, wherein upon rehydration, said extract is capable of synthesizing a target protein from a template nucleic acid encoding the target protein.

50. 50. The spray-dried extract of claim 49, wherein the composition comprises trehalose or lactose.

51. 50. The spray-dried extract of claim 49, wherein the composition comprises about 25-200 g / kg of trehalose.

52. 50. The spray-dried extract of claim 49, wherein the composition comprises about 50-100 g / kg of trehalose.

53. 53. The spray-dried extract of claim 51 or claim 52, wherein the trehalose is trehalose dihydrate (TDH).

54. 50. The spray-dried extract of claim 49, wherein the composition comprises about 25-200 g / kg lactose.

55. 55. The spray-dried extract of claim 54, wherein the composition comprises about 50-100 g / kg lactose.

56. 56. The spray-dried extract of claim 54 or claim 55, wherein the lactose is lactose monohydrate (LMH).

57. 50. The spray-dried extract of claim 49, wherein the composition comprises about 5-10 g / L of leucine.

58. 50. The spray-dried extract of claim 49, wherein the composition comprises about 25-200 g / L of raffinose.

59. One or more high glass transition temperatures (T ｇ 59. The spray-dried extract of any one of claims 49 to 58, further comprising a non-polar, uncharged amino acid.

60. the one or more high T ｇ 60. The spray-dried extract of claim 59, wherein the non-polar, uncharged amino acids are selected from valine, tryptophan, isoleucine, leucine, alanine, glycine, proline, and any combination thereof.

61. the one or more high T ｇ 60. The spray-dried extract of claim 59, wherein the non-polar, uncharged amino acid is selected from the group consisting of L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, L-proline, and any combination thereof.

62. 62. The spray dried extract of any one of claims 59 to 61, comprising 5 to 15 g / kg of amino acids.

63. 63. The spray dried extract of any one of claims 49 to 62, further comprising one or more, any combination of, or any subset of, amino acids selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine.

64. 64. The spray-dried extract of claim 63, comprising about 5 to 15 g / L of said amino acid.

65. 65. The spray-dried extract of any one of claims 49 to 64, further comprising maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), or 2-hydroxypropyl-β-cyclodextrin.

66. 66. The spray dried extract of any one of claims 49 to 65, containing no more than about 15% (w / w) residual water.

67. 66. The spray dried extract of any one of claims 49 to 65, containing no more than about 10% (w / w) residual water.

68. 66. The spray dried extract of any one of claims 49 to 65, containing no more than about 5% (w / w) residual water.

69. 69. The spray-dried extract of any one of claims 49 to 68, which is stored at 2°C to 8°C for at least 6 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

70. 70. The spray-dried extract of claim 69, wherein the extract is stored at 2°C to 8°C for at least 12 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

71. 70. The spray-dried extract of claim 69, wherein the extract is stored at 2°C to 8°C for at least 18 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

72. 69. The spray-dried extract of any one of claims 49 to 68, which is stored at about -20°C for at least 6 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

73. 73. The spray-dried extract of claim 72, wherein the extract is stored at about -20°C for at least 12 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

74. 73. The spray-dried extract of claim 72, wherein the extract is stored at about -20°C for at least 18 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

75. 69. The spray-dried extract of any one of claims 49 to 68, which is stored at about room temperature (20°C) for at least 6 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

76. 76. The spray-dried extract of claim 75, wherein the extract is stored at about room temperature (20°C) for at least 12 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

77. 77. The spray-dried extract of claim 75 or claim 76, which is stored at about room temperature (20°C) for at least 18 months and is capable of synthesizing the target protein at a titer of at least 80% compared to a control extract.

78. 75. The spray-dried extract of any one of claims 69 to 74, wherein the titer is determined by the Phytip® method.

79. 79. The spray-dried extract of any one of claims 69 to 78, wherein the control extract does not contain trehalose, lactose, leucine or raffinose.

80. 79. The spray-dried extract of any one of claims 69 to 78, wherein the control extract comprises trehalose, lactose, leucine or raffinose.

