Spray-dried extracts

Abstract

The present disclosure provides an improved method of stabilizing a spray-dried bacterial extract with a composition comprising a stabilizer, such that the extract can be used for cell-free protein synthesis. Also provided herein are formulations for a stable spray-dried bacterial extract having an increased protein synthesis activity compared to a spray-dried bacterial extract without a stabilizer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 396,143, filed on August 8, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Background of the Invention

[0004] The present disclosure provides compositions and methods for producing spray-dried bacterial extracts with enhanced stability after long-term storage at various temperatures. The spray-dried extracts can be used in commercial-scale cell-free protein synthesis reactions. Summary of the invention

[0005] Provided herein are compositions and methods for preparing spray-dried bacterial extracts for cell-free protein synthesis reactions. In one aspect, the method comprises: i) combining a bacterial extract comprising lysed bacterial components with a composition comprising trehalose, lactose, leucine or raffinose to produce a mixture, wherein the bacterial extract is capable of synthesizing the 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 the stable spray-dried bacterial extract.

[0006] In another aspect, the present disclosure provides a method for expressing a target protein in a cell-free protein synthesis reaction, the method comprising: i) combining a bacterial extract comprising lysed bacterial components with a composition comprising trehalose, lactose, leucine or raffinose to produce a mixture, wherein the lysed bacterial components can synthesize the 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 the stable spray-dried bacterial extract, iii) rehydrating the spray-dried bacterial extract; 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.

[0007] In another aspect, the present disclosure provides a method for expressing a target protein in a cell-free protein synthesis reaction, the method 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.

[0008] In some embodiments, the bacterial extract comprising lysed bacterial components is a liquid or rehydrated bacterial extract.

[0009] In some embodiments, the composition comprises trehalose or lactose. In some embodiments, the mixture comprises about 25 to 200 g / kg trehalose. In some embodiments, the mixture comprises about 50 to 100 g / kg trehalose. In some embodiments, the trehalose is trehalose dihydrate (TDH).

[0010] In some embodiments, the mixture comprises about 25 to 200 g / kg lactose. In some embodiments, the mixture comprises about 50 to 100 g / kg lactose. In some embodiments, the lactose is lactose monohydrate (LMH).

[0011] In some embodiments, the mixture comprises about 5 to 10 g / L leucine.In some embodiments, the mixture comprises about 25 to 200 g / L raffinose.

[0012] In some embodiments, the stable spray-dried bacterial extract comprises about 40 to 70 g / L of the bacterial extract solids. In some embodiments, the stable spray-dried bacterial extract comprises about 50 to 60 g / L of the bacterial extract solids.

[0013] In some embodiments, the bacterial extract is also combined with one or more of 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.

[0014] In some embodiments, the bacterial extract is further combined with one or more amino acids selected from the group consisting of leucine, glycine, alanine, valine, isoleucine, proline, tryptophan, serine, threonine, methionine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid, histidine, lysine, arginine, and any combination thereof.

[0015] In some embodiments, the mixture comprises 5 to 15 g / kg of amino acids.

[0016] In some embodiments, in step (i), the mixture further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 ( 80), polyvinylpyrrolidone (PVP or Kollidon 12PF) or 2-hydroxypropyl-β-cyclodextrin.

[0017] In some embodiments, the spray-dried bacterial extract comprises less than or equal to about 15% (w / w) residual water. In some embodiments, the spray-dried bacterial extract comprises less than or equal to about 10% (w / w) residual water. In some embodiments, the spray-dried bacterial extract comprises less than or equal to about 5% (w / w) residual water.

[0018] In some embodiments, after storage at 2°C to 8°C for at least 6 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract. In some embodiments, after storage at 2°C to 8°C for at least 12 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract. In some embodiments, after storage at 2°C to 8°C for at least 18 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract.

[0019] In some embodiments, after storage at about -20 ° C for at least 6 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract. In some embodiments, after storage at about -20 ° C for at least 12 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract. In some embodiments, after storage at about -20 ° C for at least 18 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract.

[0020] In some embodiments, after storage at about room temperature (20°C) for at least 6 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract. In some embodiments, after storage at about room temperature (20°C) for at least 12 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract. In some embodiments, after storage at about room temperature (20°C) for at least 18 months, the spray-dried bacterial extract is capable of synthesizing the target protein with a titer of at least 80% relative to the control extract.

[0021] In some embodiments, the titer is determined by Method determination.

[0022] In some embodiments, the spray-dried extract and the control extract comprise trehalose, and the spray-dried extract is capable of synthesizing the target protein with at least 80% of the titer of the control extract reconstituted at time zero after storage at 2° C. to 8° C. for at least 12 months. 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 decreases by less than about 5% per month compared to the control extract.

[0023] In some embodiments, the control extract does not comprise trehalose, lactose, leucine, and raffinose. In some embodiments, the control extract comprises trehalose, lactose, leucine, or raffinose.

[0024] In some embodiments, the spray-dried bacterial extract comprises an active oxidative phosphorylation system in cell-free protein synthesis. In some embodiments, the bacterial extract is from an Escherichia species.

[0025] 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.

[0026] In some embodiments, the spray drying in step (ii) comprises: atomizing the mixture to produce droplets; contacting the droplets with a gas to evaporate liquid from the droplets; separating the dried extract from the gas and smaller particles; and collecting the spray-dried extract.

[0027] In some embodiments, greater than or equal to 90% (w / w) of the liquid is removed from the mixture. In some embodiments, greater than or equal to 95% (w / w) of the liquid is removed from the mixture.

[0028] In some embodiments, the method further comprises (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 accounts for about 20% to 60% by volume of the cell-free protein synthesis reaction. In some embodiments, the rehydrated bacterial extract accounts for about 30% to 40% by volume of the cell-free protein synthesis reaction.

[0029] In another aspect, the present 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 is capable of synthesizing a target protein from a template nucleic acid encoding the target protein upon rehydration.

[0030] In some embodiments, the composition comprises trehalose or lactose. In some embodiments, the composition comprises about 25 to 200g / kg of trehalose. In some embodiments, the composition comprises about 50 to 100g / kg of trehalose. In some embodiments, the trehalose is trehalose dihydrate (TDH). In some embodiments, the composition comprises about 25 to 200g / kg of lactose. In some embodiments, the composition comprises about 50 to 100g / kg of lactose. In some embodiments, the lactose is lactose monohydrate (LMH). In some embodiments, the composition comprises about 5 to 10g / L of leucine.

[0031] In some embodiments, the composition comprises about 25 to 200 g / L raffinose.

[0032] 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 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 L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine, L-proline, and any combination thereof. In some embodiments, the extract comprises 5 to 15 g / kg of amino acids.

[0033] In some embodiments, the extract further comprises one or more amino acids, a combination of all amino acids, 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 comprises about 5 to 15 g / L of the amino acids.

[0034] In some embodiments, the extract further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 ( 80), polyvinylpyrrolidone (PVP or Kollidon 12PF) or 2-hydroxypropyl-β-cyclodextrin.

[0035] In some embodiments, the spray-dried extract comprises less than or equal to about 15% (w / w) residual water. In some embodiments, the spray-dried extract comprises less than or equal to about 10% (w / w) residual water. In some embodiments, the spray-dried extract comprises less than or equal to about 5% (w / w) residual water.

[0036] In some embodiments, the spray-dried extract is stored at 2°C to 8°C for at least 6 months and is capable of synthesizing a target protein having a titer of at least 80% relative to a control extract. In some embodiments, the spray-dried extract is stored at 2°C to 8°C for at least 12 months and is capable of synthesizing a target protein having a titer of at least 80% relative to a control extract. In some embodiments, the spray-dried extract is stored at 2°C to 8°C for at least 18 months and is capable of synthesizing a target protein having a titer of at least 80% relative to a control extract.

[0037] In some embodiments, the spray-dried extract is stored at about -20°C for at least 6 months and is capable of synthesizing the target protein with a titer of at least 80% relative to the 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 the target protein with a titer of at least 80% relative to the 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 the target protein with a titer of at least 80% relative to the control extract.

[0038] 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 the target protein with a titer of at least 80% relative to 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 the target protein with a titer of at least 80% relative to 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 the target protein with a titer of at least 80% relative to a control extract.

[0039] In some embodiments, the titer is determined by Methods for determination. In some embodiments, the control extract does not contain trehalose, lactose, leucine, and raffinose. In some embodiments, the control extract contains trehalose, lactose, leucine, or raffinose.

[0040] 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 is capable of synthesizing the target protein having at least 80% of the titer of the control extract rehydrated at time T=zero after rehydration.

[0041] In some embodiments, when the extract is stored at 2°C to 8°C before rehydration, the protein synthesis activity of the rehydrated spray-dried extract decreases by less than about 5% per month. In some embodiments, when the extract is stored at 2°C to 8°C for greater than or equal to 8 months before rehydration, the protein synthesis activity of the rehydrated spray-dried extract is greater than or equal to the protein synthesis activity of the rehydrated control spray-dried extract. 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.

[0042] In some embodiments, the spray-dried extract comprises trehalose or lactose, is stored at 2° C. to 8° C. for at least 4 months, and is capable of synthesizing the target protein with a titer of at least 75% relative to a control extract not comprising trehalose or lactose stored at −20° C. In some embodiments, the spray-dried extract comprises about 75 g / kg to 105 g / kg trehalose or about 100 g / kg lactose.

[0043] In some embodiments, the rehydrated extract stored at 2°C to 8°C for at least 18 months prior to rehydration has greater than or equal to 80% of the initial protein synthesis activity compared to the protein synthesis activity of the rehydrated extract at T=0.

[0044] In some embodiments, the spray-dried bacterial extract has an active oxidative phosphorylation system in cell-free protein synthesis. In some embodiments, the extract is from Escherichia species. In some embodiments, the extract is a powder. In some embodiments, the extract does not have a block appearance or is not a dry block.

[0045] In another aspect, the present disclosure provides a method for preparing a spray-dried extract, the method comprising the steps of: (i) providing a liquid bacterial extract comprising 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 liquid from the droplets; (iv) separating the dried extract from the gas and smaller particles; and (v) collecting the spray-dried extract.

[0046] 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 heating.

[0047] In some embodiments, prior to step (ii), a composition comprising trehalose, lactose, leucine or raffinose is added to the activated sterile filtered liquid bacterial extract. In some embodiments, the composition comprises about 25 to 200 g / kg trehalose, about 25 to 200 g / kg lactose, about 5 to 10 g / L leucine or about 25 to 200 g / L raffinose.

[0048] 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, and the high Tg non-polar uncharged amino acids are selected from 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.

[0049] In some embodiments, maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 ( 80), polyvinyl pyrrolidone (PVP or Kollidon 12PF), 2-hydroxypropyl-β-cyclodextrin or any combination thereof are added to the activated sterile filtered liquid bacterial extract.

[0050] In some embodiments, step (ii) comprises atomizing the liquid bacterial extract to produce the droplets. In some embodiments, the atomizing comprises passing the liquid bacterial extract through an atomizing device selected from a nozzle or a rotary atomizer. In some embodiments, the median droplet size (Dv50) is about 20 to 100 microns at an atomizing gas pressure of 10 to 50 psig.

[0051] In some embodiments, step (iii) comprises contacting the droplets with a drying gas through a drying chamber, wherein the drying gas has 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 container.

[0052] In some embodiments, the collected spray-dried extract comprises less than or equal to about 15%, 10% or 5% (w / w) residual water. In some embodiments, greater than or equal to 85%, 90% or 95% (w / w) of the liquid is removed from the spray-dried extract.

[0053] 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 prior to rehydration. In some embodiments, the protein synthesis activity of the rehydrated extract is greater than or equal to the protein synthesis activity of the rehydrated control spray-dried extract when the extract is stored at 2°C to 8°C for greater than or equal to 8 months prior to rehydration. In some embodiments, the spray-dried extract is stored at 2°C to 8°C for 13 months prior to rehydration.

[0054] In some embodiments, the spray-dried bacterial extract has an active oxidative phosphorylation system in cell-free protein synthesis. In some embodiments, the liquid extract is from an Escherichia species.

[0055] In another aspect, the present 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 wherein the concentration of the stabilizer in the liquid extract prior to spray drying is about 5 g / L to 200 g / L or about 25 g / kg to 200 g / kg.

[0056] In some embodiments, the stabilizer is selected from trehalose, lactose and leucine. In some embodiments, the stabilizer comprises about 25 to 200g / kg trehalose, about 25 to 200g / kg lactose or about 5 to 10g / L leucine. In some embodiments, the trehalose is trehalose dihydrate (TDH), and the lactose is lactose monohydrate (LMH). In some embodiments, the liquid greater than or equal to 85%, 90% or 95% (w / w) is removed from the mixture.

[0057] 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 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, a combination of all amino acids, or a subset of amino acids. In some embodiments, the extract further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 ( 80), polyvinylpyrrolidone (PVP or Kollidon 12PF), 2-hydroxypropyl-β-cyclodextrin or any combination thereof.

[0058] In another aspect, a method for producing a target protein from a spray-dried extract is provided, the method comprising: recombining the spray-dried extract of the present disclosure; providing a template nucleic acid encoding the target protein; and producing the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Data from bench-scale spray drying are shown. Drying was performed on a bench-scale Buchi B-290 spray dryer, with the corresponding inlet and outlet temperatures for the dried samples noted in parentheses as (inlet temperature / outlet temperature). The (XCF) test used 30% (by volume) of Batch reactions were performed to express the product anti-CD74 antibody. Source liquid and dried From batch DR 3c H4B5.

