High cell density fed-batch fermentation

Abstract

Disclosed herein are methods, cells, and compositions for the production of bacterial cell extracts. The bacterial cell extracts are used in cell-free protein synthesis of recombinant proteins and large quantities of bacterial cell extracts are typically needed. The present disclosure relates to the production of high cell density fermentations that provide large quantities of bacteria for the manufacturing of bacterial cell extracts. The present disclosure relates to high density fed batch fermentation for the production of bacterial cell extract. Also provided herein are cell-free protein synthesis systems and methods that use the bacterial cell extracts.

Description

PATENT Attorney Docket No.: 091200-1532416-007410PC Client Reference No.: 0221WOHIGH CELL DENSITY FED-BATCH FERMENTATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U. S. Provisional Application No. 63 / 737,543, filed on December 20, 2024, the disclosure of which is hereby incorporated by reference in its entirety herein for all purposes.BACKGROUND

[0002] Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have increased CFPS productivity and reduced the operating costs of its systems, such that they are competitive with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics.

[0003] CFPS systems are useful for incorporating nnAAs into proteins and can be used for the rapid production of a wide variety of therapeutic proteins, including antibodies and antibody fragments. For example, the an titumor properties of monoclonal antibody drugs can be significantly improved by tethering a cytotoxic small molecule to the antibody to form a derivative called an antibody drug conjugate (ADC) (Jin S et al. Emerging New Therapeutic Antibody Derivatives for Cancer Treatment. Sig Transduct Target Ther. 2022;7:1-28).). Sitespecific protein bioconjugation using CFPS allows researchers to build highly tunable protein constructs capable of improving the specificity and efficacy of targeted drug delivery while minimizing negative effects on therapeutic activity (Kline T, Steiner et al. Methods to Make Homogenous Antibody Drug Conjugates. Pharm Res. 2015;32(11):3480-93; Panowski S et al. Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014;6(1):34-45)..

[0004] CFPS systems are made possible by the availability of reagent bacterial cell extracts, which contain all the necessary biochemical components for energy production, transcription, and translation. Large quantities of bacterial cell extracts are used in the manufacturing of desired protein therapeutics from specific DNA sequences. There is a need to manufacture bacterial cell extracts at large scale to support the development and production scale-up of1KILPATRICK TOWNSEND 80240015.2protein therapeutics. Disclosed herein are methods and compositions for producing high cell density fermentations that provide large quantities of bacteria for the manufacturing of reagent bacterial cell extracts.SUMMARY

[0005] In a first aspect, the present disclosure provides a method of producing a bacterial cell extract, comprising: (a) culturing bacterial cells in a culture medium in a bioreactor of at least 250 milliliters in total volume, (b) isolating the bacterial cells, and (c) lysing the bacterial cells to produce the bacterial cell extract. In some embodiments, the bacterial cells in step (a) are cultured to a cell density of about 55 to about 150 as measured using optical density at about 595-600 nm.

[0006] In some embodiments, the bacterial cells are cultured using a fed-batch fermentation process.

[0007] In some embodiments, the maximum fermentation volume is determined by multiplying about 0.4 to 0.8 with maximum bioreactor volume. The method of any one of claims 1-3, wherein fermentation volume is at least 1,000 L and at least 700 L of bacterial extract is produced.

[0008] In some embodiments, the oxygen uptake rate (OUR) during the fermentation is at least about 500 mmol / L / hour. In some embodiments, the OUR is at least about 200 mmol / L / hour.

[0009] In some embodiments, the bacterial cells in step (a) are cultured to a cell density of about 100 to about 150 as measured using optical density at about 595-600 nm. In some embodiments, the bacterial cells in step (a) are cultured to a cell density of at least 60-130 as measured using optical density at about 595-600 nm.

[0010] In some embodiments, the method further comprises, prior to step (a), a step of initially culturing the bacterial cells in culture medium of no greater than 1 liter in volume.

[0011] In some embodiments, the method further comprises, prior to step (a), a step of initially culturing the bacterial cells in a volume that is no greater than 3% of the culture medium of step a).

[0012] In some embodiments, the culture medium comprises amino acids, salts, glucose, trace metals, and vitamins.2KILPATRICK TOWNSEND 80240015.2

[0013] In some embodiments, the feed rate of the culture is from about 12 mL / L / hour to about 95 mL / L / hour.

[0014] In some embodiments, after step (b) and before step (c) of the method, the isolated bacterial cells are washed with a buffer. In some embodiments, the buffer comprises tris base, magnesium acetate, and potassium acetate.

[0015] In some embodiments, the isolating step comprises centrifugation.

[0016] In some embodiments, the bacterial cell extract is activated with heating comprising about 35-40°C for about 20-60 minutes.

[0017] In some embodiments, the bacterial cells are E. coli cells.

[0018] In some embodiments, the method further comprises, after step (c), freezing the bacterial cell extract in liquid nitrogen for storage.

[0019] In some embodiments, the method further comprises, a step of performing cell-free recombinant protein synthesis using the bacterial cell extract.

[0020] In some embodiments, the method further comprises, after step (c), (d) combining the bacterial cell extract with a composition comprising trehalose, lactose, leucine, and / or raffinose; and (e) spray-drying the combination to produce a spray-dried bacterial extract. In some embodiments, the method further comprises, after step (e), freeze-drying the spray-dried bacterial extract for storage. In some embodiments, the method further comprises a step of reconstituting the spray-dried bacterial extract. In some embodiments, the method further comprises, after the reconstitution step, a step of performing cell- free recombinant protein synthesis using the reconstituted spray-dried bacterial extract.

[0021] In another aspect, the present disclosure provides a cell-free synthesis system comprising (i) a bacterial cell extract produced according to a method of the present disclosure and (ii) a nucleic acid encoding a protein of interest, wherein the composition comprises an active oxidative phosphorylation system containing biologically functioning tRNA, amino acids, and ribosomes for cell-free recombinant protein synthesis.3KILPATRICK TOWNSEND 80240015.2

[0022] In some embodiments, the cell-free synthesis system is capable of producing the protein of interest, wherein the amount or the activity of the protein of interest is at least 65% of a control protein.

[0023] In another aspect, the present disclosure provides a method of cell-free protein synthesis comprising incubating the cell-free synthesis system of the present disclosure under conditions permitting the expression of the protein of interest.

[0024] In some embodiments, the amount or the activity of the protein of interest is at least 65% of a control protein, In some embodiments, the protein of interest is an antibody, an antibody fragment, an antibody light chain, an antibody heavy' chain, a cytokine, a cytokine fragment, an immunogenic polypeptide, or a carrier protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows a representative process flow diagram for fermentation and extract recovery.

[0026] Figure 2 shows data for pH, pO2, and oxygen uptake rate (OUR) for representative fermentations in a IL bioreactor for F019, F020, F021 and F022.

[0027] Figure 3 shows extract recovery processes for representative fermentations F019 and F020 (target OD 150), and F021 and F022 (target OD 50).

[0028] Figure 4 shows pH trends for the extract recovery of representative fermentations F019 and F020 (target OD 150), and F021 and F022 (target OD 50).

[0029] Figure 5 shows exemplary Ambr®15 XtractCF® results for expression of an anti¬ folate antibody intermediate (SP8166), an IgG containing amino acid para-azidomethyl-L- phenylalanine (pAMF) from representative fermentations F019 and F020 (target OD 150), and F021 and F022 (target OD 50).

[0030] Figure 6 shows SPIO activity for bacterial cell extracts generated from representative chemostat and fed-batch OD 150 fermentations.

[0031] Figure 7 shows growth and metabolite data for representative fermentations F027 and F028.4KILPATRICK TOWNSEND 80240015.2

[0032] Figure 8 shows online data for pH, pO2, and OUR for representative fermentations F027 and F028,

[0033] Figure 9 shows SP10 activity for bacterial cell extracts generated from representative fermentations F027 and F028.DETAILED DESCRIPTIONI. Introduction

[0034] This application relates to the production of bacterial cell extracts that are useful in cell- free protein synthesis (CFPS) reactions. Disclosed herein are fermentation methods, bacterial cells, and related compositions and methods for producing high cell density fermentations and harvesting the cells to produce bacterial cell extracts. The present disclosure provides fed-batch fermentation processes that enable shorter bacterial cell extract manufacturing timelines and simplify liquid handling by reducing processing volumes, which positively impact recombinant protein development and manufacturing using CFPS systems.II. Definitions

[0035] As used herein, the following terms have the following meanings ascribed to them unless specified otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0036] As used herein, the terms “bacterial cell extract,” “bacterial extract,” and “extract” refer to a bacterial cell lysate or a fraction thereof (e.g., clarified and / or filtered) that can be used to synthesize protein from a nucleic acid template. The majority of the biological components in a bacterial cell extract are present in concentrations resulting from the lysis of the cells rather than having been reconstituted. A bacterial cell extract can be a portion of a lysate from which other cellular components of the lysate have been separated or removed by centrifugation, filtration, selective precipitation, selective immunoprecipitation, chromatography, or other methods. It also includes lysates or fractions thereof that contain exogenous material such as preservatives, stabilizers and reagents that enhance cell-free protein synthesis (CFPS). A bacterial cell extract may be further altered such that the extract is supplemented with additional cellular components, e.g., amino acids, nucleic acids, enzymes, etc. The bacterial cell extract 5KILPATRICK TOWNSEND 80240015.2may also be altered such that additional cellular components are removed or degraded following lysis. The terms “bacterial cell extract,” “bacterial extract,” and “extract” can refer to a preparation of an in vitro reaction mixture able to transcribe DNA into mRNA and / or translate mRNA into polypeptides. The mixture may include ribosomes, an energy source (such as ATP, GTP, glucose, glutamate, or pyruvate), amino acids, and tRNAs. The mixture may be derived directly from lysed bacteria, from purified components or combinations of both. In some cases, the bacterial extract contains all the necessary bacterial components needed to synthesize a protein of interest from a template nucleic acid encoding the target protein in a CFPS reaction without the addition of other components of a bacterial extract.

[0037] As used herein, the term “lysate” is any cell-derived preparation comprising the components required for protein synthesis machinery, wherein such cellular components are capable of expressing a nucleic acid encoding a desired protein where a majority of the biological components are present in concentrations resulting from the lysis of the cells rather than having been reconstituted. A lysate may be further altered such that the lysate is supplemented with additional cellular components, e.g., amino acids, nucleic acids, enzymes, etc. The lysate may also be altered such that additional cellular components are removed or degraded following lysis.

[0038] As used herein, the term “cell-free protein synthesis” or “CFPS” refers to the in vitro synthesis of polypeptides and / or proteins in a reaction mix comprising biological extracts and / or defined reagents. The CFPS reaction mix can comprise a template for production of the macromolecule, e.g., DNA, mRNA, and etc. monomers for the macromolecule to be synthesized, e.g., amino acids, nucleotides, etc.; and co-factors, enzymes and other reagents that are necessary for the synthesis, e.g., ribosomes, uncharged tRNAs, tRNAs charged with natural and / or unnatural amino acids, polymerases, transcriptional factors, tRNA synthetases, etc.

[0039] As used herein, the term “spray-dried bacterial extract” refers to a bacterial extract that has been spray-dried as described herein and, for example, U. S. Patent App. Pub. No. 2024 / 0043895. A “spray-dried bacterial extract” is different from a “freeze-dried bacterial extract,” which refers to a bacterial extract that has been subjected to freeze drying, lyophilization, in situ vaporization, microwave radiation sublimation, and the like as described herein and, for example, in U.S. Patent No. 10,648,01.6KILPATRICK TOWNSEND 80240015.2

[0040] As used herein, the term “control bacterial extract” can refer to an equivalent bacterial extract that was produced using an unmodified production process. For example, where a bacterial extract is produced using a modified method, composition, and / or system, that bacterial extract is analyzed for protein synthesis activity in a CFPS reaction, and the control bacterial extract is an extract prepared using an unmodified or established method, composition, and / or system. In some instances, the control bacterial extract has not been frozen, thawed, rehydrated, or modified with the addition of any additive. For example, where a freeze-dried or spray-dried bacterial extract is analyzed for protein synthesis activity in a CFPS reaction, the control bacterial extract is an isolated liquid bacterial extract (that has not been previously frozen and thawed). In another example, where an additive is added to a freeze-dried or spray-dried bacterial extract and the impact of the additive on protein synthesis in a CFPS reaction is analyzed, the control extract can be a freeze-dried or a spray-dried extract without an additive that is reconstituted prior to testing.

[0041] As used herein, the term “lysed bacterial components” refers to cellular components of a lysed bacterium. For example, the term can include bacterial components needed to synthesize a protein of interest from a template nucleic acid encoding the protein in a cell-free reaction, such as ribosomes, amino acids, polymerases, and tRNAs, and the components of an active oxidative phosphorylation system. Additional components can be added to the lysed bacterial components, which in some cases are added to provide an energy source, such as exogenous ATP, GTP, glucose, glutamate, and / or pyruvate.

[0042] As used herein, the term “carbohydrate” refers to a macromolecule consisting of carbon, hydrogen, and oxygen atoms and having an empirical formula Cn(H2O)m, wherein m and n may be different numbers. Carbohydrates include monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0043] As used herein, the term “rehydrating” or “reconstituting,” in the context of a spray-dried bacterial extract, refers to suspending a spray-dried bacterial extract in a diluent such as water or a buffer to disperse the components of the bacterial extract. In the context of a freeze- dried bacterial extract, “rehydrating” or “reconstituting,” refers to suspending a freeze-dried bacterial extract in a diluent such as water or a buffer to disperse the components of the bacterial7KILPATRICK TOWNSEND 80240015.2extract. In some cases, the components of the spray / freeze-dried bacterial extract are solubilized after rehydration or reconstitution.

[0044] As used herein, the term “protein synthesis activity” refers to the capability of a protein synthesis system, which may be reflected or expressed in the protein yield (e.g., the amount of protein) from a protein synthesis reaction, e.g., a CFPS reaction, to produce a target protein relative to a control protein synthesis reaction. It may be expressed in an absolute term or in a relative term in comparison to a control protein synthesis reaction. In some cases, protein synthesis activity is indicated by the protein yield of the bacterial cell extract that was used to produce a recombinant protein in the reaction. In some cases, the reaction is a cell-free protein synthesis (CFPS) reaction.

[0045] As used herein, the term “control protein synthesis reaction” or “control reaction” can refer to an equivalent protein synthesis reaction that uses a control bacterial cell extract as a reagent in the reaction. For example, where a bacterial cell extract is produced using a modified method, composition, and / or system, and then used as a reagent in a protein synthesis reaction, the control protein synthesis reaction comprises a bacterial cell extract that is prepared using an unmodified method, composition, and / or system. The control bacterial cell extract is typically an extract that is known to previously and reliably produce an acceptable level of protein synthesis activity in a reaction. The meaning of the terms “control bacterial extract” and “protein synthesis activity” are provided above.

[0046] A nucleic acid is “operably linked” to another nucleic acid when it is placed into a functional relative location to another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0047] As used herein, the terms “nucleic acid” and “polynucleotide” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. The terms also include,8KILPATRICK TOWNSEND 80240015.2but are not limited to, single-stranded and double-stranded forms of DNA. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.

[0048] A “nucleic acid sequence” or a “polynucleotide sequence” includes a DNA or R A sequence which can function as a template from which a polypeptide will be translated. It will be understood by those of skill in the art that a DN A polynucleotide sequence must first be transcribed into RNA, and that the RNA is translated into a polypeptide. DNA can be transcribed into RNA either in vivo or in vitro. The methods of in vitro transcription of a DNA template are well known in the art. In some embodiments, the DNA template is subject to simultaneous in vitro transcription and translation, such as in a cell-free protein synthesis reaction.

[0049] As used herein, the term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a cell (i.e., a “host cell”) into which it has been introduced. In some cases, the vector is a plasmid or is derived from a plasmid, A “vector” as used here refers to a recombinant construct hi which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0050] An “expression cassette” refers to a polynucleotide sequence that directs bacterial cell machinery7cell i.e., “host cell” machinery7) to produce gene products. An expression cassette may contain one or more genes of interest and 5’ and 3’ regulatory sequences. The cassette may additionally contain at least one additional gene or genetic element to be co-transformed into the organism (i.e., a cell, plurality of cells, tissue, or animal). Where additional genes or elements are included, the components are operably linked. Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of the polynucl eotides to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene. The expression cassette will include in the 5’ to 3’9KILPATRICK TOWNSEND 80240015.2direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a polynucleotide of the disclosure, and a transcriptional and translational termination region ((i.e., termination region) functional in the cell or organism of interest. The promoters are capable of directing or driving expression of a coding sequence in a cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the cell or to each other.

[0051] As used herein, the term “non-natural amino acid,” “non-native amino acid,” or “nnAA” refers to amino acids that are not among the twenty naturally occurring amino acids that are the building blocks for all proteins that are nonetheless capable of being biologically engineered such that they are incorporated into proteins. Non-native amino acids (nnAAs) may include D-amino acids or any post-translational modifications of one of the twenty naturally occurring amino acids. A wide variety of non-native amino acids can be used in the methods of the disclosure. The non-native amino acid can be chosen based on desired characteristics of the non-native amino acid, e.g., function of the non-native amino acid, such as modifying protein biological properties such as toxicity, biodistribution, or half-life, structural properties, spectroscopic properties, chemical and / or photochemical properties, catalytic properties, ability to react with other molecules (either covalently or noncovalently), or the like. Non-native amino acids that can be used in the methods of the disclosure may include, but are not limited to, a non- native analogue of a tyrosine amino acid: a non-native analog of a glutamine amino acid; a non- native analog of a phenylalanine amino acid; a non-native analog of a serine amino acid; a non-native analog of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotin-analog containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an10KILPATRICK TOWNSEND 80240015.2elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar -containing amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an alpha-hydroxy containing acid; an amino thio acid containing amino acid; an alpha,alpha-disubstituted (a,a-disubstituted) amino acid; a beta-amino acid (p-amino acid); a cyclic amino acid other than proline, etc. As discussed herein, nnAAs can be used to introduce bioconjugation handles at genetically defined locations in a protein, such as in an antibody. For example (without limitations), the nnAA para-azidomethyl-l-phenylalanine (pAMF) is useful for strain-promoted azide-alkyne cycloaddition-based (SPAAC) copper-free click conjugation chemistry.

