Flocculation process for purifying crude fermentation broth

Abstract

The present invention relates to a process of flocculation to purify recombinant proteins from crude fermentation broth of insulin and insulin analogs or derivatives at a manufacturing scale in the presence of urea and TritonX-100 at a specific pH. The flocculation process is followed by at least one centrifugation and pH adjustment step to further clarify the broth. It is further followed by a filtration step to finally remove any aggregates present, and a chromatography step to capture the pure protein. Using this process, a high product recovery rate of more than 95% has been achieved.

Description

[Technical field]

[0001] The process as disclosed herein falls in the field of downstream processing. The process relates to the purification of proteins from fermentation broth. In particular, a process of flocculation for the purification of proteins from a protein suspension containing soluble and insoluble components is provided. [Background technology]

[0002] The following background discussion is provided merely to aid the reader in understanding the invention and is not admitted to describing or constituting prior art to the invention.

[0003] The production of recombinant proteins using Pichia pastoris as a host cell has been widely and industrially carried out. Therapeutic recombinant proteins such as insulin, its analogs and derivatives are peptides with a size of about 5-7 kDa. These peptides are expressed in Pichia pastoris in the form of precursor molecules and are secreted externally in the fermentation supernatant. After harvesting the fermentation, the cells are separated by continuous centrifugation. The supernatant after centrifugation contains significant amounts of Pichia cells, cell debris, Pichia-associated pigments, and other medium-associated impurities. This crude cannot be directly loaded onto a chromatographic column. Insoluble solids and many soluble impurities can severely damage the purification column and, if carried all the way from capture to the purification step, can seriously interfere with the purification process, resulting in a shortened column life and poor purification performance.

[0004] To clarify the fermentation supernatant, physical separation methods such as membrane filtration were first used. Microfiltration is another technique that is widely used to clarify feedstocks with high solids content. Extensive development has been done to develop a clarification process using microfiltration. A process using a 0.1 micron microfiltration membrane has been developed. After microfiltration, the filtrate was further concentrated by ultrafiltration to overcome the dilution encountered during microfiltration. This process was carried out in a 100 m2 clarification tank with a total area of ​​100 m2.2 and has been scaled up to filter supernatant volumes of 20-22 KL. Ionic polymers have also been used to modify fermentation media to enhance the removal of impurities from process streams in applications such as depth filtration and membrane absorbents.

[0005] As described in EP 1934242, traditional biopharmaceutical protein purification methods used to remove cells and cell debris are not always effective and sometimes bind significantly to the product of interest, increasing overall process time, which can be difficult during scale-up of the operating procedure. Any improvements that allow for faster recovery times and / or greater recovery are desirable as they reduce the costs associated with the production of protein therapeutics. Therefore, there remains a need for efficient purification processes. Summary of the Invention

[0006] Summary of the invention: A process for purifying a recombinant protein in a fermentation broth is provided, which process involves aggregation of impurities.

[0007] The process disclosed is a downstream protein recovery process by providing a flocculation step after harvesting of the culture broth.

[0008] In some embodiments, the process is a process for purifying a protein of interest from a yeast fermentation broth, comprising: a) flocculating the fermentation supernatant; and b) performing at least one separation step.

[0009] A process for purifying a recombinant protein in a fermentation broth is provided. The process includes adding urea and a non-ionic detergent to the fermentation broth. The process further includes adjusting the pH of the fermentation broth to a value in the range of pH 2 to 4.5 or to a value in the range of pH 7.5 to 8.5. In some embodiments, the urea and non-ionic detergent are added prior to adjusting the pH value. In some embodiments, the pH value is adjusted prior to adding the urea and non-ionic detergent. The process also includes incubating the fermentation broth for 30 minutes or more. The process further includes separating insoluble matter from the fermentation broth. Separating the insoluble matter results in a supernatant.

[0010] In some embodiments, the process further includes adding urea and a non-ionic detergent to the supernatant. In such embodiments, the process also includes adjusting the pH of the supernatant to a value in the range of pH 2 to 4.5, or to a value in the range of pH 7.5 to 8.5. In some embodiments, the urea and the non-ionic detergent are added prior to adjusting the pH value. In some embodiments, the pH value is adjusted prior to adding the urea and the non-ionic detergent. Such embodiments of the process further include incubating the supernatant for at least 30 minutes. The process in such embodiments also includes separating insoluble matter from the supernatant.

[0011] Urea, in some embodiments, is added to a final concentration of 0.1 to 0.3 M. In some embodiments, urea is added to a final concentration of 0.15 to 0.25 M. In some embodiments, the non-ionic detergent is added at a concentration ranging up to 1% (v / v). Separating the insoluble matter from the fermentation broth and / or supernatant is, in some embodiments, performed by centrifugation or filtration. Separating the insoluble matter from the fermentation broth and / or supernatant includes subjecting the broth to depth filtration.

[0012] In some embodiments, the non-ionic detergent is based on polyoxyethylene as the polar portion and contains an alkylphenyl moiety as the non-polar portion. In some embodiments, the non-ionic detergent is based on a fatty acid ester having a polyoxyethylene chain with a terminal hydroxyl group as the polar portion, with the alkyl chain of the fatty acid defining the non-polar portion. In some embodiments, the non-ionic detergent is based on a maltoside or glucoside as the polar portion and contains an alkyl chain as the non-polar portion. In some embodiments, the non-ionic detergent is selected from the group of Triton, Tween or Brij series.

[0013] In some embodiments, the non-ionic detergent is Triton X-100 (IUPAC name: 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol). In some embodiments, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol is added to a final concentration of 0.1-0.4%, e.g., 0.15%.