[0060] Figure 2A Data from a pilot scale spray dried formulation and drying process are shown. Samples formulated with 100 g / L, 50 g / L and 25 g / L trehalose were prepared and dried. Drying was performed on a pilot scale Mobile Minor PSD-1 spray dryer. XCF testing was performed using a 30% (by volume) Batch reaction in Micro-24 microbial reactor to express product anti-CD74 antibody. Source liquid and dry From batch ER 15-4.

[0061] Figure 2BData from a pilot scale spray dried formulation and drying process are shown. Samples formulated with 100 g / L, 50 g / L and 25 g / L trehalose were prepared and dried. Drying was performed on a pilot scale Mobile Minor PSD-1 spray dryer. XCF testing was performed using a 30% (by volume) Batch reaction in Micro-24 microbial reactor to express product anti-CD74 antibody. Source liquid and dry From batch ER 15-4.

[0062] Figure 3A , Figure 3B and Figure 3C The spray-dried trehalose dihydrate was prepared Long term stability data at 2 to 8°C, -20°C and room temperature. Samples were spray dried on a Mobile Minor (PSD-1) with 25 g / L, 50 g / L and 100 g / L amounts of trehalose dihydrate formulation and associated residual moisture levels (batch and % residual moisture noted in the legend). The rate of potency loss (% / month) was calculated from a linear fit of the data from 6 to 24.5 months. The potency of the antibody product anti-CD74 antibody was measured for the initial time point (t0), and the trastuzumab antibody was used at all other times in this study.

[0063] Figure 4A Data from a 100L demonstration run of a 100L spray drying study is shown. Drying was performed using a modified PSD-2. The XCF test used a batch reaction in a Micro-24 microbial reactor with 30% (by volume) extract to express the product trastuzumab. Source liquid and dried They are (1) Demonstration Run (DR) 1, extract batch ER11:ER17 80%:20%, (2) DR 2, extract batch ER17, (3) DR 3, extract batch ER 18.

[0064] Figure 4B Data from a demonstration run of a 100L spray drying study is shown. Drying was performed using a modified PSD-2. The XCF test used a batch reaction in a Micro-24 microbial reactor with 30% extract to express the product trastuzumab. Source liquid and dried It is (1) DR 1, ER11: ER17 80%: 20%, (2) DR 2, ER17, (3) DR 3, ER 18.

[0065] Figure 5ALong term stability data from a 100 L spray drying study at 2 to 8° C. are shown. The XCF test used a batch reaction in a Micro-24 microreactor with 30% (by volume) extract to express the product trastuzumab.

[0066] Figure 5B Long term stability data at -20°C from a 100L spray drying study is shown. The XCF test used a batch reaction in a Micro-24 microreactor with 30% (by volume) extract to express the product trastuzumab.

[0067] Figure 5C Long term stability data at room temperature from a 100 L spray drying study is shown. The XCF test used a batch reaction in a Micro-24 microreactor with 30% (by volume) extract to express the product trastuzumab.

[0068] Fig. 6A Long term stability data from a 100L spray drying study at -20°C, 2 to 8°C and room temperature are shown. The XCF test 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 preformed light chain (PFLC).

[0069] Figure 6B Long term stability data from a 100L spray drying study at -20°C, 2 to 8°C and room temperature are shown. XCF testing 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.

[0070] Fig. 7A and Figure 7B Showing Mobile from February 2020 Comparative data of different trehalose spray-dried formulations from the spray drying study. Fig. 7A Shown is a comparison of the initial activity of 100 g / L trehalose and 75 g / kg trehalose under the same drying process conditions (inlet T / outlet T; 168°C / 80°C). The XCF test used 37.5% (by volume) of the extract and expressed anti-folate receptor alpha antibodies with PFLC. Figure 7B Shown is a comparison of the initial activity of 100 g / L trehalose and 75 g / kg trehalose under the same drying process conditions (inlet T / outlet T; 168°C / 80°C). The XCF test used 37.5% (by volume) of the extract and expressed anti-CD74 antibody.

[0071] Fig. 8AShowing Mobile from February 2020 Spray Drying Studies Comparative data on different drying conditions for a lactose spray-dried formulation. Fig. 8A A comparison of the initial activity of 100 g / L lactose is shown for three different drying process conditions (inlet T / outlet T; 168°C / 80°C, 150°C / 70°C, 117°C / 60°C). The XCF test used 37.5% (by volume) extract and expressed anti-folate receptor alpha antibody with PFLC.

[0072] Figure 8B Showing Mobile from February 2020 Spray Drying Studies Comparative data on different drying conditions for a lactose spray-dried formulation. Fig. 9 B shows the comparison of the initial activity of 100 g / L 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 test used 37.5% (by volume) of the extract and expressed anti-CD74 antibody.

[0073] Fig. 9 The results show that the trehalose dihydrate formulation and lactose monohydrate as a single component Long term stability data at 2 to 8°C. Fig. 9 Mobile phase data from trehalose dihydrate formulations with 75 g / kg and 100 g / L amounts and lactose monohydrate at 100 g / kg and the associated residual moisture levels are shown. Data for samples spray dried on (PSD-1). Potency loss (% / month) was calculated from a linear fit of the data from 1 to 5 months. The potency of the trastuzumab antibody was measured in this study.

[0074] Fig.10 Activity data for PSD-3 are shown. XCF tests were all in discs with 30% extract expressing trastuzumab. DETAILED DESCRIPTION

[0075] The present disclosure provides compositions and methods for preparing spray-dried bacterial extracts for cell-free protein synthesis reactions. The spray-dried bacterial extracts include additives (e.g., one or more additives) that increase the stability of the extracts during long-term storage compared to spray-dried extracts that do not include additives. Compared to spray-dried extracts that do not contain additives, the spray-dried extracts provide the following unexpected advantages, i.e., when the spray-dried extracts are stored at different temperatures for relatively long periods of time, , still retain their ability to produce biomolecules (e.g., proteins). For example, the spray-dried extracts can unexpectedly be stored for 6 months to 18 months (or longer) at about -20°C (minus 20°C), at about 2°C to 8°C, or at about room temperature / ambient temperature (e.g., about 20°C), and still retain their 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 a few months, while liquid bacterial extracts stored at about 2°C to 8°C without additives typically lose about 25% to 50% of their activity after one day.

[0076] For long-term storage for commercial scale production, spray-dried bacterial extracts also offer the advantages of both reducing the volume of liquid extracts and increasing the stability of bacterial extracts.

[0077] I. Definitions

[0078] As used herein, the following terms have the meanings ascribed to them unless otherwise stated.

[0079] It should be understood that the present disclosure is not limited to the specific methods, experimental protocols, cell lines, animal species or genera, and reagents described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims.

[0080] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled 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 expressly indicated otherwise.

[0081] In the claims, the transitional term "comprising" is a term of art and is considered to be inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The term "consisting essentially of refers to the specified materials or steps and those materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter. The term "consists of" or "consisting of" excludes any element, step, or ingredient not specified in the claim.

[0082] As used herein, the term "about" when modifying any amount refers to the variation of the amount that is commonly encountered by those skilled in the art in, for example, protein synthesis experiments. For example, the term "about" refers to the normal variation encountered in the measurement of a given analytical technique within the same batch or sample and between different batches or samples. Therefore, the term about can include a variation of 1% to 10% of the measured value, such as a variation of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the measured value. The amount disclosed herein includes equivalents of those amounts, including amounts modified or not modified by the term "about".

[0083] The term "additive", also referred to as "stabilizer" or "excipient", refers to a compound or composition that is added to or combined with the liquid bacterial extract prior to spray drying.

[0084] The term "bacterial extract" refers to a bacterial cell lysate or a fragment thereof, wherein the cell extract can synthesize protein from a nucleic acid template. In other words, the bacterial extract contains an energy source, such as ATP, GTP, etc. The bacterial extract can be a part of a lysate, which is separated from other cellular components of the lysate by centrifugation, filtration, selective precipitation, selective immunoprecipitation, chromatography or other methods. The bacterial extract also includes a lysate or a fragment thereof containing an exogenous substance (such as a preservative, a stabilizer and a reagent for enhancing cell-free protein synthesis (CFPS)). The term "bacterial extract" can refer to a preparation of an in vitro reaction mixture that can transcribe DNA into mRNA and / or translate mRNA into polypeptide. The mixture can include ribosomes, an energy source (such as ATP, GTP, glucose, glutamic acid or pyruvic acid), amino acids and tRNA. The mixture can be directly derived from cracked bacteria, purified components or a combination of the two.

[0085] The term "the extract is capable of synthesizing a target protein from a template nucleic acid encoding the target protein in a cell-free protein synthesis" means that the lysed bacterial extract contains the essential bacterial components required for synthesizing the target protein in a cell-free protein synthesis reaction.

[0086] "Cell-free protein synthesis" or "CFPS" refers to the in vitro synthesis of nucleic acids, polypeptides, small molecules and / or viral particles in a reaction mixture containing biological extracts and / or defined reagents. The reaction mixture will contain templates for the production of macromolecules, such as DNA, mRNA, etc.; monomers of the macromolecules to be synthesized, such as amino acids, nucleotides, etc.; and cofactors, enzymes, and other reagents necessary for synthesis, such as ribosomes, unloaded tRNAs, tRNAs carrying natural and / or unnatural amino acids, polymerases, transcription factors, tRNA synthetases, etc.

[0087] The term "spray-dried bacterial extract" refers to a bacterial extract that has been spray-dried as described herein."Spray-dried bacterial extract" is different from "freeze-dried bacterial extract", which refers to a bacterial extract that has been subjected to freeze drying, lyophilization, in situ vaporization, microwave radiation sublimation, etc.

[0088] The term "stable spray-dried bacterial extract" refers to a spray-dried bacterial extract that substantially maintains its physical and chemical stability and integrity after storage, e.g., at -20°C, 2°C to 8°C, or at room temperature / ambient temperature (e.g., about 20°C) for 6 months or more. For example, a stable spray-dried bacterial extract refers to an extract that retains at least 75% of its initial capacity to synthesize a target protein when stored at -20°C, 2°C to 8°C, or at room temperature / ambient temperature (e.g., about 20°C) for 6 months or more.

[0089] The term "control bacterial extract" refers to a bacterial extract without a formulation additive (such as those described herein), or an extract containing additives described herein but tested at time T=0. Therefore, 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 additives described herein that is reconstituted before testing. The control extract can be a prepared spray-dried extract that includes additives described herein and is tested at T=0.

[0090] The term "lysed bacterial components" refers to cellular components of lysed bacteria. For example, the term may include bacterial components required for synthesizing a target protein from a template nucleic acid encoding a protein in a cell-free reaction, such as ribosomes, amino acids, polymerases, and tRNAs, and components of an active oxidative phosphorylation system. Additional components (such as exogenous ATP, GTP, glucose, glutamate, or pyruvate) may be added to the lysed bacterial components to provide an energy source.

[0091] The term "carbohydrate" refers to carbohydrates composed of carbon, hydrogen and oxygen atoms and having the empirical formula C m (H2O) n A macromolecule in which m and n can be different numbers. Carbohydrates include monosaccharides, disaccharides, oligosaccharides and polysaccharides.

[0092] In the context of a spray-dried bacterial extract, the term "rehydration" or "reconstitution" refers to suspending the spray-dried bacterial extract in a diluent (such as water) or a buffer to disperse the components of the bacterial extract.

[0093] The term "water content" refers to the amount of water contained in a material, and may be expressed as a relative amount in weight percent or volume percent.

[0094] The term "residual water" or "residual moisture" refers to the amount of water contained in a material after the material has been processed, such as spray dried, and includes residual water / residual moisture of about 1% to 15%.

[0095] The term "protein synthesis activity" refers to the protein yield (eg, the amount of protein) produced from a protein synthesis reaction to produce a target protein relative to a control protein synthesis reaction.

[0096] The term "lysate" is any cell-derived preparation containing components required by a protein synthesis system, wherein such cellular components are capable of expressing nucleic acids encoding the desired protein, wherein most of the biological components are present in concentrations resulting from cell lysis, rather than in concentrations that have been reconstituted. The lysate can be further modified so that the lysate is supplemented with additional cellular components, e.g., amino acids, nucleic acids, enzymes, etc. The lysate can also be modified so that additional cellular components are removed or degraded after lysis.

[0097] The terms "polypeptide", "peptide" or "protein" are used interchangeably herein to refer to polymers of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring 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 comprising L-amino acids, polymers comprising D-amino acids, or polymers comprising both L- and D-amino acids.

[0098] "Non-natural" or "non-native" amino acids refer to amino acids that are not one of the twenty naturally occurring amino acids that are the building blocks of all proteins, but can be bioengineered so that they are incorporated into proteins. Non-natural amino acids can include the D-peptide enantiomer or any post-translational modification of one of the twenty naturally occurring amino acids. A wide variety of non-natural amino acids can be used in the methods of the present disclosure. Non-natural amino acids can be selected based on the desired properties of the non-natural amino acids, for example, based on the function of the non-natural amino acids, such as modifying protein biological properties (such as toxicity, biodistribution or half-life), structural properties, spectral properties, chemical and / or photochemical properties, catalytic properties, the ability to react (covalently or non-covalently) with other molecules, etc. Unnatural amino acids that can be used in the methods of the present disclosure can include, but are not limited to, unnatural analogs of tyrosine amino acids; unnatural analogs of glutamine amino acids; unnatural analogs of phenylalanine amino acids; unnatural analogs of serine amino acids; unnatural analogs of threonine amino acids; alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, sulfhydryl, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocycle, 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; covalently linked to another molecule. or non-covalently interacting amino acids; metal-binding amino acids; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; amino acids containing biotin or biotin analogs; 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, for example, sugar-substituted serine, etc.; carbon-linked sugar-containing amino acids, for example, sugar-substituted serine, etc.; carbon-linked sugar-containing amino acids; redox-active amino acids; α-hydroxy-containing acids; aminothio groups containing amino acids; α,α-disubstituted amino acids; β-amino acids; cyclic amino acids other than proline, etc.