[0052] As used herein, the term “active oxidative phosphorylation system” in the context of a bacterial extract, refers to a bacterial extract that exhibits active oxidative phosphorylation during protein synthesis. For example, the bacterial extract can generate ATP using ATP synthase enzymes and reduction of oxygen. It will be understood that other translation systems known in the art can also use an active oxidative phosphorylation during protein synthesis. The activation of oxidative phosphorylation can be demonstrated by inhibition of the pathway using specific inhibitors, such as electron transport chain inhibitors.

[0053] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. All three terms apply 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, wherein 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.

[0054] A polypeptide “variant,” as die term is used herein, is a polypeptide diat typically differs from one or more polypeptide sequences specifically disclosed herein in one or more substitutions, deletions, additions and / or insertions.

[0055] A “substitution,” as used herein, denotes die replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively. A “conservative substitution” refers to a substitution of an amino acid such that charge, polarity, hydropathy 11KILPATRICK TOWNSEND 80240015.2(hydrophobic, neutral, or hydrophilic), and / or size of the side group chain is maintained.Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys and Arg; and His at pH of about 6; (ii) negatively charged amino acids GIu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic hydrophobic amino acids Ala, Vai, Leu and He; ( i) hydrophobic sulfur- containing amino acids Met and Cys, which are not as hydrophobic as Vai, Leu, and lie; (vii) small polar-uncharged amino acids Ser, Thr, Asp, and Asn (viii) small hydrophobic or neutral amino acids Gly, Ala, and Pro; (ix) amide-comprising amino acids Asn and Gin; and (xi) betabranched amino acids Thr, Vai, and He. Reference to the charge of an amino acid in this paragraph refers to the charge at pH 6-7.

[0056] As used herein, the term “recombinant gene” refers to a gene that (1) is not associated with ail or a portion of a polynucleotide in which die gene is found in nature, (2) is operatively- linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature. In some cases, a “recombinant gene” can refer to a gene that has been modified from its wild-type sequence. The term “recombinant gene” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by7such nucleic acids. As used herein, the term “recombinant protein” refers to (1) a protein that is expressed from a recombinant gene, or (2) a protein that is expressed in an environment where it is not naturally found, e.g., the protein is expressed from a gene that is cloned into a vector and expressed in a model organism or a host cell wherein the model organism or host cell does not naturally express that protein.

[0057] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Thus, a host cell is a recombinant host cell and includes the primary- transformed cell and progeny derived therefrom without regard to the number of passages.

[0058] As used herein, the term “fermentation” refers to any process or any culture that can be used to cultivate cells of the present disclosure for the production of cell extract. In this context, the term “fermentation” may be used interchangeably with the terms “production,12KILPATRICK TOWNSEND 80240015.2“cultivation,” and “culture.” The term “fermentation” does not strictly mean an anaerobic chemical process performed by cells to convert a substrate to a product. As used herein, “fermentation” includes all culture processes and parameters that may be used for the production of cell extract, including cultures where oxygen is present. Although, in some cases, the fermentation is an anaerobic fermentation.

[0059] As used herein, an “increase” or a “decrease” refers to a detectable positive or negative change in quantity from a comparison control, e.g., an established standard control (such as bacterial extract that is capable of cell-free protein synthesis). An increase is a positive change that is typically at least 10%, or at least 20%, or 50%, or 100%, and can be as high as at least 2-fold or at least 5-fold or even 10-fold of the control value. Similarly, a decrease is a negative change that is typically at least 10%, or at least 20%, 30%, or 50%, or even as high as at least 80% or 90% of the control value. Other terms indicating quantitative changes or differences from a comparative basis, such as “more,” “less,” “higher,” and “lower,” as well as terms indicating an action to cause such changes or differences, such as “increase,” “promote,” “enhance,” “decrease,” “inhibit,” and “suppress,” are used herein in the same fashion as described above.

[0060] As used herein, the term “comprise,” including variations such as “comprises” or “comprising,” is used to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0061] As used herein, the term “consists of’ or “consisting of’ is used to mean that a method, process or composition of matter (e.g., amino acid sequence) has the recited steps and / or components and no additional steps or components.

[0062] As used herein, the term “consists essentially of’ or “consisting essentially of’ refers to the possibility of including additional steps or elements that are not recited in the claim, so long as the additional steps or elements do not materially affect the basic and novel characteristics of the invention.

[0063] As used herein, the term “about,” when modifying any amount, refers to the variation in that amount typically encountered by one of skill in the art, e.g., in protein synthesis13KILPATRICK TOWNSEND 80240015.2experiments. For example, the term “about” refers to the normal variation encountered in measurements for a given analytical technique, both within and between batches or samples. Thus, the term about can include variation of + / - 1-10% of the measured value, such as + / - 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% variation of the measured value. The amounts disclosed herein include equivalents to those amounts, including amounts modified or not modified by the term “about.”

[0064] As used herein the singular forms “a,” “and,” and “the” include plural referents unless die context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality' of such cells and reference to “die 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 to one of ordinary skill in the ait to which this disclosure belongs unless clearly indicated otherwise.HI. Bacterial Cells

[0065] Hie present disclosure provides bacterial cells for use in high cell density fermentations, e.g., in fermentations for producing bacterial cell extract.

[0066] Suitable bacteria include gram-negative bacteria and gram-positive bacteria, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.. Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, and Pseudomonas such as P. aeruginosa, and Steptomyces. In some embodiments, the bacterial cell is from an Escherichia species, such as Escherichia coli or a derivative thereof. In some embodiments, the bacterial cell is any E. coli strain that is known to one of skill in the art. hi some embodiments, the E. coli strain is an A (K-12), B, C or D strain.

[0067] The bacterial cell may be used to produce a bacterial cell extract for use hi CFPS reactions. See, e.g., Figure 1. In some embodiments, the bacterial cell may have reduced nuclease and / or phosphatase activity which increases cell-free synthesis efficiency. For example, the bacterial cell can have mutations in the genes encoding the nucleases RNase E and RNase A.

[0068] In some embodiments, the bacterial cell may also have mutations to stabilize components of the CFPS reaction, such as deletions in genes such as tnaA, speA, sdaA, gshA,14KILPATRICK TOWNSEND 80240015.2and sdaB, which prevent degradation of the amino acids tryptophan, arginine, serine, cysteine, and serine, respectively, in a CFPS reaction.

[0069] In some embodiments, the bacterial cell may also have mutations to stabilize the protein products of cell-free synthesis such as knockouts in the proteases OmpT or LonP.

[0070] In some embodiments, the bacterial cell may also have a wild-type OmpT. In some embodiments, the bacterial cell may also have a mutant OmpT which has altered, enhanced, or diminished OmpT activity.

[0071] In some embodiments, the bacterial cell may also comprise genes that encode 1, 2, or more exogenous protein chaperones. Examples of exogenous protein chaperones include disulfide isomerases (e.g., DsbA, DsbB, DsbC, and DsbD), prolyl isomerases or peptidyi-prolyl cis-trans isomerases (e.g., FkpA and SlyD). In some cases, chaperone proteins can help improve the folding and / or solubility of a desired protein that is produced in a CFPS reaction, thereby- increasing their yield in the CFPS reaction.

[0072] In some embodiments, the bacterial cell may comprise modifications or mutations as discussed in Groff, Dan et al. “Development of an E. colt strain for cell-free ADC manufacturing.” Biotechnology and bioengineering vol. 119,1 (2022): 162-175; or Cai, Qi et al. “A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems.” Biotechnology progress vol. 31,3 (2015): 823-31.

[0073] hi some embodiments, the bacterial cell is an auxotrophic cell that is unable to synthesize one or more amino acids required for its growth, proliferation or survival, e.g., glutamine, cysteine, and / or arginine. Accordingly, an auxotroph may be disabled or deficient for one or more target genes involved in the biosynthesis of an amino acid or in the regulation of such a biosynthetic pathway. In many embodiments, the one or more target genes is rendered disabled or deficient by gene deletion (partial or complete deletion), gene knockout, gene substitution, or introduction of one or more amino acid mutations in the protein. Alternatively, the one or more target genes is inactivated by antisense RNA, inhibitory RNA, or other RNA interference methods. In some cases, the gene that is disrupted in an auxotrophic cell is glnA, cysE, and / or argA. hi some embodiments, the auxotrophic bacterial strain is transformed with a plasmid having an expression cassette comprising a gene that restores the ability of the15KILPATRICK TOWNSEND 80240015.2auxotrophic strain to grown in the absence of the one or more amino acids required for its growth, proliferation or survival. For example, a cell that has a glnA deletion is transformed with a plasmid having an expression cassette comprising a functional glnA gene.

[0074] In some embodiments, the bacterial cell lacks a functional peptide chain Release Factor 1 (RF1 ) protein. In some cases, the deletion or depletion of RF1 is useful in the production of proteins that comprise miAAs. In some cases, to maintain the translational termination activities required for growth and function of RF1 -deficient cells, e.g., E. coli cells, the stop codons that are normally recognized by RF1 are converted to stop codons that can be recognized by RF2, which means the TAG stop codons are mutated to either TGA or TAA; both can be recognized by Release Factor 2 (RF2). In some embodiments, the gene encoding RF1, prfA, is knocked out from die genome of the E. coll strain. In some embodiments, the gene encoding RF1, prfA, is mutated to encode additional cleavage sites in RF1, and RF1 is cleaved during the bacterial cell extract manufacturing process.

[0075] In some embodiments, the bacterial cells also have an inactivated gene encoding a protein selected from tryptophanase, arginine decarboxylase, L-serine deaminase and gamma¬ glutamylcysteine synthase. Performing CFPS with extracts from genetically modified cells that are deficient in one or more amino acid metabolizing enzymes can improve protein yield.Descriptions of such E. coll cells and extracts thereof are found in, for example, U. S. Patent No.7,312,049.

[0076] Standard methods for engineering bacterial cells and for expressing proteins from bacterial cells are known to one of ordinary skill in the art. 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, N. Y.; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA.); Ausubel, F. M., et al., Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology (Volume 152 Academic Press, Inc., San Diego, Calif. 1987); and PCR Protocols: A Guide to Methods and Applications (Academic Press, San Diego, Calif. 1990). Standard methods also appear in Bindereif, Schon, & Westhof (2005) Handbook of RNA Biochemistry, Wiley-VCH,16KILPATRICK TOWNSEND 80240015.2Weinheim, Germany, which describes detailed methods for RNA manipulation and analysis, and Walker, J. M., (2009), and The Protein Protocols Handbook, 3rd ed., Humana Press, New York, N. Y., which describes detailed methods for protein manipulation and analysis. These disclosures are incorporated by reference herein.IV. Methods for Producing Bacterial Cell ExtractsA. Processes for Culturing Bacteria

[0077] Disclosed herein are bioreactor process methods for producing a high cell density (also referred to as “biomass”) fermentation that provides a large amount of bacteria for producing bacterial cell extracts. A bioreactor can be run in any mode, such as batch, fed-batch, or continuous, or a combination thereof, and the mode (also referred to as “process”) is typically selected in accordance with the application purpose.

[0078] In general, a continuous process is used to produce large amounts of bacteria. While a continuous process has many advantages, including the ability to achieve a state of sustained cell growth or sustained production of a desired recombinant protein, the process has challenges as wrell, such as increased risk of contamination in the culture, potential genetic drift over the course of a long culture period, and challenges with cell ha est related to the handling of large volumes and multiple lots of liquid culture.

[0079] In contrast, provided herein are fed-batch processes, wdiich reduce fermentation duration and processing volume (thereby simplifying liquid handling) while providing cultures with high cell densities. Further, fed-batch processes mitigate the risk of contamination and genetic drift. In some embodiments, bacterial cells are cultured to a cell density of at least 50, at least 60, at least 85, at least 100, at least 150, about 70 to about 150, about 80 to about 150, about 90 to about 1 0, about 100 to about 150, about 110 to about 150, about 120 to about 150, about 125-140, about 50 to about 130, about 60 to about 130, about 70 to about 130, about SO to about 130, as measured using optical density at about 595-600 nm (OD595-600).

[0080] A continuous process is an open system where a growth-limiting reagent is provided and / or metabolic waste is removed over time. In some cases, cell growth can be maintained at a constant cell density for extended periods. Two well-known continuous systems are chemostats and turbidostats. In the chemostat system, sterile media is fed in at a constant rate while media containing bacteria is removed at the same rate. The turbidostat system uses a photocell to 17KILPATRICK TOWNSEND 80240015.2measure absorbance or turbidity and regulates the inflow of sterile media and outflow of bacteria according to preset signals.

[0081] In contrast, a batch process is a closed system where bacteria are cultured for a period of time and no reagents are added once the media has been inoculated with bacteria (i.e., after the culture or fermentation has started). In a fed-batch process, additional reagents are added to the culture to extend the duration of the batch culture to achieve higher cell densities, or, in some cases, as a trigger to switch cell metabolism from cell growth to recombinant protein production. Although a fed-batch system is not strictly a closed system, die risk of contamination in the culture is reduced compared to a continuous (open) system, hi a batch or fed-batch system, the step where cells are harvested from the bioreactor indicates that the fermentation has terminated, hi a batch or fed-batch system, cells that are harvested are not further cultured,

[0082] In some cases, the total amount of cells diat is harvested, from a batch or fed-batch system may be less than the total amount of cells that is harvested from a continuous system. In some embodiments, at least 80%, at least 90%, or all of the cells in a batch or fed-batch system are harvested at the end of the fermentation. In contrast, in a continuous system, less than 70%, less than 80%, less than 90%, or less than 95% of the cells are harvested, for example, to produce a sublot, and the cells that remain in the bioreactor are cultured in the continuous system. After a period of time in culture, again, less than 70%, less than 80%, less than 90%, or less than 95% of the cells are harvested to produce anodier sublot, and the cells that remain the cells can be cultured in the continuous system. The accumulation of sublots from a continuous system may in some cases result in harvesting a greater total amount of cells than in a batch or fed-batch system. In spite of this, it can still be advantageous to perform a batch or fed-batch process instead of a continuous process because a fed-batch process, in general, has a shorter fermentation duration from start (i.e., inoculation) to finish (i.e., harvest) and a small volume of liquid that needs to be handled. In some embodiments, the fermentation duration is less than about 160 hours, less than about 120 hours, less than about 100 hours, less than about 50 hours, less than about 20 hours, less than about 18 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, or about 10 hours.18KILPATRICK TOWNSEND 80240015.2

[0083] In a batch or fed-batch system, liquid handling is generally simpler than in a continuous system. For example, a batch or fed-batch system typically comprises only I lot of liquid culture while a continuous system can comprise more than 1 lot, and can include, in some cases, 10, 10-15, 12-15, or more sublots of culture.

[0084] In some embodiments, a batch or fed-batch system of about 10-20 kL, e.g., 17 kL, can produce a total dry cell weight (DCW) of at least 900 kg, at least 1,000 kg, at least 2,000 kg, at least 3,000 kg, about 900 kg to about 3,000 kg, about 1,000 kg to about 3,000 kg, or about 2,000 kg to about 3,000 kg. In contrast, for example, a continuous system of 200 L (e.g., a 200 L fermenter with a 130 L working volume (L wv)) can produce about 290-500 kg, about 300-350 kg, about 290 kg, about 300 kg, about 310 kg, about 320 kg, about 330 kg, about 340 kg, about 350 kg, about 360 kg, about 370 kg, about 380 kg, about 390 kg, or about 400 kg of wet cell weight (WCW). Wet cell weight (WCW) refers to the weight of the cells obtained after die liquid culture of cells is centrifuged to produce a pellet of cells and the supernatant (e.g., fermentation broth) is discarded. In determining WCW, the pellet of cells is wet. In contrast, dry cell weight (DC ) refers to the weight of the cells obtained after the wet cell pellet is dried to remove all the liquid. In another example, a continuous system of 3 kL (e.g., a 3 kL fermenter with a 1.3 kL working volume (L wv)) can produce about 3000-5000 kg, about 3800-4600 kg, about 3900 kg, about 4000 kg, about 4100 kg, about 4200 kg, about 4300 kg, about 4400 kg, about 4500 kg, about 4600 kg, about 4700 kg, or about 4800 kg of WCW.

[0085] The culturing methods disclosed here produce large amounts of bacteria, from which large amounts of bacterial cell extract can be harvested for use in cell-free protein synthesis (CFPS) reactions. In some embodiments, a batch or fed-batch system of about 10-20 kL, e.g., 17 kL, can produce at least 1,000 kg, at least 1,100 kg, at least 1,200 kg, at least 1,300 kg, at least 1.400 kg, at least 1,500 kg, at least 1,600 kg, at least 1,700 kg, at least 1,800 kg, at least1,900 kg, at least 2,000 kg, at least 2, 100 kg, at least 2,200 kg, at least 2,300 kg, at least 2.400 kg, at least 2,500 kg, at least 2,600 kg, at least 2,700 kg, at least 2,800 kg, at least 2.900 kg, at least 3,000 kg, at least 3,100 kg, at least 3,200 kg, at least 3,300 kg, at least 3.400 kg, at least 3,500 kg, at least 3,600 kg, at least 3,700 kg, at least 3,800 kg, at least 3.900 kg, at least 4,000 kg of bacterial cell extract. In contrast, for example, a continuous system19KILPATRICK TOWNSEND 80240015.2of 200 L can produce less than 600 kg, less than 800 kg, or less than 1,000 kg of bacterial cell extract.

[0086] In some embodiments, a batch or fed-batch fermentation produces a yield of bacterial cell that is at least 0.3 L / L, at least 0.35 L / L, at least 0.4 L / L, at least 0.45 L / L, at least 0.5 L / L, at least 0.55 L / L, at least 0.6 L / L, at least 0.65 L / L, at least 0.7 L / L, at least 0.75 L / L, at least 0.8 L / L, at least 0.85 L / L, at least 0.9 L / L, at least 0.95 L / L, at least 1 L / L; wherein the unit “L / L” corresponds to L of bacterial cell per L of fermentation broth or liquid culture.