81. 75. The spray-dried extract of any one of claims 69 to 74, wherein the spray-dried extract and the control extract comprise trehalose, and wherein the spray-dried extract has been stored at 2°C to 8°C for at least 12 months and, after rehydration, is capable of synthesizing the target protein at a titer of at least 80% compared to the control extract rehydrated at time (T)=0.

82. 69. The spray-dried extract of any one of claims 49 to 68, wherein the protein synthesis activity of the rehydrated spray-dried extract declines by less than about 5% per month when the extract is stored at 2°C to 8°C before rehydration.

83. 69. The spray-dried extract of any one of claims 49 to 68, wherein the protein synthesis activity of the rehydrated spray-dried extract is equal to or greater than the protein synthesis activity of a rehydrated control spray-dried extract when the extract is stored at 2°C to 8°C for 8 months or more before rehydration.

84. 84. The spray-dried extract of claim 83, stored at 2°C to 8°C for up to 13 months before rehydration.

85. 85. The spray-dried extract of claim 83 or claim 84, wherein the control spray-dried extract does not contain trehalose.

86. 69. The spray-dried extract of any one of claims 49 to 68, which contains trehalose or lactose and is stored at 2°C to 8°C for at least 4 months and is capable of synthesizing the target protein at a titer of at least 75% compared to a control extract not containing trehalose or lactose and stored at -20°C.

87. 85. The spray-dried extract of claim 84, comprising about 75 g / kg to 105 g / kg trehalose or about 100 g / kg lactose.

88. 69. The spray-dried extract of any one of claims 49 to 68, wherein the rehydrated extract, when stored at 2°C to 8°C for at least 18 months prior to rehydration, has 80% or more of the initial protein synthesis activity when compared to the protein synthesis activity of the rehydrated extract at T=0.

89. 89. The spray-dried extract of any one of claims 49 to 88, wherein the spray-dried bacterial extract has an active oxidative phosphorylation system for cell-free protein synthesis.

90. 90. The spray-dried extract of any one of claims 49 to 89, derived from Escherichia sp.

91. 91. The spray-dried extract of any one of claims 49 to 90, which is a powder.

92. 91. The spray dried extract of any one of claims 49 to 90, which does not have a cakey appearance or is not a dry cake.

93. (i) providing a liquid bacterial extract containing components for cell-free synthesis of a target protein from a template nucleic acid encoding the target protein; (ii) generating droplets of said liquid bacterial extract; (iii) contacting the droplets with a gas to evaporate liquid from the droplets; (iv) separating the dried extract from the gases and smaller particles; and (v) collecting the spray-dried extract; A method for preparing a spray-dried extract, comprising:

94. 94. The method of claim 93, wherein prior to step (i), the liquid bacterial extract is sterile filtered.

95. 95. The method of claim 94, wherein the sterile-filtered liquid bacterial extract is activated by heat.

96. 96. The method of claim 95, wherein a composition comprising trehalose, lactose, leucine, or raffinose is added to the activated sterile-filtered liquid bacterial extract prior to step (ii).

97. 97. The method of claim 96, wherein the composition comprises about 25-200 g / kg trehalose, about 25-200 g / kg lactose, about 5-10 g / L leucine, or about 25-200 g / L raffinose.

98. 98. The method of any one of claims 95 to 97, wherein step (i) further comprises adding one or more amino acids to the activated sterile-filtered liquid bacterial extract.

99. The one or more amino acids may be selected from the group consisting of L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, L-proline, and any combination thereof. ｇ 99. The method of claim 98, wherein the amino acid sequence comprises a non-polar, uncharged amino acid.

100. 99. The method of claim 98, wherein the one or more amino acids are selected from leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine.

101. 101. The method of any one of claims 95 to 100, wherein one or more of maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), 2-hydroxypropyl-β-cyclodextrin, or any combination thereof, is added to the activated sterile-filtered liquid bacterial extract.

102. 102. A method according to any one of claims 93 to 101, wherein step (ii) comprises atomising the liquid bacterial extract to produce the droplets.