[0099] 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, a bacterial extract can use ATP synthase and the reduction of oxygen to produce ATP. It should be 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).

[0100] As used in this application, "increase" or "decrease" refers to a detectable positive or negative change compared to a comparative control, such as an established standard control (such as an extract not containing additives or stabilizers) in quantity. An increase is a positive change, which is typically at least 10% of the control value, or at least 20%, or 50%, or 100%, and can be as high as at least 2 times, or at least 5 times, or even 10 times the control value. For example, the term "increased stability", when used with respect to the bacterial extract described herein, refers to an extract with greater or more stability (i.e., greater or more protein synthesis activity) relative to a control extract when a given time period is stored at a given temperature. Similarly, a decrease is a negative change, which is typically at least 10% of the control value, or at least 20%, 30% or 50%, or even as high as at least 80% or 90% of 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 less stability (i.e., less protein synthesis activity) when stored at a given temperature for a given period of time, and is generally associated with an extract that does not contain an additive or stabilizer of the present disclosure. Other terms that indicate quantitative changes or differences compared to a comparison basis, such as "more," "less," "higher," and "lower," and terms that indicate actions that cause these changes or differences, such as "increase," "promote," "enhance," "reduce," "inhibit," and "suppress," are used in the same manner as described above in this application. In contrast, the term "substantially the same" or "substantially unchanged" means that there is little or no change in quantity compared to a standard control value, typically within ±10% of the standard control, or within ±5%, ±2%, or even less of the standard control.

[0101] The term gram per liter (g / L) refers to a unit of measurement for mass concentration that shows how many grams of a substance are present in one liter of a liquid mixture.

[0102] The term g / kg refers to the unit of mass fraction expressed as grams of a substance per kilogram of a mixture.

[0103] II. Detailed Description of Embodiments

[0104] 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 also appear in Binder et al., & Westhof (2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany, which describes detailed methods for RNA manipulation and analysis, and Walker, JM, (2009) The Protein Protocols Handbook, 3rd edition, Humana Press, New York, NY, which describes detailed methods for protein manipulation and analysis.

[0105] A. Cultivate bacteria

[0106] Bacterial cultivation is well known to those skilled in the art. Bacterial lysates derived from any bacterial strain can be used in the methods of the present disclosure. Suitable bacteria for use in cell-free synthetic systems include Gram-negative and Gram-positive bacteria, for example, Enterobacteriaceae such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescanns), and Shigella, as well as Bacilli (e.g., Bacilli subtilis and Bacillus licheniils), and Pseudomonas (e.g., Pseudomonas aeruginosa) and Steptomyces. In preferred embodiments, the bacteria used in the formulations and methods provided herein are from an Escherichia species, such as Escherichia coli or a derivative thereof.

[0107] The bacterial strain for the preparation of cell extract can have the nuclease and / or phosphatase activity of reduction, which increases the cell-free synthesis efficiency.For example, the bacterial strain for the preparation of cell-free extract can have sudden change in the gene of encoding nuclease RNase E and RNase A.Bacterial strain can also have sudden change with the component of stable cell synthesis reaction, and the sudden change is for example the disappearance in the gene such as tnaA, speA, sdaA or gshA, and it prevents the degraded of amino acid tryptophan, arginine, serine and cysteine ​​respectively in the cell-free synthesis reaction.In addition, bacterial strain can have sudden change with the protein product of stable cell-free synthesis, such as the knockout in protease ompT or lonP.

[0108] Bacterial cultures can be obtained as follows. The selected bacteria are grown overnight in any of a variety of growth media and under growth conditions well known in the art and easily optimized by the practitioner for the growth of a particular bacterium. Typically, isolated bacterial strains are grown in the culture medium until they reach a balanced exponential growth phase or stationary phase. This can be 10 6 Up to 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. The bacterial culture may be grown to an OD of595-600 In some embodiments, the bacteria are cultured at a growth rate of about 0.06 to about 0.6 to about 0.8 doublings per hour.

[0109] Bacterial cells can be grown in a medium containing glucose and phosphate, wherein glucose is present at a concentration of at least about 0.25% (weight / volume), more typically at least about 1%; and typically no more than about 4%, more typically no more than about 2%. An example of such a medium is 2YTPG medium, but it will be appreciated by those skilled in the art that many mediums may be suitable for this purpose, as there are many disclosed mediums using defined and undefined nutrient sources suitable for the growth of bacteria such as Escherichia coli (E. coli). For a particular species, optimal medium and growth conditions are known. For example, E. coli is typically grown in YT medium (yeast extract and tryptone) or variants thereof. The medium may be defined (synthetic) or composite (undefined).

[0110] Bacterial cells can be transfected or transformed with expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting for transformants, and preparing bacterial extracts, as described herein.

[0111] In some cases, bacteria are cultured under aerobic conditions to induce protein expression and then the culture is switched to anaerobic conditions, for example by bubbling nitrogen, argon, or the like, through the culture medium.

[0112] When large quantities of bacteria are needed, continuous culture means are used, rather than closed batch systems. These continuous systems involve the continuous introduction of nutrients and the removal of waste. Optimally, this allows cells to grow for extended periods of time at a constant biomass concentration. Two well-known systems are chemostat and turbidostat. In a chemostat system, sterile culture medium is fed at a constant rate, while culture medium containing bacteria is removed at the same rate. Turbidistat systems utilize photocells to measure absorbance or turbidity, and regulate the inflow of sterile culture medium and the outflow of bacteria according to a preset signal.

[0113] 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, JA and Jackson, RH (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).

[0114] In some cases, about 0.7 h -1 The engineered E. coli strain (e.g., engineered K-12-derived E. coli strain KGK10) was grown to mid-log phase (OD 595 The amount of glucose in the culture medium is about 45 OD or about 140 g / L of wet cell weight. Glucose can be increased during the culture so that there is excess glucose during the harvest. See, e.g., Zawada et al., Biotechnol. Bioeng., 108(7): 1570-1578 (2011).

[0115] B. Preparation of Bacterial Extracts

[0116] Once the bacterial culture is ready to be harvested, it can be cooled to 2°C to 8°C, typically on ice or by a heat exchanger when the culture is large-scale. The culture can be centrifuged to separate the spent culture medium from the cell paste (cytoplasm). Preferred centrifuges include disc centrifuges, tubular drum centrifuges, and other centrifuges for large-scale or small-scale bacterial culture. The cell paste is typically resuspended in S30 buffer, any equivalent buffer solution, or water. S30 buffer contains 10mM Tris acetate, 14mM magnesium acetate, and 60mM potassium acetate. In some embodiments, a dilution of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or more (liquid: solid; ml of buffer: cell gram weight) is used 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 washing step is typically performed. Upon washing, the cell paste (cell pellet) can be stored at -80°C for later use or further processed by homogenization to lyse the cells.

[0117] Cell extracts can be prepared from cultured bacteria, as described above. Cells that have been fermented overnight can be suspended in a suitable cell suspension buffer by suspending the cell mass and breaking the suspended cells by ultrasound, in a French press or with glass beads, continuous flow high pressure homogenization, or any other method known in the art for effective cell lysis. The cell lysate is then centrifuged or filtered to remove large cell debris, including DNA and cells that have not yet cracked.

[0118] In some embodiments, the bacterial culture is precipitated by centrifugation at about 8 to 20° C. for about 45 min twice at greater than 14,000 x g in a tubular drum centrifuge in continuous or batch mode, or precipitated in a disc-type continuous centrifuge at a maximum drum speed of about 12,000 rpm and a feed flow rate of about 3.0 L / min to 3.3 L / min. The pelleted cells are resuspended and pelleted repeatedly with S30 buffer. In some embodiments, the cells are stored at -80° C. for later use or processed by homogenization.

[0119] Prior to homogenization, the cell mass can be resuspended in S30 buffer or equivalent to produce a cell suspension. In some embodiments, a dilution of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or more is produced (liquid: solid; ml of buffer: gram weight of cells). Preferably, a 2:1 dilution is performed so that 2 ml of S30 buffer is used per gram weight of cell mass.

[0120] 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 produce a lysate. The homogenization step lyses the bacteria to release the essential components required for protein synthesis, and in some aspects, the inverted membrane vesicles formed provide energy for protein synthesis via respiration.

[0121] In some embodiments, the homogenizer pressure is about 3,000psi to 20,000psi. In some embodiments, the homogenizer pressure is set at about 20,000psi. In some embodiments, the speed (frequency setting) of the homogenizer is about 20Hz to about 60Hz, to produce a flow rate of about 340ml / min to 1.0L / min. Generally, the flow rate is proportional to the frequency setting, and can vary independently of the homogenization pressure. Preferably, the minimum speed of the homogenization step is set at about 20Hz, with a flow rate of about 340mL / min.

[0122] 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.

[0123] Next, the lysate can be clarified by centrifugation so that at least about 45% to about 85% or more, for example, about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of the cell solids are separated from the collected cell-free extract. In some embodiments, at least about 70%, 75%, 80%, 85%, 90% or 95% of the cell solids are separated by centrifugation. In some embodiments, centrifugation is performed by a continuous centrifuge, for example, a disc centrifuge, a tubular drum centrifuge or an appropriate centrifuge. In some embodiments, a 200L fermentation produces greater than 1.1L clarified extract / kg cell wet weight, and a total protein concentration of about 20g / L to 25g / L.

[0124] The extract may be filtered through one or more sterilizing grade filters (eg, a 0.45 μm filter and / or a 0.22 μm filter). A 0.45 μm filter may be used first, followed by a 0.22 μm filter.

[0125] In some embodiments, the filtered extract is activated or preincubated at 30° C. for about 2 to 5 hours, preferably about 2.5 hours. After preincubation, the particles can be separated from the extract by centrifugation (eg, spinning at at least 14,000×g for about 35 minutes).

[0126] The lysed bacterial extract can be aliquoted and 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 prior to freezing.

[0127] 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, JA and Jackson, RH (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).

[0128] C. Activation of bacterial extracts

[0129] The lysed bacterial extract prepared as 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 before activation (heat treatment). The 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).

[0130] After activation, the liquid bacterial extract is formulated by adding one or more additives as described below.

[0131] D. Preparation of Pre-Spray-Dried Bacterial Extracts

[0132] The present disclosure is based in part on the unexpected results of maintaining the protein synthesis activity of the extract in a cell-free protein synthesis reaction when a specific additive, excipient or stabilizer is added to the bacterial extract before spray drying. Without being bound by theory, additives can prevent protein denaturation during spray drying and / or long-term storage. In some embodiments, a specific formulation of a stable spray-dried bacterial extract described herein can be stored at -20°C, 2°C to 8°C, or room temperature for at least 8 months and has at least about 60% protein synthesis activity compared to a control extract with or without additives, excipients or stabilizers. The experiments described in the Examples (see below) show that the formulations with the additives described herein have long-term storage stability.

[0133] In some embodiments, the formulation comprises a single additive that is added to the bacterial extract prior to spray drying. Formulations comprising a single additive are referred to as "single component" spray-dried extracts.

[0134] In some embodiments, the formulation comprises a carbohydrate additive. In some embodiments, the carbohydrate additive is selected from trehalose, lactose, raffinose, maltodextrin or cyclodextrin, or a combination thereof.

[0135] 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 trehalose, for example, about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / L trehalose. In some embodiments, the formulation comprises about 50 g / L to about 100 g / L trehalose. In some embodiments, the formulation comprises about 25 g / L to about 75 g / L trehalose. In some embodiments, the formulation comprises about 75 g / L to about 125 g / L trehalose. In some embodiments, the formulation comprises about 50 g / L trehalose. In some embodiments, the preparation comprises about 75g / L trehalose. In some embodiments, the preparation comprises about 100g / L trehalose. In some embodiments, the preparation comprises about 125g / L trehalose. In some embodiments, the preparation comprises about 25g / kg to about 200g / kg trehalose, for example, about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200g / kg trehalose. In some embodiments, the preparation comprises about 50g / kg to about 110g / kg trehalose. In some embodiments, the preparation comprises about 25g / kg to about 75g / kg trehalose. In some embodiments, the preparation comprises about 75g / kg to about 125g / kg trehalose. In some embodiments, the formulation comprises about 50 g / kg trehalose. In some embodiments, the formulation comprises about 75 g / kg trehalose. In some embodiments, the formulation comprises about 100 g / kg trehalose. In some embodiments, the formulation comprises about 125 g / kg trehalose.

[0136] 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 lactose, for example, about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / L lactose. In some embodiments, the formulation comprises about 50 g / L to about 100 g / L lactose. In some embodiments, the formulation comprises about 100 g / L lactose. In some embodiments, the formulation comprises about 25 to about 200 g / kg lactose, for example, about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / kg lactose. In some embodiments, the formulation comprises about 50 g / L to about 100 g / kg lactose. In some embodiments, the formulation comprises about 100 g / kg lactose.

[0137] In some embodiments, the additive comprises raffinose. In some embodiments, the formulation comprises about 25 to 200 g / L raffinose, for example, about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / L raffinose. In some embodiments, the formulation comprises about 25 to 200 g / kg raffinose, e.g., about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 g / kg raffinose.

[0138] In some embodiments, the additive comprises maltodextrin. In some embodiments, the formulation comprises about 100 g / L maltodextrin.