[0087] In some cases, it may be advantageous to use a semi-continuous process, which is a hybrid of a batch or fed-batch process and a continuous process. The semi-continuous process comprises harvesting all but a small amount of a completed batch or fed- batch fermentation and leaving a small amount of cells available to use as an inoculum for the next filling of the vessel. The potential accumulation of toxins and unwanted metabolites is prevented due to the medium exchange. The semi-continuous process can also allow for the segregation of cells or product into sublots categorized by time, which can aid in quality control and troubleshooting efforts.

[0088] The bacterial culture can be obtained as follows. The bacterial cells of choice are grown up overnight in any of a number of growth media and under growth conditions that are well known in the art and easily optimized by a practitioner for growth of the particular bacteria. In general, isolated strains of bacteria are grown in media until they reach balanced exponential growth phase or stationary phase. This can be between 106to 109cells per mL. In some embodiments, the culture is harvested when the pH of the culture rises above a set point indicating the depletion of glucose in the media. Tire bacterial culture can be grown to an optical density at 595-600 nm (OD595-600) of 10 to 60, depending on the bacterial strain used. 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. In some embodiments, the target cell density of a culture at OD595-600, such as a high cell density fed-batch culture, that is at least 55, at least 60, at least 100, about 55 to about 150, about 60 to about 150, 80 to about 150, about 100 to about 150, or about 120 to about 150.

[0089] The bacterial cells can be grown in medium containing glucose and phosphate, where the glucose is present at a concentration of at least about 0.25% (weight / volume), more usually at least about 1%; and usually not more than about 4%, more usually not more than about 2%. An 20KILPATRICK TOWNSEND 80240015.2example of such media is 2YTPG medium, however one of skill in the art will appreciate that many culture media can be adapted for this purpose, as there are many published media suitable for the growth of bacteria such as E. coli, using both defined and undefined sources of nutrients. Optimal media and growth conditions are known for specific species. For example, E. coli are commonly grown in YT broth (yeast extract and tryptone) or variants thereof. The media can be defined (synthetic) or complex (undefined). In some embodiments, the medium comprises a mixture of amino acids (including asparagine, methionine, and / or proline), glucose, trace metals (including sodium citrate, ferric chloride, sodium molybdate, cobalt chloride, zinc sulfate, cupric sulfate, manganese sulfate, and / or boric acid) sulfate, vitamins (including nicotinic acid (niacin), para-aminobenzoic acid, pantothenic acid (PABA), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), riboflavin (vitamin B2), and / or thiamine (vitamin Bl)), potassium phosphate, ammonium sulfate, potassium chloride, sodium citrate, magnesium sulfate, and / or potassium hydroxide, dissolved in water. In some embodiments, the medium is as discussed below in the Examples section, for example, without limitations, the 1-17 medium in Example 1.

[0090] Detailed descriptions of culturing bacteria, for example for producing bacterial cell extracts, are discussed in, for example, U. S. Patent No. 10,190,145 and U. S. Patent App. Publ. Nos. 2024 / 0043895. Methods of culturing bacteria are described in, e.g., Zawada et al., Biotechnol. Bioeng., 108(7):1570-1578 (2011); Zawada, J. “Preparation and Testing ofE.coli S30 In vitro Transcription Translation Extracts”, Douthwaite, J. A. and Jackson, R. H. (eds.). Ribosome Display and Related Technologies: Methods and Protocols, Methods in Molecular Biology^, vol. 805, pp. 31-41 (Humana Press, 2012); Jewett et al, Molecular Systems Biology: 4, 1-10 (2008); Shin J. andNorieaux V., J. Biol. Eng., 4:8 (2010). Suitable media for culturing bacteria are well-known to one of ordinary skill in the art. Examples of media recipes may be found, for example, in Allikian et al., (2019). Fundamentals of Fermentation Media. In:Berenjian, A. (eds) Essentials in Fermentation Technology. Learning Materials in Biosciences. Springer, Cham; doi.org / 10.1007 / 978-3-030-16230-6_2.

[0091] In some instances, an engineered E. coli strain (e.g., engineered K-12 derived E. coll strain KGK10) is cultured to mid-log phase (OD595 of about 45 OD or about 140 g / L of cell wet weight) using glucose and amino acid fed-batch fermentation at a maximal growth rate of about21KILPATRICK TOWNSEND 80240015.20.7 h'3. Glucose can be increased during culturing such that there is excess glucose during harvest. See, e.g., Zawada et al., Biotechnol. Bioeng., 108(7): 1570-1578 (2011).

[0092] In some embodiments, e.g., during high cell density fed-batch fermentation, the bacterial cells are fed, for example, a culture media or a reagent, at a feed rate of about 10 mL / L / hour to about 95 mL / L / hour, about 12 mL / L / hour to about 95 mL / L / hour, about 20 mL / L / hour to about 95 mL / L / hour, about 30 mL / L / hour to about 95 mL / LZhour, about 40 mL / L / hour to about 95 mL / L / hour, about 50 mL / L / hour to about 95 mL / L / hour, about 60 mL / L / hour to about 95 mL / L / hour, about 70 mL / L / hour to about 95 mL / L / hour, or about 80 mL / L / hour to about 95 mL / L / hour; wherein the unit mL / L / hour corresponds to mL of culture media or reagent for every L of culture volume per hour of fermentation, hi some embodiments, where die culture volume is 1 L, die unit that is used is mL / hour, as discussed in Example 2 below.

[0093] Dissolved oxygen (DO) is a key driver affecting culture growth in bioreactors.However, cell lines and strains can have different oxygen needs. For example, an aerobic culture of mammalian cell cultures and many microbial cultures consume oxygen and an effective bioreactor DO control system is therefore required to keep the dissolved oxygen concentration stable, hi some examples, an aerobic culture may require a DO concentration of over 30%. DO concentrations may be controlled via adjusting the agitation via impellers, the influx of gases including air and oxygen using gas spargers, and / or gas composition. In contrast, an anaerobic fermentations of certain microorganisms are performed without oxygen. In anaerobic fermentations, the removal of oxygen from bioreactors can be achieved through sparging nitrogen or other anaerobic gasses.

[0094] In some embodiments, the DO setpoint is fixed, for example, at about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In some embodiments, the DO setpoint is fixed, for example, at about 0-50%, about 10-50%, about 10%-30%, about 20-40%, or about 30-50%. In some embodiments, DO is controlled by a DO cascade. As used herein, a DO cascade refers to a specific sequence of parameter changes that are employed by the bioreactor to increase the DO levels within the bioreactor. As the cascade progresses, different parameters, e.g., increased agitation, aeration, oxygen enrichment, and back pressure, of increasing or decreasing intensity are initiated to ensure DO control is sufficient to22KILPATRICK TOWNSEND 80240015.2optimize yield, e.g., protein overexpression. In some embodiments, one parameter is increased until it reaches its maximum intensity before another parameter is increased. In some embodiments, when one parameter is increased, the remaining parameters are held constant or reduced. In some embodiments, the intensity of a parameter is increased incrementally or gradually (as in a gradient), in a stepwise fashion, or a combmation thereof.

[0095] In some embodiments, a DO cascade comprises increased agitation (also termed “cascading agitation”)- Typically, increased agitation is the first step of a DO cascade. A DO cascade increases agitation speeds in a bioreactor in order to maintain conditions for cell growth and product formation as a fermentation progresses. After agitation speeds reach a maximum intensity, die agitation speed is held at that maximum setpoint while other fermentation parameters are increased to continue maintaining conditions, including DO levels, that allow for cell growth and product formation as a fermentation continues. In some embodiments, the maximum agitation speed is the intensity that will allow the cells in the culture to achieve a target titer, productivity, and / or yield. In some embodiments, the maximum agitation speed is the maximum intensity that the cells in the culture can tolerate without unfavorable effects on cell vitality, titer, productivity, and / or yield. In some embodiments, the maximum agitation speed is the maximum intensity that the bioreactor is capable of. Thus, in some embodiments, to maintain a DO setpoint, agitation is increased until it reaches a maximum agitation speed before the other parameters are changed, e.g., before aeration is increased, before oxygen enrichment is increased, and / or before back pressure is decreased.

[0096] In contrast, in some embodiments, a DO cascade comprises maintaining lower agitation speeds. In these embodiments, to maintain a DO setpoint, agitation speeds are not increased substantially. Instead, in these embodiments, agitation speeds are maintained at a minimum agitation speed limit. Lower agitation speeds can decrease the shear stress experienced by the cells in a liquid culture, but the lower agitation speeds must still allow for proper mixing of the liquid culture. Thus, in some embodiments, a DO cascade comprises agitation speeds at a minimum agitation speed (i.e., the speed is not too slow so that the culture is mixed sufficiently), and if the agitation speed is increased, the agi tation speeds that do not exceed a minimum agitation speed limit (i.e., the speed is not too fast to avoid agitating the culture too much). In some embodiments, a DO cascade comprises maintaining agitation speeds at the minimum23KILPATRICK TOWNSEND 80240015.2agitation speed limit while other parameters are changed (e.g., aeration is increased, oxygen enrichment is increased, and / or back pressure is decreased) to maintain a DO setpoint. In general, the minimum agitation speed limit is less than die maximum agitation speed limit. In some embodiments, a DO cascade comprising a minimum agitation speed limit produces greater cell vitality, titer, productivity, and / or yield than a DO cascade comprising a maximum agitation speed limit as discussed above.

[0097] Agitation is achieved using an impeller that is moved by a motor. Tip speed is die distance that a point on the edge of an impeller travels in a set amount of time. Tip speed is a function of die impeller’s diameter and rpm. In some embodiments, the maximum agitation speed is limited to a certain value, for example, to avoid extensive shear stress to the cells and / or to provide sufficient mixing of the culture. In some embodiments, the maximum agitation speed is fixed at about 200 rpm, about 250 rpm, about 400 rpm, about 500 rpm, about 700 rpm, about 900 rpm, about 1200 rpm, about 1400 rpm, about 1600 rpm, about 1800 rpm, about 1900 rpm, about 2000 rpm, about 2100 rpm, about 2200 rpm, about 2400 rpm, about 2600 rpm, about 2800 rpm, about 3000 rpm, about 3200 rpm, about 3400 rpm, about 3500 rpm, about 3600 rpm, about 3700 rpm, or about 3800 rpm. In some embodiments, the maximum agitation speed is fixed at about 200-1200 rpm, about 200-500 rpm, about 400-700 rpm, about 600-900 rpm, about 800-1100 rpm, about 900-1200 rpm, about 1000-1300 rpm, about 1200-1500 rpm, about 1400-1700 rpm, about 1600-1900 rpm, about 1800-2100 rpm, about 2000-2300 rpm, about 2200- 2500 rpm, about 2400-2700 rpm, about 2600-2900 rpm, about 2800-3100 rpm, about 3000- 3400 rpm, about 3100-3500 rpm, about 3200-3600 rpm, about 3300-3700 rpm, or about 3400-3800 rpm. In some embodiments, the volume of the fermenter is 200 liter and the maximum agitation speed is fixed at about 400 rpm, about 410 rpm, about 420 rpm, about 430 rpm, about 440 rpm, about 450 rpm, about 460 rpm, about 470 rpm, about 480 rpm, about 490 rpm, about 400-460 rpm, about 410-470 rpm, about 420-480 rpm, or about 430-490 rpm. In some embodiments, the volume of the fermenter is 10 liter and the maximum agitation speed is fixed at 1200 rpm, about 1300 rpm, about 1400 rpm, about 1500 rpm, about 1600 rpm, about 1700 rpm, about 1800 rpm, about 1900 rpm, about 2000 rpm, about 1200-1700 rpm, about 1300- 1800 rpm, about 1400-1900 rpm, or about 1500-2000 rpm. In some embodiments, the volume of the fermenter is 1 liter and the maximum agitation speed is fixed at 1800 rpm, about 1900 rpm, about 2000 rpm, about 2100 rpm, about 2200 rpm, about 1600-1900 rpm, about 1800-2100 rpm.24KILPATRICK TOWNSEND 80240015.2about 2000-2300 rpm, or about 2200-2500 rpm. In some embodiments, the volume of the fermenter is 250 mL and the maximum agitation speed is fixed at about 3000 rpm, about 3200 rpm, about 3400 rpm, about 3500 rpm, about 3600 rpm, about 3700 rpm, about 3800 rpm, about 3000-3400 rpm, about 3100-3500 rpm, about 3200-3600 rpm, about 3300-3700 rpm, or about 3400-3800 rpm. In some embodiments, the maximum agitation speed is measured as the maximum impeller tip speed. In some embodiments, the maximum impeller tip speed is fixed not to exceed about 2.2 m / s, about 2.4 m / s, about 2.6 m / s, about 2.8 m / s, about 3.0 m / s, about 3.2 m / s, about 3.4 m / s, about 3.6 m / s, about 3.8 m / s, about 4.0 m / s, about 4.2 m / s, about 4.4 m / s, about 4.7 m / s, about 5.0 m / s, about 5.2 m / s, about 5.4 m / s, about 5.7 m / s, or about 6.0 m / s. In some embodiments, the maximum impeller tip speed is fixed at about 2.2-4.7 m / s, about 2.2-3.8 m / s, about 3.0-4.0 m / s, about 3.5-4.5 m / s, about 4.0-4.7 m / s, or about 5.0-6.0 m / s. In some embodiments, the maximum impeller tip speed limit fixed at about 5.0-6.0 m / s. In some embodiments, the maximum impeller tip speed limit does not exceed about 5.0 m / s or does not exceed about 6.0 m / s.

[0098] In some embodiments, a DO cascade comprises increased aeration or air sparging (also termed “cascading air flow,” “cascading aeration,” and “cascading air sparging”). When maximum agitation speed (discussed in the preceding paragraph) has been reached, aeration is increased. In some embodiments, aeration is increased up to its maximum rate. In some embodiments, aeration is at least 500 sLPM, at least 550 sLPM, at least 600 sLPM, at least 650 sLPM, at least 700 sLPM, at least 750 sLPM, at least 800 sLPM, at least 850 sLPM, at least 900 sLPM, at least 950 sLPM, or at least 1,000 sLPM. In some embodiments, aeration is at least 500-1000 sLPM, at least 500-750 sLPM, at least 650-900 sLPM, at least 800-1000 sLPM. In some embodiments, the volume of the fermenter is 200 liter and the maximum aeration is 40 sLPM, 40.5 sLPM, 41 sLPM, 41.5 sLPM, 42 sLPM, 42.5 sLPM, 43 sLPM, 43.5 sLPM, 44 sLPM, 44.5 sLPM, 45 sLPM, 45.5 sLPM, 46 sLPM, 46.5 sLPM, 47 sLPM, 47.5 sLPM, 48 sLPM, 48.5 sLPM, 49 sLPM, 49.5 sLPM, or 50 sLPM. In some embodiments, the volume of the fermenter is 10 liter and the maximum aeration is 4.0 sLPM, 4.1 sLPM, 4.2 sLPM,4.3 sLPM, 4.4 sLPM, 4.5 sLPM, 4.6 sLPM, 4.7 sLPM, 4.8 sLPM, 4.9 sLPM, 5.0 sLPM, 5.1 sLPM, 5.2 sLPM, 5.3 sLPM, 5.4 sLPM, or 5.5 sLPM. In some embodiments, the volume of the fermenter is I liter and the maximum aeration is 0.1 sLPM, 0.2 sLPM, 0.3 sLPM, 0.4 sLPM, 0.5 sLPM, 0.6 sLPM, 0.7 sLPM, 0.8 sLPM, 0.9 sLPM, 1.0 sLPM, 1.1 sLPM, 1.2 sLPM,25KILPATRICK TOWNSEND 80240015.21.3 sLPM, 1.4 sLPM, 1.5 sLPM, 1.6 sLPM, 1.7 sLPM, 1.8 sLPM, 1.9 sLPM, or 2.0 sLPM. In some embodiments, the volume of the fermenter is 250 mL and the maximum aeration is 0.05 sLPM, 0.1 sLPM, 0.15 sLPM, 0.2 sLPM, 0.25 sLPM, 0.3 sLPM, 0.35 sLPM, 0.4 sLPM, 0.45 sLPM, or 0.5 sLPM.

[0099] In some embodiments, a DO cascade comprises increased oxygen enrichment (also termed “cascading oxygen,” “cascading pure oxygen,” “cascading oxygen flow,” or “cascading pure oxygen flow”). The air that is sparged into the system (as discussed in the preceding paragraph) is gradually replaced by pure oxygen until only pure oxygen is sparged into the bioreactor. In some embodiments, pure oxygen is added to the system at about 0 sLPM, about 100 sLPM, 125 sLPM, about 150 sLPM, about 175 sLPM, about 200 sLPM, 225 sLPM, about 250 sLPM, about 275 sLPM, about 300 sLPM, 325 sLPM, about 350 sLPM, about 375 sLPM, about 400 sLPM, about 500 sLPM, about 600 sLPM, about 700 sLPM, about 800 sLPM, or about 900 sLPM. In some embodiments, pure oxygen is added to the system at about 0-900 sLPM, about 0-300 sLPM, about 200-500 sLPM, about 400-700 sLPM, about 600-900 sLPM, 200-250 sLPM, or 210-240 sLPM.

[0100] In some embodiments, a DO cascade comprises increased back pressure in addition to the increased agitation and aeration to maximize the dissolved oxygen in the cell culture. In some embodiments, a DO cascade comprising increased back pressure is applied where the volume of the fermenter is greater than 1 L. In some embodiments, the back pressure is about 5 psi, about 10 psi, or about 15 psi. In some embodiments, the back pressure is about 5-15 psi, about 5-12 psi, about 7-15 psi, about 5-10 psi, or about 10-15 psi. hi some embodiments, the volume of the fermenter is 250 mL to 1 L, 1 L, or 250 mL, and there is no increased back pressure.