103. 103. The method of claim 102, wherein said atomizing comprises passing said liquid bacterial extract through an atomizing device selected from a nozzle or a rotary atomizer.

104. 104. The method of claim 102 or claim 103, wherein the median droplet size is about 20 to 100 microns (Dv50) at an atomizing gas pressure of 10 to 50 psig.

105. 105. A method according to any one of claims 93 to 104, wherein step (iii) comprises contacting the droplets with a drying gas through a drying chamber, the drying gas having an outlet temperature of from about 60°C to about 90°C.

106. 106. A method according to any one of claims 93 to 105, wherein step (iv) comprises separating the dried extract from the gas and smaller particles using centrifugal force.

107. 107. The method of any one of claims 93 to 106, wherein step (v) comprises collecting the spray dried extract in a container.

108. 108. The method of any one of claims 93 to 107, wherein the collected spray-dried extract comprises no more than about 15% (w / w), no more than about 10% (w / w), or no more than about 5% (w / w) residual water.

109. 108. The method of any one of claims 93 to 107, wherein at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w) of the liquid is removed from the spray dried extract.

110. 109. The method of any one of claims 93 to 108, wherein the protein synthesis activity of the rehydrated spray-dried extract declines by less than about 5% per month when the extract is stored at 2°C to 8°C before rehydration.

111. 111. The method of any one of claims 93 to 110, wherein the protein synthesis activity of the rehydrated spray-dried extract is equal to or greater than the protein synthesis activity of a rehydrated control extract when the extract is stored at 2°C to 8°C for 8 months or more before rehydration.

112. 112. The method of claim 111, wherein the spray-dried extract is stored at 2°C to 8°C for up to 13 months before rehydration.

113. 113. The method of claim 111 or claim 112, wherein the control spray-dried extract does not contain trehalose.

114. 109. The method of any one of claims 93 to 108, wherein the rehydrated extract has 80% or more of the initial protein synthesis activity when stored at 2°C to 8°C for up to 20 months prior to rehydration, when compared to the protein synthesis activity of the rehydrated extract at T=0.

115. 115. The method of any one of claims 93 to 114, wherein the spray-dried bacterial extract has an active oxidative phosphorylation system for cell-free protein synthesis.

116. 116. The method of any one of claims 93 to 115, wherein the liquid extract is derived from Escherichia sp.

117. A spray-dried extract for cell-free protein synthesis, the spray-dried extract comprising: dried lysed bacterial components; and one or more of the stabilizers, the stabilizer has a glass transition temperature (Tg) of at least about 90°C; The spray-dried extract, wherein the concentration of the stabilizer in the liquid extract before spray drying is about 5 g / L to 200 g / L, or about 25 g / kg to 200 g / kg.

118. 118. The spray-dried extract of claim 117, wherein the stabilizer is selected from trehalose, lactose, and leucine.

119. 119. The spray-dried extract of claim 118, wherein the stabilizer comprises about 25-200 g / kg trehalose, about 25-200 g / kg lactose, or about 5-10 g / L leucine.

120. 120. The spray-dried extract of claim 119, wherein the trehalose is trehalose dihydrate (TDH) and the lactose is lactose monohydrate (LMH).

121. 121. The spray dried extract of any one of claims 117 to 120, wherein at least 85% (w / w), at least 90% (w / w), or at least 95% (w / w) of the liquid is removed from the mixture.

122. One or more high glass transition temperatures (T ｇ 122. The spray-dried extract of any one of claims 117 to 121, further comprising a non-polar, uncharged amino acid.

123. 122. The spray dried extract of any one of claims 117 to 121, further comprising one or more, any combination, or any subset of amino acids selected from: leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, and arginine.

124. 124. The spray dried extract of any one of claims 117 to 123, further comprising maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 (Tween® 80), polyvinylpyrrolidone (PVP or Kollidon 12 PF), 2-hydroxypropyl-β-cyclodextrin, or any combination thereof.

125. Reconstituting the spray-dried extract of claims 117 to 124. providing a template nucleic acid encoding a target protein; and producing the target protein; 1. A method for producing a target protein from a spray-dried extract, comprising:

Images