[0139] In some embodiments, the additive comprises cyclodextrin. Cyclodextrin helps to improve the water solubility and stability of hydrophobic compounds. In some embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, or a combination thereof. In some embodiments, the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin (HP-β-CD; a cyclic oligosaccharide containing 7 D-(+)-pyranose units. In some embodiments, the formulation comprises about 5g / kg to about 50g / kg cyclodextrin (e.g., about 5g / kg to about 50g / kg HP-β-CD).

[0140] In some embodiments, the formulation comprises an additive selected from sugar alcohols. In some embodiments, the sugar alcohol is selected from mannitol or sorbitol, or a combination thereof. In some embodiments, the formulation comprises about 50 to about 100 g / L mannitol. In some embodiments, the formulation comprises about 15 g / L sorbitol.

[0141] In some embodiments, the formulation comprises an additive selected from an amino acid. In some embodiments, the amino acid is leucine. In some embodiments, the formulation comprises about 5 g / L to about 15 g / L leucine, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 g / L leucine.

[0142] 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 with a molecular weight less than 20,000 g / mol), a polysorbate (e.g., polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80 (also known as 80), or polyvinyl pyrrolidone (PVP or Kollidon 12PF), or a combination thereof.

[0143] By combining concentrated stock solutions of one or more additives, additives can be mixed with liquid lysed bacterial extracts to achieve preferred formulations of spray-dried extracts. For example, the liquid extract containing the additive can have a total dry weight of additives per volume of lysed bacterial extract of about 5 g / L to 150 g / L, such as about 5 g / L, 10 g / L, 15 g / L, 20 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, 105 g / L, 110 g / L, 115 g / L, 120 g / L, 125 g / L, 130 g / L, 135 g / L, 140 g / L, 145 g / L or 150 g / L.

[0144] In some embodiments, the formulated bacterial extract does not comprise a composition, additive, or stabilizer selected from sucrose, mannitol, sorbitol, dextran, or a combination thereof. In some embodiments, the formulated bacterial extract does not comprise sucrose.

[0145] E. Combination with other additives

[0146] The bacterial extract formulations described herein may include other compounds, such as additives, excipients, stabilizers, chemicals, molecules or reagents, that are added to the lysed bacterial extract prior to spray drying. Thus, the bacterial extract may contain a single additive described herein, or a combination of a single additive and 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 ( 80)), polyvinyl pyrrolidone (PVP or Kollidon 12PF), leucine, amino acid mixtures and / or combinations thereof.

[0147] Table 1 provides representative examples of single component formulations and combination formulations tested for improved long term stability as described in the Examples.

[0148] Table 1: Representative single component formulations and combination formulations.

[0149]

[0150]

[0151]

[0152] The difference between g / L and g / kg is the density, so that 100 g / L TDH is equivalent to about 104 g / kg, and 75 g / kg TDH is equivalent to about 73 g / L.

[0153] In some embodiments, the bacterial extract preparation includes one or more, all combinations or subsets of natural 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 "PMA amino acid mixture"). Representative PMA amino acid mixtures are shown in Table 2.

[0154] Table 2: Representative PMA amino acid mixes.

[0155]

[0156]

[0157] In some embodiments, the amino acids (tyrosine and / or phenylalanine) are added to the formulation separately from the PMA mixed amino acids.

[0158] F. High glass transition components

[0159] In some embodiments, the bacterial extract preparation comprises one or more components with a high glass transition temperature (Tg). In some embodiments, the bacterial extract preparation comprises one or more components with a Tg of about 70°C or higher. For example, the bacterial extract preparation comprises one or more components with a Tg of about 80°C, 90°C, or 100°C or higher. In some embodiments, the bacterial extract preparation comprises trehalose. In some embodiments, the bacterial extract preparation comprises lactose. In some embodiments, the bacterial extract preparation comprises raffinose.

[0160] The Tgs of the various components tested in the formulations of the present disclosure are shown in the table below.

[0161]

[0162]

[0163] *Note that for some components there are multiple sources reporting slightly different values.

[0164] In some embodiments, the bacterial extract formulation comprises one or more, or a combination or subcombination of high Tg non-polar, uncharged amino acids. Table 3 shows representative combinations of high Tg non-polar, uncharged amino acids used in some formulations (referred to herein as "amino acid selection mixtures").

[0165] Table 3: Representative high T g Non-polar, uncharged amino acids (Amino Acid Selection Mix).

[0166]

[0167] 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 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).

[0168] 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 g / L to about 100 g / L mannitol. In some embodiments, the bacterial extract comprises about 15 g / L sorbitol.

[0169] In some embodiments, the bacterial extract comprises trehalose and an amino acid selected from (i) leucine; (ii) one or more, all combinations, or a subset of the PMA amino acid mixtures in Table 2; or (iii) one or more, all combinations, or a subset of the amino acid selection mixtures in Table 3. In some embodiments, the bacterial extract comprises trehalose and about 5 g / L to 10 g / L leucine.

[0170] 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 PEG with a molecular weight less than 20,000 g / mol), polysorbate (e.g., polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80 (also known as 80), or polyvinyl pyrrolidone (PVP or Kollidon 12PF), or a combination thereof.

[0171] In any embodiment described herein, 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, for example, about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / L. In some embodiments, the bacterial extract comprises about 50 g / L to about 100 g / L trehalose or TDH. In some embodiments, the bacterial extract comprises about 100 g / L trehalose or TDH. In some embodiments, the bacterial extract comprises about 25 g / kg to about 200 g / kg trehalose, for example, about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / kg trehalose. In some embodiments, the bacterial extract comprises about 50 g / kg to about 110 g / kg trehalose or TDH. In some embodiments, the bacterial extract comprises about 75 g / kg trehalose or TDH.

[0172] 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 concentration of trehalose present in the bacterial extract is about 25g / kg to about 200g / kg; the concentration of raffinose present in the bacterial extract is about 25g / L to 200g / L; the concentration of maltodextrin present in the bacterial extract is about 100g / L; and the concentration of cyclodextrin present in the bacterial extract is about 5g / kg to about 50g / kg (e.g., about 5g / kg to about 50g / kg HP-β-CD).

[0173] 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 g / L to about 100 g / L mannitol. In some embodiments, the bacterial extract comprises about 15 g / L sorbitol.

[0174] In some embodiments, the bacterial extract comprises lactose and an amino acid selected from (i) leucine; (ii) one or more, all combinations, or a subset of the PMA amino acid mixtures in Table 2; or (iii) one or more, all combinations, or a subset of the amino acid selection mixtures in Table 3. In some embodiments, the bacterial extract comprises lactose and about 5 g / L to 10 g / L leucine.

[0175] 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 with a molecular weight less than 20,000 g / mol), a polysorbate (e.g., polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80 (also known as 80), or polyvinyl pyrrolidone (PVP or Kollidon 12PF), or a combination thereof.

[0176] In any embodiment described herein, lactose can be lactose monohydrate (LMH). In some embodiments, the bacterial extract comprises about 25 to about 200 g / L lactose or LMH, for example, about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / L lactose or LMH. In some embodiments, the bacterial extract comprises about 100 g / L lactose or LMH. In some embodiments, the bacterial extract comprises about 25 to about 200 g / kg lactose, such as about 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / kg lactose or LMH. In some embodiments, the bacterial extract comprises about 100 g / kg lactose or LMH.

[0177] 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.

[0178] In some embodiments, the bacterial extract comprises leucine and a sugar alcohol selected from mannitol or sorbitol, or a combination thereof. In some embodiments, the bacterial extract comprises about 50 g / L to about 100 g / L mannitol. In some embodiments, the bacterial extract comprises about 15 g / L sorbitol.

[0179] In some embodiments, the bacterial extract comprises leucine and one or more, all combinations, or a subset of (i) the PMA amino acid mixture in Table 2; or (ii) one or more, all combinations, or a subset of the amino acid selection mixture in Table 3.

[0180] 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 with a molecular weight less than 20,000 g / mol), a polysorbate (e.g., polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80 (also known as 80), or polyvinyl pyrrolidone (PVP or Kollidon 12PF), or a combination thereof.

[0181] 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.

[0182] 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 g / L to about 100 g / L mannitol. In some embodiments, the bacterial extract comprises about 15 g / L sorbitol.

[0183] In some embodiments, the bacterial extract comprises raffinose and one or more, all combinations, or a subset selected from (i) the PMA amino acid mixture in Table 2; or (ii) one or more, all combinations, or a subset of the amino acid selection mixtures in Table 3. In some embodiments, the bacterial extract comprises raffinose and about 5 g / L to 10 g / L leucine.

[0184] 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 with a molecular weight less than 20,000 g / mol), a polysorbate (e.g., polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80 (also known as 80), or polyvinyl pyrrolidone (PVP or Kollidon 12PF), or a combination thereof.

[0185] In some embodiments, the bacterial extract formulated prior to spray drying does not include a composition, additive, or stabilizer selected from sucrose, mannitol, sorbitol, dextran, or a combination thereof. In some embodiments, the bacterial extract formulated prior to spray drying does not include sucrose.

[0186] G. Method for producing stable spray-dried bacterial extracts

[0187] The bacterial extract comprising the lysed bacterial components and one or more additives or stabilizer compositions described herein can then be spray dried. In some embodiments, the bacterial extract is a liquid (rehydrated or reconstituted) bacterial extract comprising lysed bacterial components, which is used for cell-free synthesis of a target protein from a template nucleic acid encoding the target protein.

[0188] In some embodiments, the method comprises: atomizing a liquid bacterial composition to generate droplets; contacting the droplets with a gas to evaporate the liquid from the droplets; separating a dried extract from the gas and smaller particles; and collecting the spray-dried extract.

[0189] In some embodiments, the present disclosure provides a method for preparing a spray-dried bacterial extract, comprising: providing a liquid bacterial extract comprising 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.

[0190] In some embodiments, the liquid bacterial extract comprising the cracked bacterial component and one or more additive compositions is atomized to produce droplets by passing the liquid bacterial extract through an atomizing device. In some embodiments, the atomizing device is a tip, a nozzle or a rotary atomizer. In some embodiments, the tip or nozzle has an opening suitable for atomizing the liquid bacterial extract, which is determined by commercially appropriate means based on the type of drying equipment used and other factors. In some embodiments, two fluid nozzle systems are used to produce droplets of the extract. The two fluid nozzle systems may include a first nozzle providing the liquid bacterial extract and a second nozzle providing pressurized gas, and when the liquid bacterial extract leaves the outlet of the first nozzle, the pressurized gas contacts the liquid bacterial extract, thereby producing droplets.

[0191] It will be appreciated by those of ordinary skill in the art that the droplet size produced by the atomizing device depends on several factors, including atomizing gas pressure, required liquid feed rate, design of the nozzle or tip, and the drying equipment used. Drying gas flow rate and the inlet and outlet temperatures of the drying gas may affect the droplet size. These parameters can be adjusted based on the required droplet size and residual moisture in the spray-dried extract. In some embodiments, the required droplet size (Dv50) is about 20 microns to 100 microns. In some embodiments, the atomizing gas pressure is about 10psig to 50psig. In some embodiments, the outlet temperature of the drying gas is about 60°C to about 90°C. In some embodiments, the outlet temperature of the drying gas is about 65°C to about 80°C. For example, the outlet temperature of the drying gas can be about 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C or 80°C. In some embodiments, the outlet temperature of the drying gas is from about 65°C to about 76°C.

[0192] It will be appreciated by those skilled in the art that because the activity of the extract is temperature sensitive, the temperature used to dry the extract may vary based on the size (volume) of the dryer used. For example, in larger industrial-scale dryers, the temperature may need to be lowered due to the longer residence time of the extract droplets in the larger dryer compared to a dryer of smaller volume. It will be appreciated by those skilled in the art that there are a variety of ways to reduce heat exposure in large dryers by special equipment design to shorten the holding time and / or cool the powder particles faster once they are dried. Representative laboratory-scale dryers include the Buchi B-290 spray dryer. Representative pilot-scale dryers include the Mobile Minor PSD-1 spray dryer. Representative industrial-scale dryers include improved PSD-2 dryers and PSD3 dryers, as well as IFC-Welko spray dryers.

[0193] In some embodiments, the gas is dehumidified air. In some embodiments, the gas is nitrogen.

[0194] Centrifugal force can then be used to separate the spray-dried extract particles / droplets produced in the above contacting step (ii) from the gas and any smaller particles. In some embodiments, a cyclone separator is used to separate the spray-dried extract particles / droplets from the gas and smaller particles. In some embodiments, the smaller particles are about 1 micron to 10 microns and leave the cyclone separator in a separate stream from the desired spray-dried extract particles / droplets.

[0195] 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.

[0196] As described above, the liquid bacterial extract may be sterile filtered and activated by heating prior to spray drying. In some embodiments, the liquid bacterial extract is activated by heating the extract to about 20°C to 45°C for about 30 minutes to about 10 hours.

[0197] In some embodiments, prior to spray drying (e.g., prior to the step of generating droplets of the liquid bacterial extract), an additive or stabilizer composition comprising trehalose, lactose, leucine or raffinose is added to the activated sterile filtered liquid bacterial extract. In some embodiments, prior to spray drying, about 25 to 200 g / kg trehalose, about 25 to 200 g / kg (or about 25 to 200 g / L) lactose, about 5 to 10 g / L leucine and / or about 25 to 200 g / L raffinose are added to the activated sterile filtered liquid bacterial extract.

[0198] In some embodiments, before spray drying, one or more amino acids or a combination of amino acids are added to the activated sterile filtered liquid bacterial extract. In some embodiments, one or more amino acids include high glass transition temperature (Tg) non-polar uncharged amino acids, and the high glass transition temperature (Tg) non-polar uncharged amino acids are selected from L-valine, L-tryptophan, L-isoleucine, L-leucine, L-alanine, glycine and L-proline and combinations thereof. In some embodiments, one or more amino acids or a combination of 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 / or arginine.