[0101] The oxygen uptake rate (OUR) is the amount of oxygen consumed by microorganisms per unit of time. OUR is typically measured in mmoles of oxygen that is consumed per liter of the culture per hour (mmol / L / h). In some embodiments, the OUR is at least about 200 mmol / L / h, at least about 220 mmol / L / h, at least about 240 mmol / L / h, at least about 260 mmol / L / h, at least about 280 mmol / L / h, at least about 300 mmol / L / h, at least about 320 mmol / L / h, at least about 340 mmol / L / h, at least about 360 mmol / L / h, at least about 380 mmol / L / h, at least about 400 mmol / L / h, 420 mmol / L / h, at least about 440 mmol / L / h, at least about 460 mmol / L / h, at least about 480 mmol / L / h, at least about 500 mmol / L / h, about 250 mmol / L / h, about 300 mmol / L / h,26KILPATRICK TOWNSEND 80240015.2about 350 mmol / L / h, about 400 mmol / L / h, about 450 mmol / L / h, about 500 mmol / L / h, about 550 mmol / L / h, about 200 mmol / L / h to about 550 mmol / L / h, about 300 mmol / L / h to about 550 mmol / L / h, about 400 mmol / L / h to about 550 mmol / L / h, or about 500 mmol / L / h to about 550 mmol / L / h.

[0102] The oxygen transfer rate (OTR) is rate of oxygen delivery from gas into liquid, such as the transfer of oxygen gas into the liquid culture in die bioreactor. The oxygen transfer is typically achieved via agitation of the liquid culture and is typically measured in mmoles of oxygen that is transferred per liter of the culture per hour (mmol / L / h). In some embodiments, die OTR is at least about 200 mmol / L / h, at least about 220 mmol'L / h, at least about 240 mmol / L / h, at least about 260 mmol / L / h, at least about 280 mmol / L / h, at least about 300 mmol / L / h, at least about 320 mmol / L / h, at least about 340 mmol / L / h, at least about 360 mmol / L / h, at least about 380 mmol / L / h, at least about 400 mmol / L / h, 420 mmol / L / h, at least about 440 mmol / L / h, at least about 460 mmol / L / h, at least about 480 mmoVL / h, at least about 500 mmol / L / h, about250 mmol / L / h, about 300 mmol / L / h, about 350 mmol / L / h, about 400 mmol / L / h, about450 mmol / L / h, about 500 mmol / L / h, about 550 mmol / L / h, about 200 mmol / L / h to about 550 mmol / L / h, about 300 mmol / L / h to about 550 mmol'L / h, about 400 mmol / L / h to about 550 mmol'L / h, or about 500 mmol / L / h to about 550 mmolL / h.

[0103] Bacterial growth rate refers to how quickly a population of bacteria increases in number over time. This rate can be influenced by various factors such as nutrient availability, temperature, pH, and oxygen levels. Bacterial growth rate can be expressed as the amount of time it takes for the bacteria to double in quantity, such as in units of per hour (h‘l) or the number of doublings per hour. In some embodiments, e.g., during high cell density fed-batch fermentation, the bacterial cells are fed, for example, a culture media or a reagent, at a feed rate that achieves a growth rate of about 0,2 h’!, about 0.25 h’1, 0.3 h1, about 0.35 h"3, 0.4 h’!, about 0.45 h’3, about 0.5 h"1, about 0.55 h'!, about 0.6 IL, about 0.65 h“3, about 0.7 hr. about 0.75 h"1, about 0.8 h"3, about 0.85 h’1, about 0.9 h"!, about 0.95 h"1, or about 1 hL In some embodiments, e.g., during high cell density fed-batch fermentation, the bacterial cells are fed, for example, a culture media or a reagent, at a feed rate that achieves a growth rate of about 0.5 doublings per hour, about 0.55 doublings per hour, about 0.6 doublings per hour, about 0.65 doublings per hour, about 0.7 doublings per hour’, about 0.75 doublings per hour, about 0.8 doublings per hour.27KILPATRICK TOWNSEND 80240015.2about 0.85 doublings per hour, about 0.9 doublings per hour, about 0.95 doublings per hour, about 1.0 doublings per hour, about 1.05 doublings per hour, about 1.1 doublings per hour, or about 1.15 doublings per hour.

[0104] Specific growth rate is a useful parameter for monitoring or evaluating the behavior of a bacterial cell in a liquid culture. Specific growth rate can also be modulated in a fed-batch operation to achieve maximal protein expression. Specific growth rate, which may be expressed in units of p or h’1, is the rate of increase of biomass of a cell population per unit of initial biomass, and can be determined according to the formula below (Equation 1).Equation 1:sO’ mas# sf prsd&cW 1 ^ 1a / * Sims * Uiw I

[0105] In some embodiments, e.g„, during high cell density fed -batch fermentation, the bacterial cells are fed, for example, a culture media or a reagent, at a feed rate that achieves a specific growth rate of at least 0.05 p, at least 0.06 p, at least 0.07 p, at least 0.08 p, at least 0.09 p, at least 0.10 p, at least 0.11 p, at least 0.12 p, at least 0.13 p, at least 0.14 p, at least 0.15 p, at least 0.16 p, at least 0.17 p, at least 0, 18 p, at least 0.19 p, at least 0.20 p, at least 0.21 p, at least 0.22 p, at least 0.23 p, at least 0.24 p, at least 0.25 p. at least 0.26 p, at least 0.27 p, at least 0.28 p, at least 0.29 p, or at least 0.30 p.

[0106] 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 transformations and preparing bacterial extracts, as described herein.

[0107] In some instances, the bacteria are cultured in aerobic conditions to induce protein expression, and then the culture is switched to anaerobic conditions, for example by bubbling nitrogen, argon, etc. through the culture medium.

[0108] In some embodiments, a method of producing a bacterial cell extract comprises (a) culturing bacterial cells as disclosed herein according to fermentation processes as disclosed herein. The method comprises (b) isolating the cultured bacterial cells and (c) lysing the bacterial cells to produce bacterial cell extract. In some embodiments, the bacterial cells are cultured to a28KILPATRICK TOWNSEND 80240015.2high cell density, for example, a cell density of about 55 to about 150, or a cell density of at least 60-130, as measured using optical density at about 595-600 nm (OD595-600). In some embodiments, the bacterial cells are cultured using a batch fermentation process. In some embodiments, the bacterial cells are cultured using a fed-batch fermentation process.

[0109] In some embodiments, an initial culture (also referred to as a “seed culture”) is prepared prior to the culturing of the bacterial cells for the production of bacterial cell extract ( / .<?., prior to step (a) of the method disclosed in the preceding paragraph). The medium for the initial culture may be inoculated, for example, with a single colony picked from an agar plate, or an inoculum from a cell bank. Generally, this medium is a nutrient- rich liquid medium or broth. Once the initial culture has a sufficient number of cells (as can be determined at an optical density’ at 595-600 nm; OD595-600), the initial culture is used to inoculate a large and fixed volume of liquid media for batch or fed-batch fermentation.

[0110] In some embodiments, in this initial step of culturing bacterial cells, the volume of the initial culture is no greater than 0.1 liter, 0.25 liter, 0.5 liter, 0.75 liter, I liter, 1.25 liters, 1.5 liters, 1.75 liter, or 2 liters. In some embodiments, the volume of the initial culture is no greater than 0.5% 0.75% 1%, 1.25% 1.5%, 1.75% 2%, 2.25%, 2.5%, 2.75% 3%, 3.25%, 3.5%, 4%, 4.25%, 4.5%, 4.75% or 5% of the fermentation culture medium of step (a) of the method disclosed in the preceding paragraph.

[0111] In some embodiments, the maximum fermentation working volume (wv) is determined by multiplying the maximum bioreactor volume with a value ranging from about 0.4 to about 0.8. For example, if the maximum bioreactor volume is 200 L, the maximum fermentation wv ranges from about 80-160 L. In another example, if the maximum bioreactor volume is 3 kL, the maximum fermentation wv ranges from 1.2-2.4 kL. In another example, if the maximum bioreactor volume is 17 kL, the maximum fermentation w ranges from 6.8-13.6 kL. In some embodiments, the maximum fermentation wv is determined by multiplying the maximum bioreactor volume with about 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, or 0.8. In some embodiments, the volume of the bacterial cell culture or fermentation does not exceed the calculated maximum fermentation wv.

[0112] In some cases, it is useful to calculate the volume of bacterial cell extract that can be generated from a bacterial cell culture based on the formula below (Equation 2).29KILPATRICK TOWNSEND 80240015.2Average % solids obtained from fermentation x Bioreactor volumeTarget % solids in extractThe term “average percent (%) solids obtained from fermentation” in the formula above corresponds to wet cell weight (WCW7), which is the weight of the cell pellet that remains after the culture is centrifuged and the fermentation broth (i.e., supernatant) is removed. The WCW is dependent on the OD595-6oonm of the fermentation; a high ODs95-600nm will produce a higher WCW. The “average % solids obtained from fermentation” can be determined by multiplying OD595~600nm With 0.18.

[0113] In some embodiments, the ODsgs-eofem is about 50-140. In some embodiments, the OD595-600nm is about 50, 60, 70, 80, 90, 100, 110, 120, 125, 126, 127, 128, 129, 130, 131, 132, 133, 1 4, 135, or 140. For example, an ODs95-600nm of about 133 is multiplied with 0.18 to produce an “average % solids obtained from fermentation” of about 24.

[0114] In some embodiments, the “average % solids obtained from the fermentation” is about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, or about 27. The pelleted cells are resuspended in appropriate solutions, such as an appropriate buffer, for further processing and generation of bacterial cell extract.

[0115] In some embodiments, the pelleted cells are resuspended to achieve a new WCW, i.e., “target percent (%) solids in the extract” in the formula above. In some embodiments, the “average % solids obtained from the fermentation” is about 18, about 19, about 30, about 31, about 32, about 33, about 34, about 35, or about 36.

[0116] In some cases, the term “fermentation volume” is the maximum fermentation working volume (wv), which is limited by the maximum volume of the bioreactor. Thus, in some embodiments, the “fermentation volume” is the “bioreactor volume.” In some embodiments, the “fermentation volume” or “bioreactor volume” is at least 100 L, at least 200 L, at least 300 L, at least 400 L, at least 500 L, at least 600 L, at least 700 L, at least 800 L, at least 900 L, at least 1,000 L (1 kL), at least 3,000 L (3 kL), at least 5,000 L (5 kL), at least 8,000 L (8 kL), at least 10,000 L (10 kL), at least 13,000 L (13 kL), at least 15,000 L (15 kL), at least 17,000 L (17 kL), at least 18,000 L (18 kL), or at least 20,000 L (20 kL).30KILPATRICK TOWNSEND 80240015.2

[0117] In some embodiments, the fermentation volume produces at least 100 L, at least 200 L, at least 300 L, at least 400 L, at least 500 L, at least 600 L, at least. 700 L, at least 800 L, at least 900 L, at least 1,000 L (1 kL), at least 2,000 L (2 kL), at least 3,000 L (3 kL), at least 4,000 L (4 kL), at least 5,000 L (5 kL), at least 6,000 L (6 kL), at least 7,000 L (7 kL), at least 8,000 L (8 kL), or at least 9,000 L (9 kL) of bacterial extract. In some embodiments, the fermentation volume produces 100-1,000 L, 100-500 L, 300-700 L, 400-900 L, 500-1,000 L, 1,000-1,500 L, 1,250-1750 L, or 1,500-2,000 L of bacterial extract.

[0118] In some embodiments, (i) the fermentation volume is at least 100 L, at least 200 L, at least 300 L, at least 400 L, at least 500 L, at least 600 L, at least 700 L, at least 800 L, at least 900 L, at least 1,000 L (1 kL), or at least 3,000 L (3 kL); and (ii) the fermentation volume produces at least 25 L, at least 50 L, at least 75 L, at least 100 L, at least 125 L, at least 1 0 L, at least 175 L, at least 200 L, at least 225 L, at least 250 L, at least 275 L, at least 300 L, at least 325 L, at least 350 L, at least 375 L, at least 400 L, at least 500 L, at least 600 L, at least 700 L, at least 800 L, at least 900 L, or at least 1,000 L (1 kL) of bacterial extract.

[0119] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 130 L, OD595-60(tom is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and the calculated volume of bacterial cell extract that is generated is about 95 L. In this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 95 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 130 L and the volume of bacterial extract produced is at least 95 L.

[0120] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 180-200 L, OD595-600 is about 90-110, “average % solids obtained from fermentation” is about 16-20%, “target % solids in extract” is about 30-35%, and the calculated volume of bacterial cell extract that is generated is about 125-150 L. In this example, cell solids are concentrated to 30- 35% from 16-20%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 125 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 180 L and the volume of bacterial extract produced is at least 125 L, at least 130 L, at least 135 L, at least 140 L, at least 145 L, or at least 150 L.31KILPATRICK TOWNSEND 80240015.2

[0121] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 200 L, OD595-600 is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and die calculated volume of bacterial cell extract that is generated is about 146 L. In this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 146 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 200 L and the volume of bacterial extract produced is at least 146 L.

[0122] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 500 L, OD595-600 is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and the calculated volume of bacterial cell extract that is generated is about 365 L. In this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 365 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 500 L and the volume of bacterial extract produced is at least 365 L.

[0123] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 750 L, OD595-600 is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and the calculated volume of bacterial cell extract that is generated is about 548 L. hi this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 548 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 750 L and the volume of bacterial extract produced is at least 548 L.

[0124] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 1,000 L, ODsss-eoonm is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and the calculated volume of bacterial cell ex tract that is generated is about 727 L. In this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 727 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 1,000 L and the volume of bacterial extract produced is at least 727 L.

[0125] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 2,500 L, OD595-600 is about 133, “average % solids obtained from fermentation” is about 24%,2KILPATRICK TOWNSEND 80240015.2“target % solids in extract” is about 33%, and the calculated volume of bacterial cell extract that is generated is about 1,825 L. In this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 1,825 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 2,500 L and the volume of bacterial extract produced is at least 1,825 L.

[0126] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 5,000 L, OD595-600 is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and the calculated volume of bacterial cell extract that is generated is about 3,650 L. In this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 3,650 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 5,000 L and the volume of bacterial extract produced is at least 3,650 L.

[0127] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 7.500 L, OD595-600 is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and the calculated volume of bacterial cell extract that is generated is about 5,475 L. In this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 5,475 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 7,500 L and the volume of bacterial extract produced is at least 5,475 L.

[0128] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 10,000 L, OD595-600 is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and the calculated volume of bacterial cell extract that is generated is about 7,300 L. In this example, cell solids are concentrated to 33% from 24%, the liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 7,300 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 10,000 L and the volume of bacterial extract produced is at least 7,300 L.

[0129] For example, in some cases, the “fermentation volume” or “bioreactor volume” is about 12.500 L, OD595-6ooam is about 133, “average % solids obtained from fermentation” is about 24%, “target % solids in extract” is about 33%, and the calculated volume of bacterial cell extract that is generated is about 9,091 L. In this example, cell solids are concentrated to 33% from 24%, the 33KILPATRICK TOWNSEND 80240015.2liquid media is replaced with a different buffer, and the volume of bacterial extract produced is at least 9,091 L. Therefore, in some embodiments, the “fermentation volume” or “bioreactor volume” is at least 12,500 L and the volume of bacterial extract produced is at least 9,091 L.B. Methods for Isolating and Preparing Bacterial Cell Extracts

[0130] After bacterial cells are cultured to produce bacterial cell extract (i.e., at the end of a fermentation process), the bacterial cells are isolated and lysed so that the bacterial cell extract can be harvested. In general, the bacterial cell extract comprises components that are capable of translating messenger ribonucleic acid (mRNA) encoding a desired protein, and optionally comprises components that are capable of transcribing DNA encoding a desired protein. Non-limiting examples of such components include DNA-directed RNA polymerase (RNA polymerase), any transcription activators that are required for initiation of transcription of DNA encoding the desired protein, transfer ribonucleic acids (tRNAs), aminoacyl-tRNA synthetases, 70S ribosomes, Ni0-fomiyltetrahydro folate, formylmethionine-tRNAfMetsynthetase, peptidyl transferase, initiation factors (such as IF-1, IF-2, and IF-3), elongation factors (such as EF-Tu, EF-Ts, and EF-G) release factors (such as RF-1, RF-2, and RF-3), and the like. In some cases, the bacterial cell extract is used in CFPS reactions.

[0131] A bacterial cell extract derived from any bacterial cell of the present disclosure can be isolated and lysed for use in CFPS reactions. Methods of preparing a lysed bacterial cell extract are described in, e.g., Zawada, J. “Preparation and Testing of E.coli S30 In vitro Transcription Translation Extracts”, Douthwaite, J. A. and Jackson, R. H. (eds.), Ribosome Display and Related Technologies: Methods and Protocols, Methods in Molecular Biology, vol. 805, pp. 31-41 (Humana Press, 2012); Jewett et al., Molecular Systems Biology, 4, 1-10 (2008); Shin J. and Norieaux V., J. Biol. Eng, 4:8 (2010).

[0132] When the bacterial culture is ready for harvest at the end of a fermenta tion, it can be cooled to about 2-10°C, to about 4-12°C, to about 2°C, to about 4C'C, to about 8°C, to about 10°C, or to about 12°C, usually on ice, or with heat exchangers when the culture is of a large scale. The culture can be centrifuged to separate the spent media from the cell paste (cell slurry). Preferred centrifuges include disk stack centrifuges, tubular bowl centrifuges, and other centrifuges for large or small-scale bacterial cultures. In some embodiments, the bacterial culture is pelleted by centrifugation at greater than 14,000 x g for about 45 min at about 8°C to about 20°C in a tubular34KILPATRICK TOWNSEND 80240015.2bowl centrifuge in continuous or batch mode or a disc stack continuous centrifuge with a maximum bowl speed of about 12,000 rpm and a feed flow rate of about 3.0-3.3 L / min. The centrifugation step can be performed one, two, or more times.

[0133] The cell pellet or cell paste is typically resuspended in a buffer, such as an S30 buffer (as discussed in the Examples below), or any equivalent buffer solution, or water. For example, the buffer can comprise a tris base, magnesium acetate, and potassium acetate. In some embodiments, the buffer is an S30 buffer comprising 10 mM tris acetate, 14 mM magnesium acetate and 60 mM potassium acetate. In some embodiments, a 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8: 1 or more dilution (liquid: solid; ml of buffer: gram weight of cells) is made for washing. The cell paste can be washed again in S30 buffer or any equivalent buffer and centrifuged to remove any residual buffer. For small scale cultures, a second wash step is typically performed. At washing the cell pellet can be stored at -80° C for later use or further processed by lysis to produce a bacterial cell extract.