[0199] In some embodiments, prior to spray drying, maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 ( 80), polyvinylpyrrolidone (PVP or Kollidon 12PF) and / or 2-hydroxypropyl-β-cyclodextrin or a combination thereof are added to the activated sterile filtered liquid bacterial extract.

[0200] In some embodiments, greater than or equal to about 85% to 95% (w / w) (e.g., greater than or equal to about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% w / w) of liquid is removed from the bacterial extract after spray drying. In some embodiments, the spray-dried extract comprises less than or equal to about 15% (w / w) (e.g., less than or equal to about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / w) of residual water.

[0201] H. Residual water content

[0202] Residual moisture or the amount of residual water can affect the protein synthesis activity and / or stability of the spray-dried extract preparation. In some embodiments, the spray-dried extract comprises less than about 15% residual moisture by weight, for example, less than or equal to about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% (w / w) residual moisture.

[0203] Methods for determining the water content of dry extracts include proton nuclear magnetic resonance (NMR) spectroscopy and Karl Fischer coulometric titration.

[0204] NMR spectroscopy is based on the fact that hydrogen protons have a magnetic moment and an angular momentum. When hydrogen atoms are excited by an alternating field from a transmitter in the presence of the Earth's static magnetic field, a magnetic field is generated. A relaxation field is generated by the protons excited by the excitation field. The amplitude of the relaxation field measured after switching off the excitation is directly related to the number of protons that have been excited and therefore to the water content. Time domain nuclear magnetic resonance (TD-NMR) spectroscopy and "spin trajectory" NMR spectroscopy are variants of this technique that have been applied to biological cultures and protein solutions.

[0205] For the purposes of the disclosure provided herein, one method for measuring the percentage of water in a dry extract is Karl Fischer coulometric titration.

[0206] 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 a solvent and an organic base (such as pyridine) as a buffer. Using imidazole or primary amine instead of base can be used for pyridine-free systems. For protein or sugar solutions, a 2:1 methanol: formamide mixture can be used as a solvent. Two variants of this method, volumetric titration and coulometric titration, utilize different iodine sources. In the volumetric titration, the iodine required for the reaction is dissolved in advance, and the water content is determined by measuring the amount of iodine consumed due to the reaction with water in the sample. Automatic volumetric titration systems are commercially available. In the coulometric titration, iodine is first generated by electrolysis of a reagent containing iodine ions, and then based on the quantitative reaction of the generated iodine with water, the water content is determined by measuring the amount of electricity (coulomb) required for electrolysis in the generation of iodine [=current (ampere)×time (seconds)].

[0207] The Karl Fischer titration method can be performed using a drying oven (e.g., Model D03080, Mettler Toledo, Columbus, OH) directly interconnected with a Karl Fischer coulometric titrator (e.g., Model C20 from Mettler Toledo). Typically, the set point of the oven 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 to 200 mL / min is run through the oven to facilitate the transfer of water vapor from the oven to the titration vessel. The time or mixing time between the introduction of the sample into the oven and the start of the titration is set to 120 seconds to allow the water in each sample to be completely transferred to the titration vessel. The iodine used for titration is generated electrochemically based on the drift observed by the instrument. The initial drift standard is about less than about 25 μg / min. The drift standard that should be reached at the end of the measurement is less than about 3.0 μg / min, wherein the maximum titration time is about 3600 seconds. A voltammetric sensor (eg, model DM143-SC) with a polarization current of 5.0 μA is used for detection. Each sample can be run in triplicate to capture variability in the measurements.

[0208] I. Spray-dried bacterial extracts

[0209] The spray-dried bacterial extract described herein can be used for cell-free protein synthesis reaction. In some embodiments, the spray-dried bacterial extract comprises a dry cracked bacterial component. In some embodiments, the spray-dried extract comprises a component for synthesizing a target protein from a template nucleic acid encoding a target protein. In some embodiments, the spray-dried bacterial extract has an active oxidative phosphorylation system in cell-free protein synthesis. The additional components present in the spray-dried extract that can be used for cell-free protein synthesis are described below.

[0210] 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, before spray drying, the liquid bacterial extract comprises about 25g / kg to 200g / kg trehalose, about 25g / kg to 200g / kg (or about 25g / L to 200g / L) lactose, about 5g / L to 10g / L leucine and / or about 25g / L to 200g / L raffinose. In some embodiments, trehalose is trehalose dihydrate (TDH), and lactose is lactose monohydrate (LMH).

[0211] In some embodiments, greater than or equal to about 85% to 95% (w / w) (e.g., greater than or equal to about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% w / w) of liquid is removed from the spray-dried bacterial extract. In some embodiments, the spray-dried bacterial extract comprises less than or equal to about 15% (w / w) (e.g., less than or equal to about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / w) of residual water.

[0212] In some embodiments, the spray-dried bacterial extract further comprises one or more or combinations thereof of non-polar, uncharged amino acids with high glass transition temperatures (Tg) 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 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, a combination of all amino acids or a subset of amino acids. In some embodiments, the spray-dried bacterial extract comprises about 5 g / L to 15 g / L (or about 5 g / kg to 15 g / kg) of amino acids.

[0213] In some embodiments, the spray-dried bacterial extract further comprises maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 ( 80), polyvinylpyrrolidone (PVP or Kollidon 12PF) or 2-hydroxypropyl-β-cyclodextrin.

[0214] J. Stability of spray-dried bacterial extracts

[0215] Compared with the extract not containing additives described herein or stabilizer, the spray-dried extract of the present disclosure has increased stability when stored for a long period of time (for example, 6 months to 18 months or longer). In order to measure stability, the spray-dried extract can be reorganized by adding liquid to the spray-dried extract powder, and the reorganized extract is used for cell-free protein synthesis reaction. In some embodiments, the spray-dried extract is reorganized (also referred to as rehydration) by combining the spray-dried extract with a liquid (such as buffer or water). In some embodiments, each (1) gram of dry extract powder adds 5 to 10 grams of liquid (for example, every gram of spray-dried extract powder adds 5,6,7,8,9 or 10 grams of liquid). In some embodiments, every 1g of spray-dried extract adds 5g of water. In some embodiments, every 1g of spray-dried extract adds 6g of water. In some embodiments, every 1g of spray-dried extract adds 7g of water. In some embodiments, every 1g of spray-dried extract adds 8g of water. In some embodiments, the reconstituted spray-dried extract is stored on ice or at about 0°C to 8°C prior to use in a CFPS reaction.

[0216] In some embodiments, the reconstituted or rehydrated spray-dried extract comprises about 20% to 60% (e.g., about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%) by volume of the cell-free protein synthesis reaction.

[0217] The stability of the spray-dried extract can be determined based on the yield (also expressed as titer) of the target protein produced by the cell-free protein synthesis reaction containing the spray-dried extract. In some embodiments, the yield of the target protein is determined as described below. In some embodiments, the yield of the target protein is determined by passing the cell-free protein synthesis reaction mixture through a protein A resin or column. Protein A binds to a protein such as an antibody, and the bound target protein can then be eluted from the protein A column. In some embodiments, protein A column Used to purify and quantify the amount of target protein produced during cell-free protein synthesis reactions.

[0218] In some embodiments, the spray-dried extract is stored at about -20°C for at least 3, 6, 12, 18, 24, 30, 36 months, or more than 36 months prior to rehydration. In some embodiments, the spray-dried extract is stored at about -20°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months, or more than 36 months prior to rehydration. In some embodiments, the rehydrated spray-dried extract stored at about -20°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more prior to rehydration is capable of synthesizing the target protein at a yield of at least 80% relative to a control extract. In some embodiments, the rehydrated spray-dried extract stored at about -20°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more prior to rehydration is capable of synthesizing the target protein at a yield of at least 85% relative to the control extract. In some embodiments, the rehydrated spray-dried extract stored at about -20°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more prior to rehydration is capable of synthesizing the target protein at a yield of at least 90% relative to a control extract.

[0219] In some embodiments, the spray-dried extract is stored at about 2°C to 8°C for at least 3, 6, 12, 18, 24, 30, 36 months, or greater than 36 months prior to rehydration.

[0220] In some embodiments, the spray-dried extract is stored at about 2°C to 8°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months, or greater than 36 months prior to rehydration. In some embodiments, the rehydrated spray-dried extract stored at about 2°C to 8°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more prior to rehydration is capable of synthesizing the target protein at a yield of at least 80% relative to the control extract. In some embodiments, the rehydrated spray-dried extract stored at about 2°C to 8°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more prior to rehydration is capable of synthesizing the target protein at a yield of at least 85% relative to the control extract. In some embodiments, the rehydrated spray-dried extract stored at about 2°C to 8°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more prior to rehydration is capable of synthesizing the target protein at a yield of at least 90% relative to a control extract.

[0221] In some embodiments, the spray-dried extract is stored at about room temperature (RT) (e.g., about 20° C.) for at least 3, 6, 12, 18, 24, 30, 36 months, or more than 36 months prior to rehydration. In some embodiments, the spray-dried extract is stored at about 20° C. for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months, or more than 36 months prior to rehydration. In some embodiments, the rehydrated spray-dried extract stored at about 20°C for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or more prior to rehydration is capable of synthesizing the target protein at a yield of at least 60%, 65%, 70%, 75%, 80%, 85% or 90% relative to the control extract.

[0222] In some embodiments, the control extract is a recombinant unformulated extract, that is, an extract not comprising additives or stabilizers described herein. In some embodiments, the control extract is a frozen unformulated extract (stored at about -20 ° C to -80 ° C) not comprising additives or stabilizers described herein. In some cases, the frozen unformulated control extract is a previously undried frozen liquid extract. 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 include additives or stabilizers selected from trehalose, lactose, leucine and raffinose.

[0223] In some embodiments, the control extract is a recombinant formulated spray-dried extract comprising an additive or stabilizer described herein. In some embodiments, the control extract is a recombinant formulated spray-dried extract comprising an additive or stabilizer selected from trehalose, lactose, leucine and raffinose.

[0224] 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 greater than or equal to 3 to 18 months (e.g., greater than or equal to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months). After reconstitution, the spray-dried bacterial extract is capable of synthesizing the target protein at a titer of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% relative to the control extract reconstituted at time zero. In some embodiments, the spray-dried bacteria and control extracts comprise about 100 g / L (about 105 g / kg) trehalose before rehydration, and the control extract is reconstituted at time T=0.

[0225] In some embodiments, the spray-dried extract and the control extract comprise about 100 g / L (about 105 g / kg) trehalose, the spray-dried extract is stored at about -20°C for about 13.75 months, and after reconstitution, the target protein can be synthesized 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, the spray-dried extract is stored at about 2°C to 8°C for about 13.75 months, and after reconstitution, the target protein can be synthesized 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 comprise about 100 g / L (about 105 g / kg) trehalose, the spray-dried extract is stored at about 20°C for about 13.75 months, and after reconstitution, the target protein can be synthesized at a titer greater than about 60 compared to the control extract reconstituted at time T=0.

[0226] In some embodiments, when the extract is stored at 2°C to 8°C for greater than or equal to 8 months before rehydration, the protein synthesis activity of the rehydrated spray-dried extract is greater than or equal to the protein synthesis activity of the rehydrated control spray-dried extract. In some embodiments, when the extract is stored at 2°C to 8°C for greater than or equal to 13 months before rehydration, the protein synthesis activity of the rehydrated spray-dried extract is greater than or equal to the protein synthesis activity of the rehydrated control spray-dried extract. In some embodiments, when the extract is stored at about 20°C (RT) for 8 months before rehydration, the protein synthesis activity of the rehydrated spray-dried extract is greater than or equal to the protein synthesis activity of the rehydrated control spray-dried extract. In some embodiments, the rehydrated spray-dried extract comprises trehalose. In some embodiments, the spray-dried extract comprises about 100 g / L (about 105 g / kg) trehalose before rehydration. In some embodiments, the control extract is an unformulated extract that does not comprise trehalose.

[0227] In some embodiments, the spray-dried extract has a decrease in protein synthesis activity (as measured by the rate of potency 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 decrease in protein synthesis activity (as measured by the rate of potency loss) of about 2% to about 6% per month when stored at 2° C. to 8° C., and a residual moisture content of less than or equal to about 7%, 6%, 5%, 4%, 3% or 2% (w / w) residual moisture.

[0228] In some embodiments, the spray-dried extract comprises trehalose and about 2% to 6% residual moisture, and has a decrease in protein synthesis activity 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 comprises 100 g / L trehalose and about 2% to 3% residual moisture, and has a decrease in protein synthesis activity 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 comprises 75 g / L trehalose and about 2% to 3% residual moisture, and has a decrease in protein synthesis activity of about 4% to 5% per month when stored at 2°C to 8°C before reconstitution.

[0229] In some embodiments, the spray-dried extract comprises lactose and about 3% to 4% residual moisture, and has a decrease in protein synthesis activity of about 3% to about 4% per month when stored at about 2°C to 8°C prior to reconstitution. In some embodiments, the spray-dried extract comprises about 100 g / L or about 100 g / kg lactose and about 3% to 4% residual moisture, and has a decrease in protein synthesis activity of about 3% to 4% per month when stored at 2°C to 8°C prior to reconstitution.