[0134] Harvested cells are lysed to produce a bacterial cell extract. If the harvested cells were frozen as cell pellets, the frozen cell pellets are resuspended and thawed in a suitable cell suspension buffer. The suspended cells are lysed by sonication, with a French press or with glass beads, continuous flow high pressure homogenization, or any other method known in the art usefill for efficient cell lysis.

[0135] In some embodiments, the cell suspension is homogenized or disrupted in a standard high-pressure homogenizer (e., an Avestin Emulsiflex C-55a Homogenizer) and / or microfluidizer (e.g., Microfluidics Microfluidizer) set at the appropriate pressure, such as 3,000 psi to produce a lysate. The homogenization step lyses the bacteria to release the necessary components required for protein synthesis, and in some aspects, formed inverted membrane vesicles provide energy for protein synthesis via respiration.

[0136] In some embodiments, the homogenizer pressure is at about 3,000-20,000 psi. In some embodiments, the homogenizer pressure is set at about 20,000 psi. In some embodiments, the speed (frequency setting) of the homogenizer is at about 20 Hz to about 60 Hz to produce flow rates of about 340 ml / min-1.0 L / min. Generally, flow rate is proportional to the frequency setting and can be varied independently from the homogenizing pressure. Preferably, the minimum speed setting for homogenizing steps is about 20 Hz wdth a flow rate of about 340 ml..'min 5KILPATRICK TOWNSEND 80240015.2

[0137] In some cases, the temperature of the cell lysate increases during the homogenization step. In some embodiments, during the homogenization step, the temperature of the cell lysate is lowered to about 10 C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, or about 20°C. In some embodiments, after the homogenization step, the temperature of the cell lysate is lowered to about 10°C, about 119C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 179C, about I8°C, about 19°C, or about 20°C.

[0138] The cell lysate is then centrifuged or filtered to remove large cell debris, including DNA and cells that have not been lysed. The lysate can be clarified by centrifugation such that from at least about 45% to about 85% or more, e.g., about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of the cell solids are separated from the cell- free extract which is collected. In some embodiments, at least about 70%, 75%, 80%, 85%, 90%, or 95% of the cellular solids are separated by centrifugation. In some embodiments, the centrifugation is by a continuous centrifuge, e.g., disk stack centrifuge, tubular bowl centrifuge or appropriate centrifuge.

[0139] The clarified cell lysate can be filtered through one or more sterilizing grade filter membranes, e.g., a 0.45-gm filter membrane and / or a 0.22-pm filter membrane. A 0.45-pm filter membrane can be used first, and then a 0.22-pm filter membrane afterwards. In some cases, it is beneficial to raise the temperature of the cell lysate prior to filtration, for example, to a temperature of about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 3O‘;’C, about 31" C. about 32°C, about 33°C, about 34°C, about 35°C, about 36" C, or about 37°C.

[0140] The filtered cell lysate can be aliquoted and frozen in liquid nitrogen before storing at -80°C. Optionally, a cell-free protein synthesis (CFPS) reaction mix, as described herein, can be added to the cell lysate prior to freezing.

[0141] Bacterial cell extracts of the present disclosure may also be used to supplement commercially-available bacterial cell extracts. Commercial bacterial cell extracts may be purchased from manufacturers such Promega Corp., Madison, WI; Agilent Technologies, Santa Clara, CA; GE Healthcare Biosciences, Pittsburgh, PA; Life Technologies, Carlsbad, CA; and Roche Diagnostics, Basel, Switzerland.36KILPATRICK TOWNSEND 80240015.2

[0142] Bacterial cell extracts of the present disclosure may be stored in several formats for long periods of time before use. In some embodiments, the bacterial cell extract is stored at about -20°C, about 2°C to 8°C, or about 20°C to 24°Croom temperature) for at least or equal to 3 to 18 months (e.g., at least or equal to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months).

[0143] In some embodiments, the bacterial cell extract is dried prior to storage. In some embodiments, the bacterial cell extract is subjected to freeze drying, lyophilization, in situ vaporization, microwave radiation, or sublimation. In some embodiments, the bacterial cell extract is subject to a spray-drying process where the spray-dried bacterial cell extract essentially retains its physical and chemical stability and integrity upon storage. For example, a stable, spray-dried bacterial cell extract refers to an extract that retains at least 75% of its initial capacity to synthesize a protein of interest when stored at about -20°C, about 2°C to 8°C, or about 20°C to 24°C (z.e., room / ambient temperature) for 6 months or more. An additive (also referred to as a “stabilizer” or “excipient”) can be added to a liquid bacterial cell extract prior to spray-drying. The additive may help support physical and chemical stability of the spray-dried bacterial extract during storage. Methods for preparing dried extracts, including freeze-dried extracts, spray-dried extracts, and related methods are described in, for example, U.S. Patent No. 10,648,010 and U. S. Patent App. Pub. No. 2024 / 0043895.

[0144] In some embodiments, an activation step is applied to the bacterial cell extract before it is used in a protein synthesis reaction because activation can increase protein synthesis, for example, in a CFPS reaction. In some embodiments, heat is applied in the activation step.Activation may cause ribosomes in the bacterial extract to dissociate from messenger RNAs, and the ribosomes can then be used in subsequent protein synthesis. In some embodiments, the bacterial cell extract has not been frozen or dried. In some embodiments, the bacterial extract has been frozen or dried. In embodiments where the bacterial extract was dried, the bacterial extract can be reconstituted in a buffer or other liquid to form a liquid bacterial extract prior to the heat activation step.

[0145] The bacterial extract can be activated with a heat activation step i.e., heat-treatment). In some embodiments, the bacterial cell extract is heated from about 20°C to about 45°C for about 20 minutes to about 10 hours. In some embodiments, the bacterial cell extract is sterile-37KILPATRICK TOWNSEND 80240015.2filtered before activation. Activation of bacterial cell extracts is described in Groff, D., et al. ( Development of an£. coli strain for cell-free ADC manufacturing. Biotechnology and Bioengineering, 119, 162- 175. doi.org / 10.1002 / bit.27961).

[0146] In some embodiments, a thawed, activated, or rehydrated bacterial cell extract (including a rehydrated spray-dried bacterial extract) in a CFPS reaction is able to synthesize a target protein of interest with a yield of at least 80%, at least 85%, at least 90%, at least 95% relative to a control bacterial extract. In some embodiments, the control bacterial extract is a fresh preparation of a bacterial cell lysate, hi some embodiments, the control bacterial extract is a bacterial cell lysate that was not previously frozen, hi some embodiments, the control bacterial extract is a bacterial cell lysate that was not previously dried. In some embodiments, the control bacterial extract is a bacterial cell lysate that was not previously thawed, activated, and / or rehydrated.V. Cell-Free Protein Synthesis

[0147] Bacterial cell extracts of the present disclosure may be used in cell-free protein synthesis (CFPS) reactions. CFPS systems have been used to generate 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 Groff, Dan et al., mAbs vol.6,3 (2014)) and antibody-drug conjugates (Zimmerman et al., Bioconjug Chem, 25(2):351-61 (2014)). hi a generic CFPS reaction, a gene encoding a protein of interest is expressed in a transcription buffer, resulting in mRNA that is translated into the protein of interest in a CFPS extract and a translation buffer. The transcription buffer, cell-free extract and translation buffer can be added separately, or two or more of these solutions can be combined before their addition or added contemporaneously.

[0148] To synthesize a protein of interest in vitro, the bacteri l cell extract at some point comprises an mRNA molecule that encodes the protein of interest. In some systems, mRNA is added exogenously after being purified from natural sources. In some systems, synthetic mRNA is prepared separately from cloned DNA using RNA polymerases (RNAPs) such as RNAP II, SP6 RNAP, T3 RNAP, T7 RNAP, RNAP III and / or phage derived RNAP.

[0149] In other systems where both transcription and translation occur in the same reaction, the mRNA is produced in vitro from a template DNA. In some embodiments, the transcription and 38KILPATRICK TOWNSEND 80240015.2translation systems are coupled or comprise complementary transcription and translation systems, which carry out the synthesis of both RNA and protein in the same reaction. In such in vitro transcription and translation systems, the bacterial cell extracts contain all the components (exogenous or endogenous) necessary both for transcription (to produce mRNA) and for translation (to sy nthesize protein) in a single system. In some embodiments, the bacterial cell extract is prepared according to methods as discussed in the preceding sections.

[0150] A CFPS reaction can contain the following components: a template nucleic acid, such as DNA, that comprises a gene of interest operably linked to at least one promoter and, optionally, one or more other regulatory sequences (e.g., a cloning or expression vector containing the gene of interest) or a PCR fragment; an RN A polymerase that recognizes the promoter(s) to which the gene of interest is operably linked (e.g. T7 RNA polymerase) and, optionally, one or more transcription factors directed to an optional regulatory sequence to which the template nucleic acid is operably linked; ribonucleotide triphosphates (rNTPs); optionally, other transcription factors and co-factors therefor; ribosomes; transfer RNA (tRNA); other or optional translation factors (e.g., translation initiation, elongation and termination factors) and co-factors therefore; one or more energy sources, (e.g., ATP, GTP); optionally, one or more energy regenerating components (e.g., PEP / pyruvate kinase, AP / acetate kinase or creatine phosphate / creatine kinase); optionally factors that enhance yield and / or efficiency (e.g., nucleases, nuclease inhibitors, protein stabilizers, chaperones) and co-factors therefore; and; optionally, solubilizing agents. The reaction mix can also include amino acids and other materials specifically required for protein synthesis, including salts (e.g., potassium, magnesium, ammonium, and manganese salts of acetic acid, glutamic acid, or sulfuric acids), polymeric compounds (e.g., polyethylene glycol, dextran, diethyl aminoethyl dextran, quaternary? aminoethyl and aminoethyl dextran, etc.), cyclic AMP, inhibitors of protein or nucleic acid degrading enzymes, inhibitors or regulators of protein synthesis, oxidation / reduction adjuster (e.g., DTT, ascorbic acid, glutathione, and / or their oxides), non-denaturing surfactants (e.g., Triton X-I00), buffer components, spermine, spermidine, putrescine, etc. Components of such reactions are discussed in more detail in U. S. Patent Nos. 7,338,789; 7,351,563; 8,715,958; and 8,778,631.39KILPATRICK TOWNSEND 80240015.2

[0151] Depending on the specific enzymes present in the extract, for example, one or more of the many known nuclease inhibitors, polymerase inhibitors, or phosphatase inhibitors can be selected for inclusion to advantageously improve synthesis efficiency.

[0152] Protein and nucleic acid synthesis typically requires an energy source. Energy is required for initiation of transcription to produce mRNA (e.g., when a DNA template is used and for initiation of translation high energy phosphate for example in the form of GTP is used). Each subsequent step of one codon by the ribosome (three nucleotides; one amino acid) requires hydrolysis of an additional GTP to GDP. ATP is also typically required. For an amino acid to be polymerized during protein synthesis, it must first be activated. Significant quantities of energy from high energy phosphate bonds are thus required for protein and / or nucleic acid synthesis to proceed.

[0153] An energy- source is a chemical substrate that can be enzymatically processed to provide energy to achieve desired chemical reactions. Energy sources that allow release of energy for synthesis by cleavage of high-energy phosphate bonds such as those found in nucleoside triphosphates, e.g., ATP, are commonly used. When sufficient energy is not initially present in the synthesis system, an additional source of energy is preferably supplemented. Energy’ sources can also be added or supplemented during the in vitro synthesis reaction. Any source convertible to high energy phosphate bonds is especially suitable. ATP, GTP, and other triphosphates can normally be considered as equivalent energy' sources for supporting protein synthesis. Other energy’ sources that may be considered include glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyd phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, giyceraldehyde-3-phosphate, 3-Phosphoglycerate and glucose-6-phosphate, that can generate or regenerate high-energy' triphosphate compounds such as ATP, GTP, other NTPs, etc.

[0154] In some embodiments, the CFPS reaction is performed using the PANOx-SP system comprising NTPs, E.coli tRNA, amino acids. Mg21acetate, Mg2+glutamate, K+acetate, K+glutamate, folinic acid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfide isomerase, phosphoenol pyruvate (PEP), NAD, Co, Na+oxalate, putrescine, spermidine, and S30 extract.40KILPATRICK TOWNSEND 80240015.2

[0155] In some embodiments, the CFPS reaction is performed using the Cytomim system comprising NTPs, E.coli tRNA, amino acids, Mg2+acetate, Mgi+glutamate, K' acetate, K' glutamate, folinic acid. Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfide isomerase, sodium pyruvate, NAD, CoA, Na oxalate, putrescine, spermidine, and S30 extract. The Cytomim system is defined as a reaction condition performed in the absence of polyethylene glycol with optimized magnesium concentration. This system does not accumulate phosphate, which is known to inhibit protein synthesis. Detailed descriptions of the Cytomim system are found in, for example, U. S. Patent No. 7,338,789; Jewett et al.. Mol Syst Biol, (2008), 4:220; Spirin, A. S. and Swartz, J. R. (2008) Cell-Free Protein Synthesis; Methods and Protocols, New Jersey: John Wiley & Sons. In some embodiments, the energy substrate for the Cytomim system is pyruvate, glutamic acid, and / or glucose. In some embodiments of the system, the nucleoside triphosphates (NTPs) are replaced with nucleoside monophosphates (NMPs).

[0156] In some embodiments, proteins containing a non-natural amino acid (nnAA) may be synthesized. In such embodiments, the reaction mix may comprise the nnAA, a tRNA orthogonal to the 20 naturally occurring amino acids, and a tRNA synthetase that can link the nnAA with the orthogonal tRNA. See, e.g, U. S. Patent No. 8,715,958. Alternatively, the reaction mix may contain a nnAA conjugated to a tRNA for which the naturally occurring tRNA synthetase has been depleted. See, e.g., U. S. Patent No. 8,778,631 and U. S. App. Publ. No. 2010 / 0184134. Various kinds of unnatural amino acids, including without limitation detectably labeled amino acids, can be added to cell-free protein synthesis reactions and efficiently incorporated into proteins for specific purposes. See, for example, Albayrak, C. and Swartz, JR., Biochem. Biophys Res. Cornmun., 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.

[0157] In some embodiments, the CFPS reaction includes inverted membrane vesicles to perform oxidative phosphorylation. These vesicles can be formed during the high-pressure homogenization step of the preparation of cell extract process, as described herein, and remain in the extract used in the reaction mix. The presence of an active oxidative phosphorylation pathway can be tested using inhibitors that specifically inhibit the steps in the pathway, such as electron transport chain inhibitors. Examples of inhibitors of the oxidative phosphorylation41KILPATRICK TOWNSEND 80240015.2pathway include toxins such as cyanide, carbon monoxide, azide, carbonyl cyanide?n-chlorophenyl hydrazone (CCCP), and 2,4-dinitrophenol, antibiotics such as oligomycin, pesticides such as rotenone, and competitive inhibitors of succinate dehydrogenase such as malonate and oxaloacetate.

[0158] The CFPS reaction conditions may be performed as batch, continuous flow, or semi- continuous flow, as known in the art. The reaction conditions are linearly scalable, for example, the 0.3 L scale in a 0.5 L stirred tank reactor, to the 4 L scale in a 10 L fermenter, and to the 100 L scale in a 200 L fermenter.

[0159] hi some embodiments, a protein of interest is expressed using a CFPS reaction at a concentration of at least about 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, or 1,000 mg or more per L.

[0160] The CFPS reaction can be used to produce many proteins of interest. In some embodiments, the protein of interest is an antibody, an antibody fragment, an antibody light chain, an antibody heavy chain, a cytokine, a cytokine fragment, or an immunogenic polypeptide.

[0161] In some embodiments, the protein of interest, such as an antibody or an antibody¬ fragment, can be conjugated to a biologically active adduct (also referred to as a “payload”) using a chemical reaction such as the click chemistry. In some cases, the antibody comprises one or more nnAAs (as described herein) at specific sites in the protein sequence, and the biologically active adduct can be conjugated to these nnAAs. Having antibodies containing die nnAA(s) at the desired amino acid location(s), a biologically active adduct can be conjugated to the nnAA(s) using a chemical reaction such as the click chemistry-. For instance, the pAMF containing antibody produced using the methods disclosed herein can be purified using standard procedures. Then, the purified protein is subject to a click chemistry reaction (e.g., copper(I)-catalyzed azide-alkyne 1,3-cycloaddition reaction or copper- free catalyzed azide-aklyne 1,3 -cycloaddition reaction) to directly conjugate a biologically active adduct to the pAMF residue. Exemplary biologically active adducts for use include, but are not limited to, small molecules, oligonucleotides, peptides, amino acids, nucleic acids, sugars, oligosaccharides, polymers, synthetic polymers, chelators, fluorophores, chromophores, other detectable agents, drug moieties, cytotoxic agents, detectable agents, and the like.42KILPATRICK TOWNSEND 80240015.2

[0162] In some embodiments, the protein of interest is an immunogenic polypeptide. An immunogenic peptide refers to a polypeptide comprising at least one T-cell activating epitope, wherein the T-cell activating epitope is derived from a protein capable of inducing immunologic memory in animals. The term “T-cell activating epitope” refers to a structural unit of molecular structure which is capable of inducing T-cell immunity. The function of carrier proteins which include T-cell activating epitopes is well known and documented for conjugates. Without wishing to be bound by theory a T-cell activating epitope in the carrier protein enables the covalently attached antigen to be processed by antigen-presenting cells and presented to CD4+ T cells to induce immunological memory against the antigen.