[0230] In some embodiments, the spray-dried extract comprises trehalose, is stored at 2°C to 8°C for about 4 months or at least 4 months before reconstitution, and is capable of synthesizing the target protein at a titer of at least 75% relative to an unformulated frozen control extract not comprising trehalose stored at -20°C to -80°C. In some embodiments, the spray-dried extract comprises about 70 to 100 g / L trehalose (e.g., 70, 75, 80, 85, 90, 95 or 100 g / L trehalose) before reconstitution. In some embodiments, the spray-dried extract comprises about 75 to 105 g / kg trehalose (e.g., 75, 80, 85, 90, 95, 100 or 105 g / kg trehalose) before reconstitution.

[0231] In some embodiments, the spray-dried extract comprises lactose, is stored at 2°C to 8°C for about 4 months or at least 4 months before reconstitution, and is capable of synthesizing the target protein at a titer of at least 75% relative to an unformulated frozen control extract not comprising lactose stored at -20°C to -80°C. In some embodiments, the spray-dried extract comprises 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 comprises about 75 to 105 g / kg lactose (e.g., 75, 80, 85, 90, 95, 100, or 105 g / kg lactose) before reconstitution.

[0232] K. Use of spray-dried bacterial extracts in cell-free protein synthesis

[0233] Cell-free protein synthesis systems such as open cell-free (OCFS) systems based on Escherichia coli can be used to synthesize, appropriately fold and / or assemble biologically active target proteins. In this system, cell extracts from Escherichia coli cells include template DNA (such as plasmid or linear DNA fragments), amino acids (including natural amino acids or non-natural amino acids), nucleotides, T7 RNA polymerase and energy sources. Optionally, disulfide isomerase chaperones are also added to help the formation of disulfide bonds. The CFPS system has been used to produce various 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)).

[0234] The bacterial strain for the preparation of cell extract can have the activity of the nuclease and / or phosphatase that reduce, and this increases the cell-free synthesis efficiency.For example, the bacterial strain for the preparation of cell-free extract can have sudden change in the gene of encoding nuclease RNaseE and RNase A.Bacterial strain can also have sudden change with the component of stable cell synthesis reaction, and described sudden change is for example the disappearance in the gene such as tnaA, speA, sdaA or gshA, and it prevents amino acid tryptophan, arginine, serine and cysteine ​​from being degraded respectively in cell-free synthesis reaction.In addition, bacterial strain can have sudden change with the protein product of stable cell-free synthesis, such as the knockout in protease ompT or lonP.

[0235] In a general CFPS reaction, a gene encoding a target protein is expressed in a transcription buffer, producing mRNA that is translated into the target protein in a CFPS extract and a translation buffer. The transcription buffer, the cell-free extract, and the translation buffer may be added separately, or two or more of these solutions may be combined before adding them or added simultaneously.

[0236] In order to synthesize the target protein in vitro, the bacterial extract includes the mRNA molecules encoding the target protein at some moments. In some systems, after purification from natural sources or using RNA polymerase (such as RNA polymerase II, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, RNA polymerase III and / or phage-derived RNA polymerase) to synthesize from cloned DNA in vitro, exogenous mRNA is added. In other systems, mRNA is produced in vitro by template DNA; transcription and translation both occur in this type of reaction. In some embodiments, the transcription system and translation system are coupled or include complementary transcription systems and translation systems, which perform the synthesis of both RNA and protein in the same reaction. In this in vitro transcription system and translation system, the bacterial extract contains all components (exogenous or endogenous) necessary for transcription (to produce mRNA) and translation (to synthesize protein) in a single system.

[0237] The CFPS reaction mixture can contain the following components: a template nucleic acid, such as DNA, comprising a target gene operably linked to at least one promoter and optionally one or more other regulatory sequences (e.g., a clone or expression vector containing the target gene) or a PCR fragment; an RNA polymerase that recognizes a promoter operably linked to the target gene (e.g., T7 RNA polymerase) and optionally one or more transcription factors that are directed to the optional regulatory sequence operably linked to the template nucleic acid; ribonucleotide triphosphates (rNTPs); optionally, other transcription factors and their cofactors; ribosomes; transfer RNA (tRNA); other or optional translation factors (e.g., translation initiation, elongation and termination factors) and their cofactors; one or more energy sources (e.g., ATP, GTP); optionally, one or more energy regeneration components (e.g., PEP / pyruvate kinase, AP / acetate kinase or creatine phosphokinase / creatine kinase); optionally, factors that improve yield and / or efficiency (e.g., nucleases, nuclease inhibitors, protein stabilizers, chaperone proteins) and their cofactors; and; optionally, solubilizing agents. The reaction mixture may also include 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), polymer compounds (e.g., polyethylene glycol, dextran, diethylaminoethyl dextran, quaternary aminoethyl dextran and aminoethyl dextran, etc.), cyclic AMP, inhibitors of protein or nucleic acid degrading enzymes, inhibitors or regulators of protein synthesis, oxidation / reduction regulators (e.g., DTT, ascorbic acid, glutathione and / or their oxides), non-denaturing surfactants (e.g., Triton X-100), buffer components, spermine, spermidine, putrescine, etc. The components of such reactions are discussed in more 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.

[0238] Depending on the specific enzymes present in the extract, for example, one or more of a number of known nuclease, polymerase or phosphatase inhibitors may be selected and advantageously used to improve the efficiency of synthesis.

[0239] Protein and nucleic acid synthesis generally require energy sources. The initiation of transcription requires energy to produce mRNA (e.g., when using a DNA template, a high-energy phosphate in the form of, for example, GTP is used for the initiation of translation). Each subsequent step of a codon (three nucleotides; one amino acid) by the ribosome requires the hydrolysis of additional GTP to GDP. ATP is also generally required. For amino acids to be polymerized during protein synthesis, they must first be activated. Therefore, a large amount of energy from high-energy phosphate bonds is required to perform protein and / or nucleic acid synthesis.

[0240] Energy source is a chemical substrate that can be enzymatically treated to provide energy to realize required chemical reaction.Usually use an energy source that allows to release the energy for synthesis by the cracking of high-energy phosphate bonds (such as those found in nucleoside triphosphates (ATP)).Any source that can be converted into high-energy phosphate bonds is particularly suitable.ATP, GTP and other triphosphates can be considered as the equivalent energy source supporting protein synthesis.

[0241] To provide energy for synthetic reactions, the system can include added 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 produce or regenerate high-energy triphosphate compounds such as ATP, GTP, other NTPs, etc.

[0242] When there is not enough energy initially present in the synthesis system, it is preferred to supplement with an additional energy source.The energy source may also be added or supplemented during the in vitro synthesis reaction.

[0243] In some embodiments, a system comprising NTPs, 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 + Cell-free protein synthesis reactions were performed using the PANOx-SP system with oxalate, putrescine, spermidine, and S30 extract.

[0244] In some embodiments, proteins containing non-natural amino acids (nnAA) can be synthesized. In such embodiments, the reaction mixture can include non-natural amino acids, tRNAs orthogonal to 20 naturally occurring amino acids, and tRNA synthetases that can connect nnAAs to orthogonal tRNAs. See, for example, U.S. Patent No. 8,715,958. Alternatively, the reaction mixture can contain nnAAs conjugated to tRNAs (wherein naturally occurring tRNA synthetases have been exhausted). See, for example, U.S. Patent No. 8,778,631 and U.S. Application Publication No. 2010 / 0184134. Various non-natural amino acids (including but not limited to detectably labeled amino acids) can be added to the cell-free protein synthesis reaction and effectively incorporated into the protein for a specific purpose. See, e.g., 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.

[0245] In some cases, the cell-free synthesis reaction does not need to add common secondary energy sources, but uses the co-activation of oxidative phosphorylation and protein synthesis. In some cases, CFPS is carried out in a reaction such as a Cytomim (cytoplasmic mimetic) system. The Cytomim system is defined as a reaction condition carried out with an optimized magnesium concentration in the absence of polyethylene glycol. This system does not accumulate phosphates known to inhibit protein synthesis. A detailed description of the Cytomim system can be found in, for example, 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, for all purposes, the contents of which are incorporated herein in their entirety.

[0246] 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 oxidative phosphorylation pathway inhibitors 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.

[0247] In some embodiments, a system comprising NTPs, 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 + The Cytomim system of oxalate, putrescine, spermidine and S30 extract performs cell-free protein synthesis reaction. In some embodiments, the energy substrate for the Cytomim system is pyruvic acid, glutamic acid and / or glucose. In some embodiments of the system, nucleoside triphosphates (NTPs) are replaced with nucleoside monophosphates (NMPs).

[0248] Cell extracts can be treated with iodoacetamide to inactivate enzymes that can reduce disulfide bonds and impair correct protein folding. As further described herein, cell extracts can also be treated with prokaryotic disulfide isomerases (such as, but not limited to, E. coli DsbC and PDI). Cell extracts can be treated with DsbC, FkpA, and peptidyl proline isomerases. Exogenous chaperone proteins can be expressed by bacterial strains of cell extracts. Glutathione disulfide (GSSG) and glutathione (GSH) can also be added to the extract in a ratio that promotes correct protein folding and prevents abnormal protein disulfide formation.

[0249] In some embodiments, the CFS reaction includes inverting membrane vesicles to perform oxidative phosphorylation. These vesicles can be formed during the high pressure homogenization step of the cell extract preparation process as described herein and retained in the extract used in the reaction mixture.

[0250] Cell-free synthesis reaction conditions can be carried out in batches, continuous flow or semi-continuous flow as known in the art. Reaction conditions are linearly scalable, for example, a 0.3L scale in a 0.5L stirred tank reactor can be amplified to a 4L scale in a 10L fermentor, and a 100L scale in a 200L fermentor.

[0251] The protein synthesis reaction described herein can utilize large-scale, small-scale reactors, or can be multiplexed to carry out multiple simultaneous synthesis. Continuous reactions can use a feed mechanism to introduce reagent streams, and the final product can be separated as a part of the process. Batch systems are also useful, in which additional reagents can be introduced to extend the time period of active synthesis. Reactors can be operated in any mode, such as batch, extended batch, semi-batch, semi-continuous, feed-batch and continuous, and will be selected according to the application purpose.

[0252] L. Methods for Comparing Yields of Cell-Free Protein Synthesis

[0253] The activity of the dried extract (e.g., the yield of a specific protein in a cell-free protein synthesis system) can be determined using a detection method, 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 in detail in, for example, Kim, DM and Swartz, JR Biotechnol. Bioeng. 66: 180-8 (1999); Kim, DM and Swartz, JR Biotechnol. Prog. 16: 385-90 (2000); Kim, DM and Swartz, JR Biotechnol. Bioeng. 74: 309-16 (2001); Swartz et al., Methods Mol. Biol. 267:169-82 (2004); Kim, DM and Swartz, JRB Biotechnol. Bioeng. 85:122-29 (2004); Jewett, MC and Swartz, JR, 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).

[0254] The amount of protein produced in the CFPS reaction can be measured using any method known to those skilled in the art. In some embodiments, the yield of the target protein is determined by dual flow chromatography (DFC) using protein A resin. The protein A resin can be filled between two thin sieve plates of a disposable pipette tip. In some embodiments, the product of the cell-free synthesis reaction is passed through a protein A column (e.g., column ) to determine the yield of the target protein. The yield of the target protein can be expressed as the weight / volume of the CFPS reaction (e.g., mg / L or g / L) or as a percentage of the yield of the control extract.

[0255] In some embodiments, the production of the target protein is determined by performing high performance liquid chromatography (HPLC) on the protein product from the CFPS reaction and protein standards.

[0256] In some embodiments, the output of target protein is measured by measuring the detection method of the activity of the specific protein being translated. The example of the detection method for measuring protein activity is the luciferase detection system or the chloramphenicol acetyltransferase detection system for producing related proteins. These detection methods measure the amount of functional active protein produced by the translation reaction. The detection method for measuring protein level includes but is not limited to the polyacrylamide gel of Coomassie staining, the polyacrylamide gel of silver staining, ELISA, immunoblotting, western blot, size exclusion chromatography, affinity chromatography and mass spectrometry. Any method for measuring the activity (for example, function) of the specific protein of target measurement known to those skilled in the art can be used to measure the activity of the specific protein being translated. For example, the amount of the specific kinase produced in the translation reaction can be measured by kinase detection, wherein the activity of the specific kinase is measured by quantitative kinase reaction.

[0257] Another method for measuring the amount of protein produced in a coupled in vitro transcription and translation reaction is to use known amounts of radiolabeled amino acids (such as 35 S-methionine, 3 H-leucine or 14 In vitro translation is a process for the in vitro translation of proteins. The in vitro translation of proteins is carried out by reacting with a 50% 40% 50% 50% 20% 40% 25% 50% 20% 50% 25% 30% 40% 25% 3 ...40% 25% 30% 40% 40% 25% 30% 40% 40% 25% 30% 40% 40% 25% 30% 40% 40% 25% 30% 40% 40% 25% 30% 40% 40% 40% 25% 30% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40% 40

[0258] Methods for measuring the ability of an expression system to express a protein include 14 C Leu incorporation detection method. In some embodiments, the method for measuring the protein synthesis activity of the spray-dried extract is 14 C Leu incorporation detection method.

[0259] In some embodiments, the soluble protein produced in the cell-free protein synthesis reaction is incorporated into 14The yield of soluble protein is calculated based on the amount of CLeu. The extract can be treated with 50 μM iodoacetimide (IAM) at room temperature for about 30 minutes. IAM is added to allow disulfide bonds to form within the target protein. Other thiol capping agents (such as iodoacetic acid (IAA) and N-ethylmaleimide (NEM)) can replace IAM.

[0260] Typically, the extract is then added to a mixture containing 14 C Leu protein synthesis reaction mixture in a microcentrifuge tube to start the reaction. About 60 μl of the reaction mixture was transferred to a 24-well plate and spread evenly in the wells. The mixture was incubated at 30°C for 5 hours. At the end of 5 hours, the mixture was transferred to a new microcentrifuge tube. Two 10 μl aliquots were transferred to two pieces of chromatography paper labeled "A" and "B". "A" represents the total count, while "B" represents the total protein count. The remaining mixture in the tube was centrifuged at about 13,000 rpm in a microcentrifuge tube for about 15 minutes. Two 10 μl aliquots of the supernatant were transferred to two pieces of chromatography paper labeled "C" and "D". "C" and "D" represent soluble proteins. All paper pieces were dried at about 2 inches from a heat lamp for about 15 minutes. "A" was transferred to a microcentrifuge tube. "B", "C" and "D" were washed 3 times on ice with 5% TCA for about 15 minutes, and then washed with 100% ethanol. They were then dried under a heat lamp for approximately 15 minutes. "B", "C" and "D" sheets were transferred to separate microcentrifuge tubes. Scintillation cocktail (Optiphase Supermix, PerkinElmer, Waltham, MA) was added to each microcentrifuge tube and counted in a scintillation counter for 5 minutes.

[0261] The total protein yield can be determined using the following equation:

[0262] (Counts in slice B / Counts in slice A) (Leucine concentration in cell-free / # of Leucine in target protein) (MW of target protein)

[0263] The soluble protein yields of "C" and "D" can be determined by the following equations:

[0264] (Counts in slice C or slice D / counts in slice A) (Leucine concentration in CF / # of 1eu residues in target protein) (MW of target protein)

[0265] The average yield of soluble protein can be determined by averaging the yields of "C" and "D".

[0266] Alternatively, the samples can be run on polyacrylamide gels using conventional techniques. 14The production of protein is determined by CLeu-labeled protein. Depending on the polypeptide to be detected, the gel can be denaturing or non-denaturing. When a protein containing multiple subunits is to be detected, a non-denaturing gel is preferred.

[0267] Alternatively, protein production can be determined by specific binding detection methods such as enzyme-linked immunosorbent assay (ELISA) or surface binding resonance (eg, Biacore).

[0268] Alternatively, protein yield can be determined by complete or partial purification (such as using chromatography) coupled with protein quantitation (such as UV absorbance or BCA analysis).

[0269] Although the above disclosure has been described in detail by way of examples and embodiments for purposes of clear understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made thereto in light of the teachings of this disclosure without departing from the spirit or scope of the appended claims.

[0270] Example

[0271] Example 1. Preparation of bacterial cell-free extract

[0272] 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).

[0273] For small-scale preparation of bacterial extracts, 10 μl of thawed E. coli glycerol stock 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. Then 50 mL of culture was 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 the growth rate was monitored. During the exponential phase and before the growth rate decreased during the transition to the stationary phase, the cells were harvested and cooled. Once the culture was cooled, the cells were collected by centrifugation at 8,000 x g for 20 to 30 minutes. At OD 3, about 8 g of wet cells were collected from 1 L. For each gram of wet cell weight, the cell mass was resuspended in at least 5 mL of S30 buffer. The cell suspension was centrifuged at 8,000 x g for 20 to 30 minutes. The supernatant was discarded and the washed cell mass was frozen at -80 ° C.

[0274] For large-scale preparation of bacterial extracts, the culture is harvested after fermentation for 16 to 20 hours.Then the fermentor (200L fermentor) is pressurized to 20psi, and the culture of about 200L is transferred to the 200L jacketed storage tank cooled by two parallel heat exchangers via pressure.By making glycol recirculate through the jacket, the culture temperature of about 200L in the storage tank is cooled to 2 ℃ to 8 ℃.After the transfer is completed, the culture is ready for the first centrifugation step via a disc centrifuge.During the first centrifugation step, cells are separated from the spent culture medium (supernatant).The paste (cell paste) discharged is collected in a container, weighed, and then assigned to the cooling 200L storage tank containing the S30 buffer of 100L and mixed to resuspend.Then the resuspended cells are centrifuged to agglomerate the cells.The cell agglomerates are stored at -80 ℃.

[0275] One liter of 2YTPG medium contains 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 22 mM sodium dihydrogen phosphate, 40 mM sodium dihydrogen phosphate, 100 mM glucose and optionally 100 μl antifoam 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.

[0276] The frozen S30 cell paste is broken into small pieces and thawed in 1 mL of room temperature S30 buffer per gram of cell paste. Once thawed, the cell suspension is kept on ice. Before processing the cell extract, the cell homogenizer is rinsed in S30 buffer. The cells are lysed by passing through a high pressure cell homogenizer at 17,500 psi in a single pass. The lysate is then quickly cooled by a cooling coil or a heat exchanger. The lysate is kept on ice until all the cell paste is lysed as described herein. The lysate is centrifuged at 30,000xg for 30 minutes at 4°C. The supernatant is collected in a clean tube and the centrifugation step is repeated again to collect all supernatants in the lysate.

[0277] The collected supernatant has 0.2 mL of pre-incubation mixture added for every 1 mL of supernatant. The mixture is then incubated at about 37°C for about 80 minutes. The pre-incubation mixture contains 370 mM Tris acetate (pH 8.2), 11.1 mM magnesium acetate, 16.5 mM ATP, 50 μM each of 20 amino acids or unnatural amino acids, 105 mM phosphoenolpyruvate (PEP) and 8.4 U / mL pyruvate kinase.

[0278] Example 2. Preparation of cell-free extracts with additives

[0279] This example provides a representative method for formulating a cell-free extract with additives that can be used to stabilize the spray-dried extract.

[0280] Liquid bacterial extracts are prepared by directly mixing in powdered excipients, or by adding concentrated stock solutions of additives to achieve various additive levels and combinations in the prepared extracts before spray drying. The concentrated stock solutions can be sterile filtered for bioburden control before being added to the liquid extracts. Some excipients are readily soluble in water at concentrations up to several hundred grams per liter under ambient conditions and frozen conditions, while other excipients have limited solubility. Some components show solubility up to at least 100 g / L or 100 g / kg in the extracts, and are tested at these levels, while lower extract solubility limits determine the maximum concentration that can be assessed for some other excipients. The additives used include trehalose dihydrate (TDH), lactose monohydrate (LMH), raffinose, maltodextrin, sucrose, mannitol, sorbitol, polyethylene glycol 200, polysorbate 80 ( 80), polyvinylpyrrolidone (PVP or Kollidon 12PF) and 2-hydroxypropyl-β-cyclodextrin. More than 33 formulations were produced and dried to evaluate the extract formulations. Representative single component and combination formulations are shown in Table 1.

[0281] In addition, some mixtures include all combinations or subsets of natural 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 "PMA amino acid mixtures". The PMA amino acid mixtures optionally do not include tyrosine or phenylalanine. In some embodiments, tyrosine and / or phenylalanine are added separately from the PMA mixture in the XCF reaction mixture. Representative PMA amino acid mixtures are shown in Table 2.

[0282] Example 3. Spray drying process

[0283] This example describes a representative spray drying process.

[0284] Spray drying is a scalable, continuous process that converts liquid feedstocks into fine powders and reduces material volume and bulk density. ) is achieved by: (1) atomizing the liquid feedstock material into a fine mist by pumping it through an atomizing device (e.g., a nozzle or a rotary atomizer), (2) intersecting the fine mist with convection currents of a hot drying gas in a chamber that evaporates the liquid to dry it, (3) separating the dried product material from the moist drying gas and smaller fine particles using a cyclone separator or other similar technology designed for powder classification, and (4) collecting the dried material. Spray drying from liquid to powder form reduces the volume of the material and increases storage capacity, which is necessary for commercial-scale production.

[0285] Liquid raw material material is prepared before spray drying, and the liquid raw material material is composed of extract solid, formulation solid and water. The prepared raw material is continuously pumped to the dryer at a liquid feed rate determined by the dryer process control scheme. The material is fed to a two-fluid nozzle through a pipeline. The nozzle combines the liquid with an atomizing gas flow with an atomizing gas pressure and an atomizing gas flow rate, and the atomizing gas pressure and the atomizing gas flow rate affect the droplet size formed based on the pressure and relative flow of the two streams. Once atomized into a fine mist, the droplets are in contact with the dry gas at the inlet temperature. However, evaporative cooling occurs, and the solid does not directly experience a high inlet dry gas temperature. The mist is exposed to the dry gas for a continuous residence time in the drying chamber, during which the dry gas is cooled to the outlet temperature. Initially, the solid in the droplet experiences a wet bulb temperature, but as the water evaporates, there will be a transition of the solid to experience a dry bulb temperature. There is an interdependence between the above-mentioned process parameters and the output in the spray drying process, and the balance of momentum transfer, heat transfer and mass transfer in the process defined by these parameters determines the particle size or particle size distribution that affects the yield. Water evaporates significantly during drying. The dried material contains a portion of residual moisture or water content which is negatively correlated with the observed shelf life stability. Spray dried extracts typically have a residual moisture content of less than 7% and can have a residual moisture content of less than 5%.

[0286] for Spray drying, inlet temperature and outlet temperature The spray-dried The residual moisture or water content has a significant impact on shelf life stability when stored at 2°C to 8°C and ambient temperature. Spray drying development includes laboratory scale (Buchi B-290), pilot scale (Mobile Minor PSD-1), and industrial scale (Modified PSD-2, PSD3, and IFC Welko≈PSD-4). These drying scales span batch sizes from about 0.1 L to 1000 L and throughputs from about 1 kg / hr to 100 kg / hr. Larger dryers typically have longer material residence times, which can result in Therefore, the outlet temperature is adjusted to lower the temperature set point in order to maintain the best drying performance in larger scale. The outlet temperature of the larger dryer was adjusted to about 65°C to 76°C.

[0287] Process control schemes can be configured in a variety of ways whereby some parameters are controlled around set points, while other parameters are allowed to float or adjust freely to accommodate control of the set parameters. These are not necessarily scale specific. Due to the temperature sensitivity of the drying agent, a typical drying method is to control the inlet temperature, outlet temperature, drying gas flow rate and atomizing gas pressure / flow rate at set values, while allowing the liquid feed rate to float and be adjusted to compensate.

[0288] Example 4. Testing various extract preparations and drying conditions

[0289] This example describes various extract formulations and drying conditions tested using different spray dryers.

[0290] Formulation testing was performed using a Buchi-290 lab-scale spray dryer to evaluate several common spray-dried excipients as single-component formulations. Before performance, the spray-dried powder samples were reconstituted into liquid form. Figure 1 Show from Initial potency and residual moisture results for the (XCF) test. Figure 1 In the Figures 1 and 2, the corresponding inlet and outlet temperatures of the dried samples are noted in parentheses as (inlet temperature / outlet temperature). Samples 1A to 1E are unformulated spray-dried samples, while samples 2A to 2E are spray-dried samples with varying inlet temperature / outlet temperature as shown. Protein A resin in pipette tips (also referred to herein as "Phytip") is used in a high-throughput format via laboratory automation for rapid purification and titration quantification using A280 absorbance. Flower disks, also referred to herein as "FP", are small multiwell plates (approximately 1 ml reaction) without pH and DO control. Trehalose dihydrate (100 g / L, sample 2B) is a promising excipient, followed by leucine (10 g / L, sample 2D), and then Tween 80 (0.1%, sample 2A). Low initial titer results were obtained with PVP (100 g / L, sample 2C) and maltodextrin (100 g / L, sample 2E). For the dried samples tested, the residual moisture ranged from about 3 to 6%. Showed good recovery of initial activity. Control sample was unformulated liquid extract. Some freeze-thaw effect on potency was observed for the control.

[0291] based on Figure 1 The results in the dry and unformulated dry Stability was evaluated at 2°C to 8°C and room temperature. Testing included the use of multiple extract batches and drying conditions, as shown in Table 4.

[0292] Table 4: Buchi B-290 Laboratory Spray Drying Stability

[0293]

[0294] The samples in Table 4 were prepared and dried for both the unformulated sample and the sample formulated with 100 g / L trehalose. Drying was performed on a laboratory scale Buchi B-290 spray dryer. XCF tests were performed using a 30% Batch reactions in a Micro-24 microbial reactor were used to express the products anti-CD74 antibody (for the 8-month and 13-month samples) and trastuzumab (for the 18-month sample). Source liquid and dry From DR6-2, DR7-6, and DR 8-7. Sample extraction batch, residual moisture, and dryer outlet temperature are noted for the corresponding samples. As shown in Table 4, promising stability up to 18 months was observed for storage conditions between 2°C and 8°C. In contrast, unformulated Limited shelf life stability was shown, indicating the importance of the presence of stabilizing formulation components. Higher performance of the 100 g / L trehalose formulation for room temperature storage was observed compared to the unformulated extract (even when stored at 2°C to 8°C).

[0295] The drying process was scaled up to a pilot scale on a Mobile Minor PSD-1. Several trehalose formulations were tested, including 100 g / L, 50 g / L, and 25 g / L, as well as some drying process conditions. Figure 2A and Figure 2B As shown in, comparable initial activity was obtained at the three tested trehalose formulation levels. It was observed that the moisture content of the 50 g / L and 25 g / L formulations was higher than that of the 100 g / L formulation, which was attributed to the lower total solid content of the raw material, resulting in lower drying efficiency and lower overall water evaporation. The source unformulated liquid extract was used as a control.

[0296] In long-term stability studies at storage conditions of -20°C, 2 to 8°C and room temperature, the Figure 2A and Figure 2B A subset of samples with promising initial activity. Figure 3A Table 5 and Table 6 show the stability results from the 2 to 8°C storage condition. The 2 to 8°C storage condition was evaluated for up to 24.5 months. The calculated potency loss rates are listed in Table 5.