[0163] In some embodiments, the immunogenic polypeptide is a carrier protein. The term “carrier protein” refers to a non-toxic or detoxified polypeptide containing a T-cell activating epitope which is able to be attached to an antigen (e.g., a polysaccharide) to enhance the humoral response to the conjugated antigen in a subject. The term includes any of the bacterial proteins used as epitope carriers in FDA-approved vaccines. In some embodiments, the carrier protein is Corynebacterium diphtheriae toxin, Clostridium tetani tetanospasmin, Haemophilus influenzae protein D (PD, HiD), outer membrane protein complex of serogroup B meningococcus (OMPC), CRM197, or malaria ookinete specific surface protein Pfs25. In another case, the carrier protein is BB, derived from the G protein of Streptococcus strain G148.

[0164] In some embodiments, a carrier protein comprises a polypeptide that can be conjugated to an antigen to provide a T-cell dependent immune response. In some embodiments, the antigen comprises a T-cell independent antigen selected from the group consisting of a hapten, a bacterial capsular polysaccharide, a bacterial lipopolysaccharide, or a tumor-derived glycan. In another embodiment, the antigen comprises a bacterial non-capsular polysaccharide, such as an exopolysaccharide e.g., the 5. aureus exopolysaccharide. In another embodiment, the antigen is a bacterial polysaccharide and the bacteria is selected from the group consisting of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae (e.g., Hib), Streptococcus pyogenes, and Streptococcus agalactiae.

[0165] In some embodiments, the protein of interest is a cytokine. In some embodiments, the cytokine is selected from an interleukin, an interferon, a transforming growth factor, or a chemokine. In some embodiments, the cytokine is selected from the group consisting of IL- 1-43KILPATRICK TOWNSEND 80240015.2like, IL-la, IL-lp, IL-IRA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5,, IL-16, IL- 17, IFN-a, IFN-p, IFN-y, TNF, CD154, LT-p, TNF-a, TNF-p, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-J3, TGF-pi, TGF-p2, TGF-p3, Epo, Tpo, Flt-3L, SCF, M-CSF, and MSP. In certain embodiments, the protein is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36.

[0166] In some embodiments, the protein of interest comprises one or more nnAAs. In some embodiments, the one or more nnAAs is selected from the group consisting of p-acetyl-L-phenylalanine (pAcPhe), O-methyl-L-tyrosine, an -3-(2-naphthyl)alanine, 3-methyI- phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, a tri O-acetyl-GlcNAcP-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-azido- methyl-L-phenylalanine (pA. MF), p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p- bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine. In some embodiments, at least one of the non-natural amino acids is selected from 2-amino-3-(4-azidophenyl)propaiioic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4- (azidomethyl)pyridin-2-yI)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5 -azidopentanoic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid. VI. Methods for Evaluating Protein Expression

[0167] Various methods can be used to evaluate the expression level of the various proteins in the bacterial cells or the bacterial cell lysate of the present disclosure, or in a CFPS reaction. In some cases, in CFPS reactions, it is useful to compare the amount of protein produced using a first bacteri al cell lysate to the amount of protein produced using a second bacterial cell lysate. In some cases, the first and second bacterial cell lysates are produced using different methods, compositions, and / or systems. For example, a first bacterial cell lysate is produced using an established fermentation process (i.e., the control ly sate) and a second bacterial cell lysate is produced using a modified fermentation process. Both bacterial cell lysates are used in separate CFPS reactions and then analyzed for protein expression. The protein levels are compared so that44KILPATRICK TOWNSEND 80240015.2the performance of the bac terial cell lysate from the modified fermentation process can be determined. The amount of protein produced by the first bacterial cell lysate (i.e., the control) is fixed at 100% and the amount of protein produced by the second bacterial cell lysate is calculated as a percentage of the control. In some embodiments, the amount of the protein produced using a different / modified method, composition, andfor system is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the control protein.

[0168] Methods for measuring protein levels include, but are not limited to, Coomassie-stained polyacrylamide gel electrophoresis (PAGE), silver-stained polyacrylamide gel, capillary electrophoresis (e.g., Caliper LabChip), ELISA, immunoblotting, Western blotting, size exclusion chromatography, affinity chromatography, and mass spectrometry.

[0169] hi some embodiments, the protein of interest is detected by an assay that measures the activity of that particular protein. For example, if the protein of inter est is a kinase, the kinase may be detected by performing an enzymatic assay specific to detecting the conversion of substrate to product by that kinase. Illustrative examples of assays include luciferase assays and the chloramphenicol acetyl transferase assay. These assays measure the amount of functionally active protein that is expressed. In some embodiments, the activity of the protein of interest that is produced using a modified method, composition, and / or system is compared to the activity of the same protein that is produced using an established or unmodified method, composition, and / or system (i.e., the control). The activity of the protein produced using the control bacterial cell lysate is fixed at 100% and the activity of the protein produced using a different / modified method, composition, and / or system is calculated as a percentage of the control. In some embodiments, the activity of the protein of interest is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the control protein.

[0170] In some embodiments, the protein of interest is covalently linked with a detectable label. The amount of fluorescence signal detected in a cell, culture, or reaction may indicate the amount of protein that is present. Exemplary fluorophores that may be used as a detectable label include but are not limited to, cyanine dyes (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor (AF) dyes (e.g., AF 647, AF 555, or AF 488), rhodamine dyes (e.g., fluorescein, FITC, Texas Red, ROX), ATTO dye (e.g., ATTO 532 or 655), Exemplary proteins that may be used as a detectable label include green fluorescent protein (e.g., GFP and enhanced GFP (eGFP)),45KILPATRICK TOWNSEND 80240015.2yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and Ypet ), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet, mTurquoise2) or photoactivabale fluorescent proteins, such as PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PamCherry, PAtagRFP, mMaple, mMapleZ, and mMaple3. Other suitable fluorophores are known to those of ordinary skill in the art. Methods of detecting these labels, including their excitation and emission at specific wavelengths, are known to one of ordinary-' skill in the art.

[0171] In some embodiments, the protein of interest in a fluorescent protein, such as GFP, eGFP, YFP, Citrine, Venus, Ypet, ECFP, Cerulean, CyPet, mTurquoise2, PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PamCherry, PAtagRFP, mMaple, mMaple2, and mMaple3. In some embodiments, the protein of interest is GFP.

[0172] hi some embodiments, radiolabeled amino acids are incorporated into the protein of interest. In some embodiments, the protein of interest may be monitored by the incorporation of radiolabeled amino acids, typically,j5S-labeled methionine or14C-labeled leucine. Radiolabeled proteins can be visualized for molecular size and quantitated by autoradiography after electrophoresis or isolated by immunoprecipitation.

[0173] In some embodiments, a bacterial cell of the present disclosure is cultured under conditions that permit transcription of a target polynucleotide sequence and expression of a corresponding protein. The titer of the liquid culture refers to the amount or mass (g) of the expressed protein in the total liquid volume (L) of the culture. Titer can be expressed in various units of mass per volume, e.g., mg / mL, mg / L, and g / L. Titer can also be used as a parameter for evaluating a CFPS reaction. Here, titer refers to the amount or mass (g) of the expressed protein in the total liquid volume (L) of the CFPS reaction.

[0174] When expressing a protein using a CFPS reaction, the activity of a bacterial cell extract refers to the amount of protein produced in the reaction when that particular bacterial cell extract is used as a reagent of the reaction. In some embodiments, the activity of a bacterial cell extract is the yield of an expressed protein in a CFPS reaction. The activity of the bacterial cell extract can be determined using assays such as performing CFPS to produce a model protein (a test protein) which can be measured. Methods for CFPS are described in detail in, e.g., Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng. 66:180-8 (1999); Kim, D. M. and Swartz, J. R. Biotechnol. Prog. 16:385-90 (2000); Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng. 74:309-16 (2001);46KILPATRICK TOWNSEND 80240015.2Swartz et al., Methods Mol. Biol. 267:169-82 (2004); Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng. 85:122-29 (2004); Jewett, M. C. and Swartz, J. R., Biotechnol. Bioeng. 86:19-26 (2004); Yin, G. and Swartz, J. R., Biotechnol. Bioeng. 86:188-95 (2004); Jewett, M. C. and Swartz, J. R., Biotechnol. Bioeng. 87:465-72 (2004); Voloshin, A. M. and Swartz, J. R., Biotechnol. Bioeng. 91:516-21 (2005).

[0175] In some embodiments, the yield of a protein of interest is determined by dual flow chromatography (DFC) using a Protein A resin. The Protein A resin can be packed between two thin frit screens in the tip of a single-use pipette tip. In some embodiments, the yield of a protein of interest is determined by passing the products of a CFPS reaction over a Protein A column, such as a PhyTip® column (Biotage® ). In some embodiments, the yield of a protein of interest is determined by using an IgG BioHT kit using Cedex Bio HT Analyzer. The yield of a protein of interest can be expressed as weight / volume (e.g., mg / L or g / L) of a CFPS reaction or as a percentage of the yield of a control extract.

[0176] In some embodiments, the yield of a protein of interest is determined by performing High-performance liquid chromatography (HPLC) of the protein products from a CFPS reaction along with protein standards.

[0177] Another method of measuring the amount of protein produced in coupled in vitro transcription and translation reactions is to perform the reactions using a known quantity of radiolabeled amino acid such as35S-methionine,3H-leucine or34C-leucine and subsequently measuring the amount of radiolabeled amino acid incorporated into the newly translated protein. Incorporation assays will measure the amount of radiolabeled amino acids in all proteins produced in an in vitro translation reaction including truncated protein products. The radiolabeled protein may be further separated on a protein gel, and by autoradiography confirmed that the product is the proper size and that secondary protein products have not been produced.

[0178] Methods of measuring the capacity of an expression system to express a protein includes thei4C Leu incorporation assay. In some embodiments, a method for measuring the protein synthesis activity of a spray-dried extract is the,4C Leu incorporation assay.47KILPATRICK TOWNSEND 80240015.2

[0179] Alternately, the yield of protein can be determined by running the protein labeled with14C Leu on a polyacrylamide gel using conventional techniques. The gel can be denaturing or non-denaturing, according to the polypeptide to be detected. Where a protein containing multiple subunits is to be detected, a non-denaturing gel is preferred. The yield of protein can be determined through specific binding assays such as enzyme linked immunosorbant assay (ELISA) or surface binding resonance (e.g., Biacore).

[0180] Alternatively, the yield of protein can be determined through whole or partial purification, such as using chromatography, coupled with protein quantitation, such as UV absorbance or BCA analysis.VII. Compositions and Kits

[0181] This disclosure also provides kits that comprise viable bacterial cells of the present disclosure and optionally growth media, reagents, and / or instructions for producing bacterial cell extract. In some embodiments, the kit may comprise one or more reagents for analysis of the bacterial cell extract that is produced according to the present disclosure. In some embodiments, the kit may comprise reagents and / or instructions for CFPS. In some embodiments, the kit may comprise reagents and / or instructions for producing GFP - a control protein of interest - using a CFPS reaction.EXAMPLES

[0182] The following Examples relate to production of bacterial cell extract from E. coll using a high cell density fed-batch fermentation process. The bacterial cell extract produced using the fed-batch process demonstrated cell-free protein synthesis (CFPS) activity that is comparable to the activity of bacterial cell extracts produced via the chemostat (continuous) fermentation process.Example 1: Materials and Methods

[0183] 1.1. E. coli strains. The E. coli strain used in the Examples was a modified E. coli K- 12 strain. The E. coli strain wns modified to express exogenous chaperone proteins and to include a prfA mutation. Details pertaining to these modifications are disclosed above.

[0184] 1.2. Media.1-17 (lOx) Medium. The preparation of the shake flask medium, bioreactor batch medium, and fed-batch feed solution involved utilization of an 1-17 (lOx) medium. The48KILPATRICK TOWNSEND 80240015.21-17 medium comprises amino acids, trace metals, and vitamins. The 1-17 medium and its components are known to one of ordinary' skill in the art and are readily available.

[0185] Shake Flask Medium. Seed expansion was performed using a shake flask medium. The shake flask medium comprises potassium phosphate (monobasic), ammonium sulfate, potassium chloride, sodium citrate dihydrate, magnesium sulfate heptahydrate, and 1-17 medium. The shake flask medium and its components are known to one of ordinary skill in the art and are readily available. Components of the shake flask medium v ere combined in 800 mb of deionized (DI) water. After the components had dissolved, pH of the medium was adjusted to 6.8 using IO N potassium hydroxide. DI water was added to bring the medium to a final volume of IL and the medium was sterilized.

[0186] S30 Buffer. Following cell harvest, the cells 'were washed and resuspended in S30 buffer to facilitate recovery of the extract. For preparation of 1 L of S30 buffer, 10 mL each of 1 M tris base (pH 8.2), 1.4 M magnesium acetate, and 6 M potassium acetate were added to 970 mL of deionized (DI) water. Upon achieving homogeneity, the solution was sterilized.

[0187] 1.3. Equipment and Software. Equipment and software used in these Examples are listed in Tables 1 and 2 below.Table 1. Equipment.Equipment Name SupplierShaker Incubator New Brunswick EppendorfInnova40R (1” orbitaldiameter)Bioreactor Biostat 2X BDCU-10L Sartorius Stedim Biotech Biostat® B-1LAmbr® 250Biostat DDCU-200LCentrifuge Sorvall LYNX 6000 Thermofisher Scientific Superspeed CentrifugeExhaust Gas Analyzer Blue Vary BlueSensRotor Stator Homogenizer Ultra-Turrax® T 25 1 KA WorksHigh Pressure Emulsiflex-C5 AvestinHomogenizerMicrofl uidizer M-110EH-30 MicrofluidicsWater bath HAAKE A 10B Thermofisher Scientific Immersion Circulator HAAKE AC200 Thermofisher ScientificMetabolite. Analyzer Cedex® BioHT Roche49KILPATRICK TOWNSEND 80240035.2Mixing / holding tank Palletank®-200L, 50L Sartorius Stedim Biotech Disc stack centrifuge CSC 6-06-476 GEA Westfalia SeparatorCirculating Chiller VWRTable 2. Software.Software SupplierBioPAT® MFCS EppendorfAmbr® Runtime Sartorius Stedim Biotech

[0188] 1.4. E. coli Cultivation. Seed Flask. For preculture cultivation in a shake flask, frozen cryo stock of development cell bank (DCB) for E. coli was thawed at room temperature. I mL of thawed culture was inoculated into 50 mL of shake flask media within a 250 mL baffled Erlenmeyer flask. The flask was incubated for 8-10 hours at 37°C and 250 RPM in the shaker incubator. Upon reaching an OD595-600 within the range of 2-4, the flask was harvested and subsequently transferred to the production bioreactor.

[0189] 1 L Bioslat, Fed-Batch Cultivation. Fed-batch cultivations were executed in 1 L Biostat® B bioreactors. Each reactor was equipped with probes for measuring temperature, pH, and dissolved oxygen. Air sparging was accomplished through a multi-orifice ring sparger, and mixing was achieved utilizing two 6-blade Rushton impellers. To prevent volume loss due to evaporation, an external condenser was installed, and its outlet was connected to an exhaust gas analyzer. The pH probe was externally calibrated at room temperature to determine offset and slope using pH 7 and pH 10 buffers, respectively.

[0190] The bioreactor was assembled by installing probes, a condenser, headplate, feeding and sampling ports. Prior to cultivation, the bioreactor was autoclaved at 120°C for 30 minutes with deionized (DI) water and antifoam. The amount of DI water used was determined based on the experimental design, as detailed in the following sections. Once the bioreactor cooled to 37°C, 1-17 (lOx) media was added post-sterilization to achieve the target glucose concentration specified by the experimental conditions.

[0191] Dissolved oxygen (pO or DO) probes were calibrated to estimate offset by sparging with pure nitrogen, while slope wras determined using the saturation level of air in the media. The pH was adjusted to 7.2 using 10% Ammonium Hydroxide solution, followed by inoculation with the seed inoculum. Throughout the cultivation, pH was maintained at 7.2 using 10% ammonium50KILPATRICK TOWNSEND 80240015.2hydroxide, temperature was held at 37°C, and pO? was controlled at 30% air saturation using agitation, oxygen supplementation, and aeration.

[0192] Upon depletion of the initial glucose in the batch media, 1-17 (1 Ox) media was fed to the bioreactor to supplement glucose demand using an exponential feeding strategy. The amount of 1-17 (lOx) to be fed was determined based on the target final OD595-600 assuming the I g / L of 1-17 (lOx) wras required to achieve OD595-600 of 1. Upon feeding the desired quantity of 1-17 (lOx) media, feeding was halted, and bioreactors were harvested post-glucose depletion.

[0193] 250 mL Cultivation Using the Sartorius Ambr® 250 Bioreactor. Fed-batch cultivations were conducted in 250 mL disposable Ambr® 250 microbial vessels in high throughput system. Each vessel was equipped with a pH probe and temperature probe and optical patch dissolved oxygen. The air sparging system utilized a single orifice sparger, and mixing was facilitated by two 6-blade Rushton impellers. To prevent volume loss due to evaporation, the system was equipped with condenser plate for each bioreactor holder. The outlet of die bioreactor was connected to integrated infrared exhaust gas analyzer.

[0194] The bioreactor setup involved the sterile addition of deionized (DI) water, 1-17 (lOx) media, and antifoam based on specific experimental conditions. The pH probe underwent overnight hydration and external calibration at room temperature, estimating the offset through offline pH measurement of the batch media. Upon reaching a temperature of 37°C, pO? probes were calibrated, estimating offset through sparging with pure nitrogen and determining slope using the saturation level of air in the media. Subsequently, the pH was adjusted to 7.2 using 10% Ammonium Hydroxide solution, followed by inoculation with the seed inoculum. After inoculation, fermentation was maintained.