[0297] Table 5. Potency of some trehalose preparations

[0298]

[0299] As shown in Table 5, the loss rate ranged from 0.3% / month to 1.6% / month for all TDH concentrations. However, the 50 g / L formulation and the 25 g / L formulation had higher moisture than the 100 g / L formulation, which confounds the ability to make direct comparisons based purely on formulation concentration, as moisture content may affect stability. Figure 3B As shown in , -20°C storage conditions showed minimal potency loss over the 12 months evaluated compared to 2°C to 8°C conditions, and as Figure 3C As shown in, room temperature conditions showed significant activity loss within 8 months, including a clear distinction between formulation levels, where higher levels of TDH were more stable. The control used in this test was a dry extract reconstituted at t=0, which was stored frozen (≤-65°C) and thawed when needed for testing.

[0300] The results presented above indicate that trehalose can be used as a single component stabilizer. Therefore, 100 g / L trehalose was selected for further testing.

[0301] The minimum target drying feed flow rate was 10 kg / hr. Therefore, the experiments were repeated using a PSD-2 dryer that had been modified to achieve higher gas flow rates than a typical design, which was evaluated as having the potential to meet the required throughput. Although the size and gas flow rate of the drying chamber of the modified PSD-2 is different from that of the PSD-3 from a technical perspective, i.e., the PSD-2 chamber is narrower than the PSD-3 chamber, the drying capacity can be described as similar because both the chamber size / dimensions and the gas flow rate contribute to drying performance and efficiency.

[0302] Testing involved an initial process of four ranges of conditions at 25 L each, from which a single process condition was selected. This condition was run in three consecutive 100 L runs, each using a different extract batch to demonstrate process robustness. Figure 4A and Figure 4B As shown in Figure 4A and Figure 4B For each demonstration run, the samples tested included the unformulated extract pool (UEP), the formulated extract pool (FEP), a blend of formulated liquid samples collected in each 100 L demonstration run (FEP dry feed blend), and a reconstituted dry feed blend collected in each 100 L demonstration run. Blends of samples (spray dried blends). The formulated samples contained 100 g / L trehalose. All three runs showed that the corresponding dried blends recovered approximately 100% of the activity compared to the formulated blends.

[0303] Material from the demonstration run was tested for long term stability. Figure 5A , Figure 5B and Figure 5CAs shown in the results, when the extracts were stored at -20°C and 2°C to 8°C, there was minimal if any decrease in activity over about 19.5 months, whereas when the extracts were stored at room temperature (about 20°C), the activity decreased to about 70% to 90% recovered activity over about 19.5 months. The control used for the test was the dried extract reconstituted at t=0.

[0304] Selected stability sample time points were tested in a scaled-up stirred tank reactor configuration (DASbox, 200 ml XCF reaction volume), which is considered to be a more representative large-scale bioreactor compared to the Micro-24. The DASbox, also referred to herein as "DB", is a small-scale stirred tank bioreactor system (approximately 100 ml to 200 ml reactions) with pH and DO control. Fig. 6A and Figure 6B Stability data at about 14 months showed that when the extract was stored at -20°C, the activity was restored to >90%, when the extract was stored at 2 to 8°C, the activity was restored to about 80%, and when the extract was stored at room temperature, the activity was restored to about 60% to 65%. This test utilizes anti-folate receptor alpha antibody using pre-made light chain (PFLC) expression product. The control for the test was a reconstituted dried extract at t=0.

[0305] Additional formulation screening was performed. The scope of testing included screening of multiple single-component formulations and multi-component formulations, including (1) sugar / polyol, (2) amino acid, (3) detergent / polyethylene glycol, and (4) macromolecular crowding agent. Inspired by the above data, indicating that 50 g / L trehalose may have acceptable stability compared to 100 g / L trehalose, an intermediate 75 g / kg trehalose formulation was evaluated.

[0306] Table 6 shows the list of formulations tested, the corresponding Initial activity results, the reaction configuration tested (batch or fed-batch), and the expression products used. As shown in Table 6, sample code 1119, sample code 0120, and sample code 0220 represent November 2019, January 2020, and February 2020, respectively. The XCF reaction configuration (batch or fed-batch) and expression product for each test preparation are marked. All tests were performed with 37.5% (by volume) of extract. The extract batches used were 19006-10 (November 2019), 19013-01 (January 2020), and 19011-04 (February 2020). m24, also known as micro-24, is a small porous microbial reactor system (approximately 5 ml reaction) with pH and DO control. 19013-01 was a high bioburden batch that is suspected to be at least partially responsible for the lower than expected potency from the January 2020 testing, thereby motivating additional testing in February 2020.

[0307] Table 6: Pilot scale Mobile Spray-dried preparations to investigate activity results

[0308]

[0309]

[0310]

[0311]

[0312]

[0313] The data in Table 6 show that the excipient glass transition temperature (T g ) is positively correlated with the activity recovered after drying. Fig. 7A , Figure 7B , Fig. 8A and Figure 8B As shown in , the top candidate formulations are single components (A) trehalose dihydrate 100g / L, (B) trehalose dihydrate 75g / kg and (C) lactose monohydrate 100g / kg, which show promising results for expression of two different antibody products. The initial activity with 100g / kg lactose is more favorable for 60°C and 70°C outlet temperatures compared to 80°C outlet temperature, so only these two conditions are further investigated. Long-term stability was tested for these single component formulations and shown as Fig. 9 Similar titer loss rates as shown in Table 7. Some gCombination formulations of either a mixture of nonpolar / uncharged amino acids or 2-hydroxypropyl-β-cyclodextrin showed promising initial activity, but none provided any improvement over the TDH or LMH single component options.

[0314] Table 7: Single component formulation of trehalose dihydrate and lactose monohydrate Long term stability at 2°C to 8°C.

[0315] Batch preparation Residual moisture (%) Potency loss rate (% / month) 0220A 100g / L Trehalose dihydrate 2.23 2.15 0220I 100g / L Trehalose dihydrate 2.36 5.89 0220E 75g / kg Trehalose dihydrate 2.03 4.77 0220K 75g / kg Trehalose dihydrate 2.38 4.54 0220D 100g / kg lactose monohydrate 4.02 3.27 0220J 100g / kg lactose monohydrate 3.55 3.40

[0316] PSD-3(Sutro)

[0317] A series of range runs were performed during the startup of the PSD-3 dryer. A water run was performed first to test the drying process operating range. This was followed by several iterations of testing using extracts to gradually understand the process parameter ranges and outputs. Process and analytical data from these runs were used to select GMP production run conditions. Fig.10 As shown in , a 75 g / kg TDH formulation was used in a GEA PSD-3 drying run and produced SDE with high activity recovery.

[0318] In summary, the spray-dried bacterial extract described in this example shows high activity percentage and improved stability during storage with various formulations. Compared with control extracts, such as additive-free (unformulated) extracts, this extract has a longer shelf life at room temperature, 2°C to 8°C, and -20°C. The results of the above studies show that spray-dried bacterial extracts containing additives improve the stability of bacterial extracts in CFPS reactions.

[0319] 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. A method for producing a stable spray-dried bacterial extract for cell-free protein synthesis, the method comprising: i. combining a bacterial extract comprising lysed bacterial components with a composition comprising trehalose, lactose, leucine or raffinose to produce 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 the stable spray-dried bacterial extract.

2. The method according to claim 1, wherein the bacterial extract comprising lysed bacterial components is a liquid bacterial extract or a rehydrated bacterial extract.

3. The method according to claim 1 or 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 trehalose.

5. The method of claim 4, wherein the mixture comprises about 50 to 100 g / kg trehalose. The method according to claim 4 or 5, wherein the trehalose is trehalose dihydrate (TDH).

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

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

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

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

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

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

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

14. The method according to any one of claims 1 to 13, wherein in step (i), the bacterial extract is also mixed with one or more high glass transition temperature (T g )A non-polar, uncharged amino acid combination.

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

16. The method according to claim 14, wherein the one or more high T g 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.

17. The method according to any one of claims 14 to 16, wherein the mixture comprises 5 to 15 g / kg of the high T g Nonpolar, uncharged amino acids.

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

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

21. The method of any one of claims 1 to 20, wherein the spray-dried bacterial extract comprises less than or equal to about 15% (w / w) residual water.

22. The method of any one of claims 1 to 20, wherein the spray-dried bacterial extract comprises less than or equal to about 10% (w / w) residual water.

23. The method of any one of claims 1 to 20, wherein the spray-dried bacterial extract comprises less than or equal to about 5% (w / w) residual water.

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

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

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

27. 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% relative to a control extract after storage at about -20°C for at least 6 months.

28. 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% relative to a control extract after storage at about -20°C for at least 12 months.

29. 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% relative to a control extract after storage at about -20°C for at least 18 months.

30. 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% relative to a control extract after storage at about room temperature (20°C) for at least 6 months.

31. 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% relative to a control extract after storage at about room temperature (20°C) for at least 12 months.

32. 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% relative 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 measured by Method determination.

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

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

36. 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 is capable of synthesizing the target protein having a titer of at least 80% relative to the control extract reconstituted at time zero after reconstitution.

37. The method of any one of claims 1 to 23, wherein the protein synthesis activity of the spray-dried extract decreases 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. The method of claim 37, wherein the control extract does not contain trehalose, lactose, leucine, and raffinose.

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

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

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

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. The method according to any one of claims 1 to 41, wherein the spray drying in step (ii) comprises: atomizing the mixture to produce droplets; contacting the droplets with a gas to evaporate liquid from the droplets; separating the dried extract from the gases and smaller particles; and The spray dried extract was collected.

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

45. The method of claim 43, wherein greater than or equal to 95% (w / w) of the liquid is removed from the mixture.

46. ​​The method according to 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. 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. 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. A spray-dried bacterial extract for cell-free protein synthesis comprising: dried lysed bacterial components; and A composition comprising trehalose, lactose, leucine or raffinose, Wherein, upon rehydration, the extract is capable of synthesizing the target protein from a template nucleic acid encoding the target protein.

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

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

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

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

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

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

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

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

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

59. The spray-dried extract of any one of claims 49 to 58, wherein the extract further comprises one or more high glass transition temperatures (T g )Non-polar, uncharged amino acids.

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

61. The spray-dried extract of claim 59, wherein the one or more high T g 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. The spray-dried extract of any one of claims 59 to 61, wherein the extract comprises 5 to 15 g / kg of amino acids.

63. The spray-dried extract of any one of claims 49 to 62, wherein the extract further comprises one or more amino acids, a combination of all amino acids, 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.

64. The spray-dried extract of claim 63, wherein the extract comprises about 5 to 15 g / L of the amino acids.

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

66. The spray-dried extract of any one of claims 49 to 65, wherein the spray-dried extract comprises less than or equal to about 15% (w / w) residual water.

67. The spray-dried extract of any one of claims 49 to 65, wherein the spray-dried extract comprises less than or equal to about 10% (w / w) residual water.

68. The spray-dried extract of any one of claims 49 to 65, wherein the spray-dried extract comprises less than or equal to about 5% (w / w) residual water.

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

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

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

72. The spray-dried extract of any one of claims 49 to 68, wherein the spray-dried extract 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% relative to a control extract.

73. The spray-dried extract of claim 72, wherein the spray-dried 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% relative to a control extract.

74. The spray-dried extract of claim 72, wherein the spray-dried 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% relative to a control extract.

75. The spray-dried extract of any one of claims 49 to 68, wherein the spray-dried extract 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% relative to a control extract.

76. The spray-dried extract of claim 75, wherein the spray-dried 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% relative to a control extract.

77. The spray-dried extract of claim 75 or 76, wherein the spray-dried extract 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% relative to a control extract.

78. The spray-dried extract of any one of claims 69 to 74, wherein the titer is determined by Method determination.

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

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

81. The spray-dried extract of any one of claims 69 to 74, 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 is capable of synthesizing the target protein having a titer of at least 80% relative to the control extract rehydrated at time (T) = zero after rehydration.

82. The spray-dried extract of any one of claims 49 to 68, wherein 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 prior to rehydration.

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

84. The spray-dried extract of claim 83, wherein the spray-dried extract is stored at 2°C to 8°C for 13 months prior to rehydration.

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

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

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

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

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 in cell-free protein synthesis.

90. The spray-dried extract of any one of claims 49 to 89, wherein the extract is from Escherichia species.

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

92. The spray-dried extract of any one of claims 49 to 90, wherein the extract does not have a lumpy appearance or is not a dried mass.

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

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

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

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

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

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 method of claim 98, wherein the one or more amino acids comprise a high glass transition temperature (T g ) non-polar uncharged amino acid, the high glass transition temperature (T g ) 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.

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

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

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

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

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

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

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

108. The method of any one of claims 93 to 107, wherein the collected spray-dried extract comprises less than or equal to about 15%, 10%, or 5% (w / w) residual water.

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

110. The method of any one of claims 93 to 108, wherein 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 prior to rehydration.

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

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

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

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

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

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

117. 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 (T g ),as well as 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. The spray-dried extract of claim 117, wherein the stabilizer is selected from the group consisting of trehalose, lactose and leucine.

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

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

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

122. The spray-dried extract of any one of claims 117 to 121, wherein the extract further comprises one or more high glass transition temperatures (T g ) non-polar uncharged amino acid, the high glass transition temperature (T g ) 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.

123. The spray-dried extract of any one of claims 117 to 121, wherein the extract further comprises one or more amino acids, a combination of all amino acids, 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.

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

125. A method for producing a target protein from a spray-dried extract, the method comprising: reconstitute the spray-dried extract of claims 117 to 124; Providing a template nucleic acid encoding the target protein; as well as The target protein is produced.

Images