[0195] Seed Expansion for 200 Liter Scale Up Using the Sartorius Biostat 2X BDCU-10 L Seed Bioreactor. Seed expansion cultivations were performed using VWR 250-mL vented PETG baffled flasks in a Sartorius Biostat 2X BDCU-10 L bioreactor equipped with probes for monitoring and controlling pH, temperature, and dissolved oxygen (DO). Aeration was provided via a multi-orifice ring sparger, while mixing was facilitated by two six-blade Rushton impellers. An external condenser was installed to minimize evaporative losses, with its outlet connected to an exhaust gas analyzer. Prior to operation, the pH probe was externally calibrated at room temperature using standard pH 7 and pH 10 buffers to determine offset of -1.9 mV and slope of 51KILPATRICK TOWNSEND 80240015.256.6 mV. The bioreactor was assembled by integrating all probes, the condenser, headplate, and feeding and sampling ports. The bioreactor was then sterilized in the autoclave at 121°C for 30 minutes with 4,900 mL of deionized (DI) water and 5 mL of antifoam. After cooling to 37°C, 100 mL of I- 17 (1 Ox) medium containing glucose was aseptically added to achieve a target glucose concentration of 10 g / L. Dissolved oxygen probes were calibrated by sparging with pure nitrogen to establish the offset of 0.4 mV and with air-saturated medium to define the slope of 68.2 mV.

[0196] 200 Liter Cultivation Using the Sartorius Biostat DDCU-200L Bioreactor. Industrial scale cultivation was performed in a Sartorius Biostat DDCU-200L bioreactor equipped with probes for monitoring temperature, pH, and dissolved oxygen (DO). Air sparging was facilitated through a multi-orifice ring sparger, while agitation was provided by two six-blade Rushton impellers. To minimize evaporative losses, an external condenser was installed, with its outlet connected to an exhaust gas analyzer. Two pH probes were installed in the bioreactor and were externally calibrated at room temperature using standard pH 7 and pH 10 buffers to determine offset -7.2 mV, 1 mV and slope 57.1 and 57.8 mV respectively. Prior to sterilization, 145 L of deionized (DI) water and 180 mL of antifoam were added. In situ sterilization of the bioreactor vras performed using the “Sterilization Phase” program on the DDCU. For sterilization bioreactor was held at 121°C for 30 minutes. Following sterilization, the water volume was adjusted to 140 L. After the vessel cooled to 37C'C, 9.5 kg of 1-17 (lOx) medium containing glucose was aseptically added to achieve the glucose concentration of 27.9 gzL. Two dissolved oxygen (DO) probes installed in the bioreactors were calibrated by sparging with pure nitrogen to establish the offset of 0.4 and 0.3 mV respectively and by equilibrating with air-saturated medium to determine the slope of 76.8 and 74.7 mV respectively.

[0197] 1.5. Harvest and Recovery of Extract. Upon completion of the cultivation, the bioreactor was cooled to reach a temperature of 10 C. Subsequently, aeration, dissolved oxygen (DO) control, and pH control were deactivated. Agitation settings were adjusted to 100 RPM and 300 RPM for 1 L and 250 mL bioreactors, respectively. The cultivation broth was carefully collected and immediately placed on ice for further processing.

[0198] Following this, the collected broth wras centrifuged at 18,000 g for 15 minutes to segregate the cell pellet, with the supernatant being discarded. The resulting cell pellet as52KILPATRICK TOWNSEND 80240015.2precisely weighed and washed with S30 buffer. The weight of buffer used was six times the weight of the cell pellet. The cells were resuspended using ULTRA-TURRAX® homogenizer to achieve a homogeneous suspension. Afterward, the suspension was centrifuged at 18,000 g for 15 minutes. The cell pellet was collected while the supernatant was discarded. The collected pellet was weighed and resuspended in S30 buffer at twice the weight of the cell pellet.

[0199] This homogenous resuspension was subjected to lysis using the Avestin Emulsiflex C5 homogenizer, undergoing two passes through the homogenizer. The average pressure for the initial pass was 17,000 psi, followed by 3,000 psi for the second pass. Lysate obtained out of the homogenizer was cooled down using a circulation loop kept on ice. Once the lysate was obtained, it was clarified through centrifugation at 18,000 g for 60 minutes. The resulting lysate, devoid of solids, was collected.

[0200] For the 200 L bioreactor, agitation was maintained at 50 RPM to prevent cell sedimentation. Cells were harvested using a disc stack centrifuge equipped for inline washing with S30 buffer. During harvest, the cells were continuously washed using an inline static mixer as the cells were pumped into the centrifuge bowl. Process parameters, including inline wash ratio, discharge time, centrifugation speed, and back pressure, were optimized to minimize cell lysis and maximize recovery'. The optimal conditions were determined to be an inline wash ratio of 1: 1 (harvest to buffer), a combined harvest and buffer flow rate of 1.8 L / min, a discharge time of 1.5 minutes, centrifuge speed setting of 5, and a back pressure of 40 psi.

[0201] Bacterial cell extract activation. The clarified lysate, also referred to herein as the bacterial cell extract, was then activated to yield activated extract suitable for expressing proteins using the XpressCF® platform. Heat activation dissociates ribosomes from messenger RNAs, enabling the reuse of ribosomes in subsequent protein synthesis. The duration of heat activation was contingent upon experimental conditions. The activated extract was stored in sterile falcon tubes and promptly frozen.

[0202] 1.6. Activity Assay. The ability of each preparation of the extract to support cell-free transcription and translation of exogenous plasmid DNA was tested using activity assays. Three different assays were used for this purpose: (1) a GFP assay, (2) an SP10 use test assay, and (3) an assay to express IgG containing the non-natural amino acid para-azidomethyl-L-phenylalanine (pAMF).53KILPATRICK TOWNSEND 80240015.2

[0203] GFP Assay. In this assay, a plasmid harboring the GFP gene was employed for the synthesis of the target protein, A supermix was prepared for use in the assay. A supermix is a reaction mixture containing components that are necessary for the assay, and may include salts, amino acids, nucleotide monophosphates (NMPs), T7 RNA polymerase (XpressRNAP® ), peptide deformylase (XpressPDF®), nnAA para-azidomethyi-L-phenylalanine (pAMF), an orthogonal tRNA (XpressTRNA®), and engineered aminoacyl-tRNA Synthetase (XpressRS®). The plasmid comprising the GFP gene was added to the supermix to a produce a final volume of 45 uL. The GFP and supermix solution was added to 55 pL of the extract, thereby initiating the XtractCF® reaction. This reaction was performed in clear-bottom 96-well plate and the plate was incubated in a plate reader enabled with shaking. The quantification of protein production is determined by assessing the fluorescence emitted by the reactor cell.

[0204] SPIO Use Test Assay. In this assay, plasmids incorporating the heavy and light chain genes for the IgG antibody trastuzumab are employed to facilitate the production of intact antibody monomers. This assay was performed as described in U. S. Patent No. 9,938,516. Titer measurements were performed using PhyTip® columns instead of Protein A HPLC affinity chromatography or an IgG BioHT kit using Cedex Bio HT Analyzer.

[0205] Assay to express IgG containing pAMF. In this assay, a plasmid housing the heavy and light chain genes for an IgG antibody, incorporating the non-natural amino acid pAMF, is utilized to produce intact antibody monomers. The Sartorius high-throughput Ambr® 15 system is employed for the assay, ensuring stringent control over environmental conditions such as pH, dissolved oxygen (DO), and temperature with feed supplementation. To initiate the XtractCF® reaction, 52.5% of the supermix containing the genes of interest is combined with 47.5% of the extract. Protein quantification is carried out using Protein A PhyTip® columns or an IgG BioHT kit using Cedex Bio HT Analyzer.Example 2: Fed-Batch Fermentations Achieved High OD595-600 Cell Concentrations

[0206] This Example relates to scale-up of the fed-batch process based on cell concentration. The final OD595-600 targets of the fed-batch process at 1 L scale were 50 and 150.

[0207] A fed-batch process was implemented targeting OD595-600 of 50 and 150, each in duplicate, with the 1 L Biostat® B system. The final target OD595-600 for F019 and F020 was 150, while the final target OD595-600 for F021 and F022 was 50. The total glucose required to attain the54KILPATRICK TOWNSEND 80240015.2final OD595 was calculated based on a final volume of 900 ml, excluding the total base addition for pH control. Assuming 1 g / L of glucose was needed to generate 1 OD595-600 of cell biomass, the calculated total glucose required for the final volume of 900 mL and target OD595-600 of 150 and 50 are 135 g and 45 g, respectively. The composition of the bioreactors before inoculation is outlined in Table 3.Table 3. Composition of Bioreactors F019, F020, F021, and F022 before Inoculation. Components F019 F020 F021 F022DI water (mL) 802 802 622 622 Antifoam (mL) 0.9 0.9 0.9 0.91-17 (lOx) Media (mL) 36 36 36 36

[0208] For the seed expansion phase, two 250 mL shake flasks, SF1 and SF2, each containing 1-17 shake flask media, were each inoculated with 0.5 mL of DCB. After 8.25 hours, they achieved OD595-600 of 2.70 and 2.61, respectively. For inoculation of the bioreactors, 8 mL of culture from SF1 was introduced into each of F019 and F020, while 8 mL of culture from SF2 was added to each of F021 and F022, with an inoculation OD595-600 of 0.03. Post-inoculation, the dissolved oxygen levels began to decrease, indicating the initiation of metabolic activity. As the dissolved oxygen reached 30% of air saturation, it was controlled at the same level through a cascade control system involving agitation, O flow, and aeration cascade, as described in Table 4. The setpoints for controller output were linearly interpolated based on Table 4.Table 4. Polygon Cascade for DO Control.DO Controller Agitation Aeration O2 Flow Output (%) Setpoint (RPM) Setpoint (sLPM) Setpoint (sLPM) 0 300 0.8 040 1150 0.8 080 2000 0 0.8100 2000 0 1.2

[0209] Glucose depletion in the batch media was indicated by a spike in dissolved oxygen levels, indicating the end of the batch phase. Subsequently, 1-17 media (lOx) was fed into the bioreactors to sustain growth, employing an exponential feeding strategy. The feed rates for F019 and F020 were controlled according to the feed profile outlined in Table 5. Feed rate (g / h)55KILPATRICK TOWNSEND 80240035.2was determined by multiplying feed rate setpoint (mL / hr) with the density of the feed solution (g / mL). For Tables 5 and 6 below, the density of the feed solution was 1.24 g / mL.Table 5. Feed Rate Profile for F019 and F020.Time (hr) Feed Rate Total Feed Time (min) Feed Rate Setpoint (mL / hr) Fed (mL) (g / h)0 10.79719 0 0 13.38852 0.25 11.63812 2.8031 15 14.431270.5 12.54455 5.824518 30 15.55524 0.75 13.52157 9.081256 45 16.766751 14.57468 12.59164 60 18.07261 1.25 15.70982 16.37543 75 19.480181.5 16.93337 20.45392 90 20.99738 1.75 18.25221 24.85006 105 22.632742 19.67377 29.58858 120 24.39547 2.25 21.20604 34.69616 135 26.295492.5 22.85766 40.20155 150 28.34349 2.75 24.6379 46.13571 165 30.5513 26.55681 52.53205 180 32.93044 3.25 28.62516 59.42656 195 35.49523.5 30.85461 66.85805 210 38.25971 3.75 33.25769 74.86834 225 41.239544 35.84794 83.50249 240 44.45145 4.25 38.63993 92.80911 255 47.913514.5 41.64936 102.8406 270 51.64521 4.75 44.89319 113.6533 285 55.667555 48.38966 125.3082 300 60.00318 5.25 52.15844 137.8708 315 64.676475.5 56.22076 151.4119 330 69.71374 5.75 60.59947 166.0076 345 75.143346 65.31921 181.74 360 80.99581 6.25 70.40654 198.6978 375 87.30416.5 75.89009 216.9763 390 94.103716.75 81.80072 236.6784 405 101.4329

[0210] Feed rates for F021 and F022 were controlled according to the feed rate profile in TableTable 6. Feed Rate Profile for F021 and F022.Time Feed Rate Total Feed Time Feed Rate_ Setpoint (mL / hr) Fed (mL) (min) W _56KILPATRICK TOWNSEND 80240015.20 10.81188 0 0 13.406730.25 11.65395 2.806914 15 14.4509 0.5 12.56161 5.832441 30 15.5764 0.75 13.53996 9.09361 45 16.78955 1 14.59451 12.60877 60 18.09719 1.25 15.73119 16.39771 75 19.50668 1.5 16.9564 20.48174 90 21.02594 1.75 18.27704 24.88386 105 22.66353 2 19.70053 29.62883 120 24.428662.25 21.23489 34.74336 135 26.331262.5 22.88875 40.25623 150 28.382052.75 24.67142 46.19847 165 30.592563 26.59293 52.60351 180 32.975243.05 26.99483 53.94318 183 33.47359

[0211] The feed was stopped upon completion of the prescribed profiles and fermentations were terminated when residual glucose in the bioreactor is exhausted, as indicated by dissolved oxygen levels spike. The culture broth was harvested. Data collected on the growth and metabolite concentration at the end of the fermentation are presented in Table 7.Table 7. Growth and Metab o ite Data for F019, F020, F021, and F022.Bioreactor Total Final Final Final Base Acetate Glucose fermentation OD595- Volume Addition (mmol / L) (g / L) time (hr. ) 600 (mL) (mL)F019 20.20 136 1059.9 157 15.14 0.02 F020 17.80 131 1071.6 169 32.25 0 F021 16.10 53.8 964.8 66 1.11 0F022 17.20 50.6 956.1 57 0.44 0

[0212] F021 and F022 successfully achieved the target optical density (OD595-600) of 50, while F019 and F020 exhibited approximately -11% lower than the target OD595-600 of 150. This discrepancy can be attributed to a -18% dilution effect from base addition. The final acetate concentration at the end of the fermentation v / as lower for the target OD595-600 of 50 compared to the target OD595-600 of 150. This indicates that an extended fed-batch phase resulted in an increased accumulation of acetate. The maximum base feed rate was initially set to 50% of the maximum system value. However, F019 demanded frequent base addition, more than the system could deliver, which accounted for pH decline in F019. Maximum base feed rate was increased to 100% to accommodate the elevated base consumption rate, which led to realignment of 57KILPATRICK TOWNSEND 80240035.2F019’s pH trend with the set point. Data for pH, pOz, and oxygen uptake rates (OURs) for all the four bioreactors is shown in Figure 2. Oxygen uptake rate (OUR) reached a maximum of -550 mmol / L / hr. for both F019 and F020 at the end of fermentation.

[0213] This Example demonstrates that fed-batch process with exponential feeding can achieve high cell density with slightly higher acetate accumulation than chemostat process. The oxygen requirement for fed-batch process to achieve high cell density is significantly higher. Example 3: Analysis of Recovery Process Steps for the Production of Cell Extracts with High Cell-Free Synthesis Activity

[0214] This Example relates to the process for recovery of cell extract from the fermentation discussed in Example 2 above. The impact of washing the cell paste as a step in the cell extract recovery process on the final cell extract product was evaluated.

[0215] The harvested broth from each of the four bioreactors - F019, F020, F021, and F022 -was divided into two streams, as shown in Figur e 3. In one stream (Figure 3, top portion), the broth was subjected to centrifugation and die supernatant was discarded. The cell paste was resuspended with S30 buffer. The resuspended brodi was homogenized (“lysis” in Figure 3), and die lysate was clarified by centrifugation. The solids were removed, and the clarified lysate was promptly frozen in liquid nitrogen and saved for subsequent heat activation.

[0216] In another stream (Figure 3, bottom portion), die broth was centrifuged, and the supernatant was discarded. The collected cell paste was subjected to a wash with S30 buffer, followed by a resuspension in the same buffer. Resuspended suspension was homogenized (“lysis” in Figure 3). The obtained lysate was clarified by centrifugation, and the solids were removed.

[0217] pH and Magnesium Concentrations. The pH was measured for the harvested broth, resuspended broth, clarified lysate, and activated extract. Figure 4 illustrates pH trends during the extract recovery process. Notably, the pH of the resuspended broth for washed cell pellets is higher than its unwashed counterparts. In comparing clarified lysates that were collected from cell pellets that wrere washed and cell pellets that were not washed, the pH of the clarified lysate from washed cell pellets was also higher than the pH of the clarified lysate from unwashed cell pellets.58KILPATRICK TOWNSEND 80240015.2

[0218] Magnesium concentration analysis was conducted for resuspended broth, where a sample was centrifuged, and the magnesium concentration was measured in the supernatant. The average magnesium concentration for the resuspended broth of the washed sample was ~14 mM, while for the unwashed sample, it was ~8 mM. This difference suggests potential precipitation, possibly attributed to a lower pH. Consequently, die pH of the clarified lysate is lower for the unwashed cells when compared to washed cells.

[0219] Activity Assays. The heat-activated extracts were tested for cell-free protein synthesis activity using GFP and SP10 assays, as described above in Example 1. Measured activities were compared to a reference standard and relative activities were calculated. The average titer for the standard in SP10 and GFP assays was 448 mg / 1 and 73243 RFU, respectively.

[0220] Extracts were obtained from fermentations F019, F020, F021 and F022 at a target OD595-600 of 50 or 150. In the GFP assay, the extract generated from washed cell paste exhibited higher activity than the extract from unwashed cell paste. The negative impact of not washing die cell paste was more pronounced in the extracts from fermentations with a target OD595-600 of 150 than for fermentations with a target OD595-600 of 50. For washed cell paste, a higher OD59-6005 led to increased activity of the extract in the GFP assay. ODs, and the extracts were tested for GFP activity and SP10 activity, was observed in all extracts that correlated with an increase in activation time. An increase in GFP activity also correlated with increase in activation time.

[0221] Interestingly, the negative impact of not washing the cell paste was not significant for the SP10 assay. While the peak activity of the extracts from unwashed cell paste is nearly the same for both target OD595-600 conditions, the extracts from the higher target OD595-600 condition exhibited higher activity for washed cell paste. The lower activity of the extract from unwashed cell paste could be attributed to low magnesium concentrations, which can impact the activation of the extract and the productivity of the XtractCF® reaction.

[0222] Next, extracts were tested for producing pAMF-containing IgG in a cell-free protein reaction. The extracts were activated and tested in an Arnbr® 15 bioreactor for the production of pAMF-containing IgG. Results from Ambr® 15 XtractCF® reaction are shown in Figure 5. The impact of washing cells was less significant on the extract generated from the OD595-60050 fermentation, whereas it had a substantial impact on the extract generated from the OD595-600 15059KILPATRICK TOWNSEND 80240015.2fermentation. In contrast to the GDP and SP10 assays, this assay exhibited less than 100% relative activity.

[0223] Activity data generated for washed cells from F019 and F020 were compared with the activity data for the extract generated at a bench scale from chemostat fermentation. The assay reference standard produced an absolute titer of 657 mg / L. As shown in Figure 6, the extract generated from fed-batch process with target OD595-600 of 150 had comparable SP10 activity to extract from the chemostat process. In general, GFP activity was observed to increase with activation time.Example 4: Production of a High-Activity Extract Using the Fed-Batch Fermentation Process at 250 mL Scale Using the Ambr® 250 Bioreactor System

[0224] This Example relates to replicating the results obtained at 1 L-scale (as discussed above in Example 2 above) at a smaller 250 mL-scale using the Ambr® 250 high throughput bioreactor system. Four 1-L bioreactors were available for use whereas twelve 250-mL bioreactors in the Ambr® 250 system were available. The experiment was scaled down such that the Ambr® 250 system’s twelve 250-mL bioreactors could be utilized to increase throughput of the experiment. Further, smaller fermentation volumes also resulted in less material being used and shorter processing times for bacterial extract recovery'.

[0225] A fed-batch process aiming for a final OD595-600 of 150 was implemented in disposable 250 mL microbial bioreactors. As the bioreactors were already7sterile, 156 mL of sterile DI water, 0.23 mL of antifoam, and 9.2 mL of 1-17 (lOx) media were added using an automated liquid handler robot inside the biosafety cabinet after the bioreactors were mounted in the holder and connected to the system. The amount of 1-17 (lOx) media was calculated based on the final target volume of 230 mL, excluding the base addition for pH control. Two bioreactors were designated as F027 and F028. Once all three components were added, the bioreactors were held at room temperature overnight to hydrate the pH probes preinstalled in the bioreactors.

[0226] For seed inoculation, 1 mL of culture from DCB was added to 50 mL of 1-17 shake flask media in a 250 mL baffled shake flask. At 7 hours, it reached an OD595-600 of 2.22, and 5 mL of seed from the shake flask was added to the bioreactor with an inoculation OD595-6O8 of 0.06 - this inoculation OD595-600 was twice the inoculation OD595-600 used in Example 2 above. Post inoculation, dissolved oxygen immediately started dropping. Once it reached 30% air 60KILPATRICK TOWNSEND 80240015.2saturation, it was controlled at that level using a proportional-integral-derivative (PI) controller with three levels: agitation, O2 mix, and aeration rate. Details of the PI controller parameters are described in Table 8 below.Table 8. PI Loop Control for Ambr® 250.Parameters Agitation O2 mix AerationLevel 1 2 3Minimum 500 RPM 0% 200 mL / min Maximum 3500 RPM 100% 300 mL / min Proportional term-kP 10 0.05 0.4Integral Time-tl (s) 250 30 150

[0227] DO was controlled at 30% of air saturation. The feed rate was controlled using an exponential feeding strategy, with the initial feed rate set to 2.76 niL / hr. according to Equation 3 below. Subsequently, feeding was controlled based on Equation 4 below, and the feed rate was adjusted according to the sampling volume.Fo=— - - (Equation 3)Ft=Foxe^t(Equation 4)Fsrepresents the feed rate at time t (hours) during the fed-batch phase, while Fo denotes the initial feed rate at the onset of the fed-batch operation. The parameter p corresponds to the target specific growth rate for the fed-batch phase, which was set at 0.2 h-1. So represents the initial glucose concentration in the batch medium, and SF refers to the glucose concentration in the 1-17 ( lOx) feed, which was 500 g / L.

[0228] Once the target feed rate of 30 mL / hr was achieved, feeding was halted. Upon depletion of residual glucose in the bioreactor, dissolved oxygen level s started rising. Subsequently, dissolved oxygen control was discontinued, agitation was set to 300 RPM, pH control was stopped, and the temperature was lowered to 10°C.

[0229] Throughout the fermentation process, samples were collected at four specific time points. Figure 7 illustrates the trends in growth, glucose consumption, and acetate accumulation. The optical density' data reflects the exponential growth achieved by the cells. In the batch phase,61KILPATRICK TOWNSEND 80240015.2acetate accumulated, and as the fed-batch phase commenced, it started decreasing before eventually building up again.

[0230] Figure 8 shows online data for pH, pO?., and OUR for F027 and F028. Maximum OUR was lower for F027 and F028 compared to F019 and F020. Delay in the feeding was also indicated by the dip in OUR trend around 8 hows, of cultivation. Fermentation was terminated at 15.83 hours for both bioreactors, resulting in final OD595-600 values of 131 and 127 for F027 and F028, respectively. Similar to the previous experiment (as discussed in Example 2 above), the final OD595-600 'as lower than the target OD595-600 of 150 due to dilution caused by base addition. Folkwing the termination of bioreactor control, the tanks were maintained at 10°C for approximately 3 hours before harvesting. The harvested broth was processed for extract recovery, following the procedures outlined in Example 1 above.

[0231] The clarified lysate was thawed at 20°C, and five 5 mL samples were aliquoted, and activated for 40-120 minutes at 40°C in a water bath. The activated extracts were then assessed for activity using the SP10 assay and compared to a reference standard. The absolute titer for the reference standard was 595 nig / L. Results are shown in Figure 9.

[0232] In summary, this Example demonstrates that die fed-batch fermentation process can be used to produce cell extracts with high cell-free synthesis activity.

[0233] The Examples above demonstrate that the bacterial cell extract produced using a fed- batch process had comparable cell-free synthesis activity to the cell extract produced using a chemostat, continuous, fermentation process. Successful scaling down of the process to 250 mL bioreactors was achieved.

[0234] Hie yield of cell extract per liter of culture broth is 0.8 L / L, which is a remarkable 166% increase compared to the yield of 0.3 L / L achieved in chemostat fermentation. Table 9 show's calculations for scale-up of the fed-batch process to a target extract production volume of 600 L. A bioreactor volume that is five times that of the cheniostat bioreactor volume is needed in order to achieve the same volume of extract as the cheniostat fermentation. How'ever, fed-batch process is superior because it requires less total volume to achieve a greater yield of cell extract than the chemostat fermentation process. Furthermore, a fed-batch process typically takes less than 24 hours whereas a chemostat process can range from three to five days.62KILPATRICK TOWNSEND 80240015.2Table 9. Calculations for Fed-Batch Process.Target Extract Production Vol. (L) 600Final OD595-600nm 50 133Extract Yield / Liter of Culture Broth (L / L) 0.3 0.8Final Volume of Fermentation broth required 2000 750(L)# of sublots 15 1Size of fermenter (L) 200 1000

[0235] A representative process flow diagram for fermentation and extract recovery is shown in Figure 1. Parameters and details for seed flask cultivation to bench scale fermentation and the extract recovery process are as disclosed in Example I above.Example 5: Fed-Batch Fermentation and Recovery of High-Activity' Extract at 200 Liter Scale

[0236] Hie fed-batch fermentation and cell-extract recovery' process developed and assessed in Examples 2-4 above was scaled up and tested at 200 liter scale. Materials and methods used in this Example are as disclosed in Example 1 above. An E. coli strain suitable for producing bacterial cell extract for use in CFPS reactions was used.

[0237] Seed culture was prepared from E. coll cryo stock as described in Example 1.4 above. The seed cultures were prepared in several flasks and later harvested and combined to produce a pooled culture of about 150 mL, which was transferred to a Sartorius Biostat 2X BDCU-10 L seed bioreactor. The OD595-600 at inoculation was about 0.12. The pooled culture was cultivated for approximately 12 hours and arrived at OD595-600 of about 6.

[0238] Prior to inoculation, the media pH was adjusted to about 7.2 using 10% ammonium hydroxide. During cultivation. pH was maintained at about 7.2 using 10% ammonium hydroxide, temperature was controlled at 37°C and DO was maintained at 30% air saturation through a combination of agitation and aeration. A polygon cascade for DO control was applied as follows. Agitation was increased gradually from 100 RPM to 1400 RPM as the DO controller output increased from 0% to 80%. Agitation was further increased to 1600 RPM as the DO controller output arrived at 100%. Aeration was increased from 1.18 sLPM to 3.0 sLPM between 0% and63KILPATRICK TOWNSEND 80240015.220% DO controller output. Aeration remained constant at 3.0 sLPM up to 80% DO controller output. Aeration was increased to 5.0 sLPM as the DO controller output increased from 80% to 100%.

[0239] After termination of seed cultivation, 2.5 L of the culture was used to inoculate the 200 liter bioreactor to achieve an initial inoculation OD595-600 of about 0.12. The culture was cultivated towards a target OD595 of about 100 in a final volume of about 175-190 L.

[0240] Prior to inoculation, the media pH was adjusted to about 7.2 using 28% ammonium hydroxide. During cultivation, the pH was maintained at about 7.2 using 28% ammonium hydroxide, the temperature was controlled at 37°C, and DO was maintained at 30% air saturation through a combination of aeration, agitation, and oxygen supplementation. A polygon cascade for DO control was applied as follows. Agitation was increased from 30 RPM at 0% DO controller output to 400 RPM at 80% DO controller output. Agitation was then increased to 450 RPM at 100% DO controller output. Aeration was decreased slightly from 30 sLPM at 0% DO controller output to 29.5 sLPM at 20% DO controller output. Then, aeration was increased progressively to 119.5 sLPM at 60% DO controller output, and finally arrived at 45 sLPM at 100% DO controller output. O2 flow began at 0 sLPM, increases to 10.5 sLPM at 20% DO controller output, increased further to 42.5 sLPM at 60% DO confroller output, and arrived its maximum of 225 sLPM at 100% DO controller output.

[0241] Upon depletion of the initial glucose in the batch medium at about 16 hours, 1-17 (lOx) feed medium was supplied to the bioreactor using an exponential feeding strategy to meet the increasing glucose demand governed by Equations 3 and 4 disclosed in Example 4 above. A total of about 28 L of 1-17 (lOx) medium was calculated and used based on the target final OD595-600 of 100, with the assumption that 1 g / L of 1-17 (lOx) corresponds to an OD595 increase of 1. Upon completion of feed addition to reach the desired volume, glucose feeding was terminated. After glucose in the bioreactor was depleted (as indicated by a sudden increase in DO), cultivation was terminated. Cultivation was terminated at about 25 hours achieving final OD595-600 of about 100. Growth of the E. coli strain at OD595-600 was tracked from the beginning to the end of the fed- batch cultivation - from 0 hours to about 25 hours, an exponential growth curve was observed for the E. coli from OD595 of about 0 to about 100. Total acetate and glucose accumulated at the end of cultivation was about 0.4 mM and about 0.024 g / L respectively.64KILPATRICK TOWNSEND 80240015.2

[0242] After cultivation was terminated, the cells were harvested using a disc stack centrifuged as discussed above in Example 1.5. Under those conditions, a final slurry of about 38% cell solids was collected in a Palletank® connected to a chilling loop. S30 buffer was added to die cell solids to achieve a target concentration of about 33%. The cells were resuspended and lysed using a micro fluidizer. The resuspended cells were pumped into the microfluidizer with a diaphragm pump operating at an inlet pressure of 20 psi. Cell lysis was carried out at a target pressure of 20,000 psi.

[0243] The resulting cell lysate was immediately cooled to about 10-14°C and transferred to a disc stack centrifuge for clarification. The centrifuge was operated at speed setting 1 with a back pressure of 70 psi, and solids were discharged 'when the centrate OD595-600 reached approximately 1.6. The solids fraction wras discarded, while the clarified centrate was directed through a heat exchanger to raise the temperature to 30°C prior to filtration using a 0.45 / 0.22 pm Durapore® filter. The filtered clarified lysate was collected in a Palletank® connected to a chilling loop. The final clarified lysate contained magnesium ions at a concentration of about 10 mM and a total protein concentration of about 35 g / L. More magnesium acetate solution was added to the filtered clarified lysate to achieve a target magnesium ion concentration of about 10-20 mM. The lysate was then heat activated using a water bath. After heat activation, the activated extract was tested for activity or rapidly frozen in liquid nitrogen and stored at -80 °C.

[0244] The activated extract was tested using the SP10 use test assay described in Example 1.6 above. Titer measurement was assessed using an IgG BioHT kit with the Cedex Bio HT Analyzer. The activated extract derived using the 200 L scale fed-batch process demonstrated greater than 100% activity relative to the activity for activated extract produced using the chemostat (continuous fermentation) process. This activity result significantly exceeds the established performance threshold of 65% activity, indicating successful scale-up and effective translation of process performance at industrial L production scale.

[0245] The activated extract was also tested using the for producing IgG containing pAMF as described in Example 1.6 above. The activity of the produced extract was evaluated relative to the GMP reference lot generated using the chemostat process with the S251 strain for the expression of pAMF -containing IgGl. Titer measurement was assessed using an IgG BioHT kit with the Cedex Bio HT Analyzer. Again, the activated extract achieved a titer of greater than65KILPATRICK TOWNSEND 80240015.2100% activity relative to the activity for activated extract produced using chemostat (continuous fermentation) process. The result confirms that the 200 L fed-batch process produces extracts with superior performance over extracts produced using the chemostat process. The titer result is consistent with titer results at 1 L and 10 L scales. The IgGl titer from a 10 L fermentation was about 1290 mg / L and the IgGl titer from a 1 L fermentation was about 1200 nig / L at the 1 L scale. These results confirm robustness and scalability" of the fed-batch process at industrial scale.

[0246] Ail publications, issued patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0247] It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims.66KILPATRICK TOWNSEND 80240015.2

Claims

WHAT IS CLAIMED IS:

1. A method of producing a bacterial cell extract, comprising:a) culturing bacterial cells in a culture medium in a bioreactor of at least 250 milliliters in total volume,b) isolating the bacterial cells, andc) lysing the bacterial cells to produce the bacterial cell extract; and whereinthe bacterial cells in step a) are cultured to a cell density of about 55 to about 150 as measured using optical density at about 595-600 nm.

2. The method of claim 1, wherein the bacterial cells are cultured using a fed-batch fermentation process.

3. The method of claim 1 or 2, wherein maximum fermentation volume is determined by multiplying about 0.4 to 0.8 with maximum bioreactor volume.

4. The method of any one of claims 1 -3, wherein fermentation volume is at least 1,000 L and at least 700 L of bacterial extract is produced.

5. The method of claim any one of claims 1 -4, wherein oxygen uptake rate (OUR) is at least about 500 mmol / L / hour.

6. The method of claim any one of claims 1 -5, wherein oxygen uptake rate (OUR) is at least about 200 mmol / L / hour.

7. The method of any one of claims 1-6, wherein the bacterial cells in step a) are cultured to a cell density of about 100 to about 150 as measured using optical density at about 595-600 nm.

8. The method of any one of claims 1-7, wherein the bacterial cells in step a) are cultured to a cell density of at least 60-130 as measured using optical density at about 595-600 nm.67KILPATRICK TOWNSEND 80240015.2PATENT Attorney Docket No.:: 091200-1532416-0074I0PC Client Reference No.: 0221 WO 9. The method of any one of claims 1 -8, further comprising, prior to step a), a step of initially culturing the bacterial cells in culture medium of no greater than 1 liter in volume.

10. The method of any one of claims 1-8, further comprising, prior to step a), a step of initially culturing the bacterial cells in a volume that is no greater than 3% of the cul ture medium of step a).

11. The method of any one of claims 1-10, wherein the culture medium comprises amino acids, salts, glucose, trace metals, and vitamins.

12. The method of any one of claims 1-11, wherein the feed rate of the culture is from about 1 mL / LZhour to about 95 mL / L / hour.

13. The method of any one of claims 1-12, wherein after step b) and before step c), the isolated bacterial cells are washed with a buffer.

14. The method of claim 13, wherein the buffer comprises tris base, magnesium acetate, and potassium acetate.

15. The method of any one of claims 1-14, the isolating step comprises centrifugation.

16. The method of any one of claims 1-15, wherein the bacterial cell extract is activated with heating comprising about 35-40°C for about 20-60 minutes.

17. The method of any one of claims 1-16, wherein the bacterial cells are E. coli cells.

18. The method of any one of claims 1-17, further comprising, after step c), freezing the bacterial cell extract in liquid nitrogen for storage.

19. The method of any one of claims 1-18, further comprising a step of performing cell- free recombinant protein synthesis using the bacterial cell extract.68KILPATRICK TOWNSEND 80240015.2PATENT Attorney Docket No.:: 091200-1532416-007410PC Client Reference No.: 022 i WO 20. The method of any of claims 1-17, further comprising, after step c), d) combining the bacterial cell extract with a composition comprising trehalose, lactose, leucine, and / or raffinose; ande) spray-drying the combination to produce a spray-dried bacterial extract.

21. The method of claim 20, further comprising, after step e), freeze-drying the spray-dried bacterial extract for storage.

22. The method of claim 20 or 21, further comprising a step of reconstituting the spray-dried bacterial extract.

23. The method of claim 22, further comprising, after the reconstitution step, a step of performing cell-free recombinant protein synthesis using the reconstituted spray-dried bacterial extract.

24. A cell-free synthesis system comprising a bacterial cell extract produced according to the method of any one of claims 1 -23 and a nucleic acid encoding a protein of interest, wherein the composition comprises an active oxidative phosphorylation system containing biologically functioning tRNA, amino acids, and ribosomes for cell-free recombinant protein synthesis.

25. The cell- free synthesis system of claim 24, wherein the cell-free synthesis system is capable of producing the protein of interest, wherein the amount or the activity of the protein of interest is at least 65% of a control protein.

26. A method of cell-free protein synthesis comprising incubating the cell-free synthesis system of claim 24 or 25 under conditions permitting the expression of the protein of interest.

27. The method of claim 26, wherein the amount or the activity of the protein of interest is at least 65% of a control protein.69KILPATRICK TOWNSEND 80240015.2PATENT Attorney Docket No.: 091200-1532416-007410PC Client Reference No.: 0221 WO 28. The method of cl aim 26 or 27, wherein the protein of interest is an antibody, an antibody fragment, an antibody light chain, an antibody heavy chain, a cytokine, a cytokine fragment, an immunogenic polypeptide, or a carrier protein.70KILPATRICK TOWNSEND 80240015.2

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