[0014] In some embodiments, the recombinant protein is insulin or an insulin analog or derivative. The insulin analog may be, by way of example, insulin glargine, insulin lispro, insulin aspart or oral insulin tregopir.

[0015] In some embodiments, the recombinant protein is produced by yeast, which may be, for example, Pichia pastoris.

[0016] Adjusting the pH of the fermentation broth or supernatant is performed by adding a suitable base, such as sodium hydroxide or potassium hydroxide, which may be added in the form of a solution that is, for example, 2.5 M.

[0017] In some embodiments, the process is part of a purification process that includes cation exchange chromatography as the final capture step. In some embodiments of such purification processes, more than 99% of urea and non-ionic detergents are removed after cation exchange chromatography. In some embodiments of such purification processes, more than 95% of the produced protein is recovered.

[0018] In some embodiments, the process is a flocculation process for purification of a recombinant protein from a crude fermentation broth, comprising the following steps: a. Production of recombinant proteins using Pichia pastoris as a suitable host; b. flocculating impurities in the fermentation broth by adding urea and Triton X-100 at a pH range of 2-4.5 and / or at a pH range of 7.5-8.5; c. removal of aggregates by centrifugation or filtration; d. Readjust the pH to the range of 2 to 2.5; e. Final capture of the protein by chromatography; Includes.

[0019] In some embodiments, the process relates to the purification of recombinant proteins such as insulin or insulin analogs such as insulin glargine, insulin lispro and insulin aspart.

[0020] In some embodiments of the aggregation process, aggregates are removed by centrifugation and depth filtration, and final capture of the protein is achieved by cation exchange chromatography. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 depicts a flow chart of an exemplary aggregation process applied to primary processing of glargine supernatant. [Figure 2A] FIG. 2A shows the NTU stability of glargine when kept at room temperature. [Figure 2B] FIG. 2B shows the NTU stability of glargine when kept at low temperature. [Diagram 3] FIG. 3 depicts a flow chart of an exemplary process of flocculation applied to primary processing of Lispro supernatant. [Figure 4A] FIG. 4A shows the NTU stability data for Lispro at pH 2.0±0.2 and kept at room temperature. [Figure 4B] FIG. 4B shows NTU stability data for Lispro at pH 2.0±0.2, kept at low temperature. [Figure 5A] FIG. 5A shows the NTU stability data for Lispro at pH 4±0.2 and kept at room temperature. [Figure 5B] FIG. 5B shows NTU stability data for Lispro at pH 4±0.2 and low temperature storage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description If the recombinant protein is secreted into the cell culture medium, downstream processing of the recombinant protein begins with harvesting the respective medium and separating it from the cells that expressed the protein. This recovery step includes the removal of cell debris, as well as the removal of any particulate and colloidal material. Then, bulk contaminants (mainly proteins) can be removed, and polishing steps that remove trace contaminants can be applied. The process provided herein relates to a purification step that follows the initial removal of the cells that expressed the protein.

[0023] The disclosed process is the result of an effort to address the problems faced by methods existing in the prior art. The impurities present in the supernatant were screened for a wide range of flocculation pH ranges. During such studies, it was observed that these impurities tend to aggregate in the pH ranges of 2-4.5 and 7.5-8.5. The flocculation occurred primarily due to changes in pH levels and was partially facilitated by lyotropic salts formed in situ. Attempts have been made to promote or increase the extent of flocculation using external flocculating agents such as calcium chloride, but it has been observed that pH-based flocculation is sufficient and causes a significant change in the distribution of particulate matter present in the supernatant. The finer colloidal particles all coalesce to form larger aggregates, thus improving their removal by centrifugation or other filtration techniques. However, along with the flocculated impurities, the product also precipitates or becomes physically attached to the aggregates and is lost during centrifugation or filtration.

[0024] A strategy was devised to keep the protein in solution but still not dissolve the aggregated solids. This was a bit challenging since solubilizing the protein could potentially also dissolve the solids. However, a fine balance was achieved by optimizing the concentrations of urea and TritonX-100 to keep the protein in solution without dissolving the aggregated solids. This ensured optimal protein recovery and supernatant samples with NTU turbidity of 20-100 immediately after centrifugation. After such clarification the samples remain stable in terms of turbidity for more than 7-8 days when stored at 2-8°C.

[0025] definition Unless otherwise defined herein, scientific and technical terms used in the context of the present invention shall have the meanings generally understood by those skilled in the art.Furthermore, unless otherwise specified by the context, singular terms shall include plural terms and plural terms shall include singular terms.The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art.The nomenclature used in the context of this specification and the techniques described herein are those generally used in the art.The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art. The term "Pichia pastoris" refers to a species of methylotrophic yeast that is frequently used as an expression system for the production of proteins.

[0026] The term "recombinant protein" refers to an altered / modified gene sequence that has been cloned and expressed in a suitable host system.

[0027] The terms "primary recovery" or "primary treatment" refer to the process during clarification of the fermentation supernatant, where the harvested broth is treated with chemicals such as urea, TritonX-100 and / or co-solubilizers to undergo flocculation, followed by several pH adjustment and centrifugation steps to remove the aggregates.

[0028] The term "downstream purification" refers to the recovery and purification of a biosynthetic pharmaceutical product from associated impurities and waste products generated during production.

[0029] The term "human insulin" refers to a human hormone whose structure and properties are well known. Human insulin has two polypeptide chains, the A chain and the B chain, connected by disulfide bridges between cysteine ​​residues. The A chain is a peptide of 21 amino acids, and the B chain is a peptide of 30 amino acids, and the two chains are connected by three disulfide bridges: one between cysteines at positions 6 and 11 of the A chain, a second between cysteine ​​at position 7 of the A chain and cysteine ​​at position 7 of the B chain, and a third between cysteine ​​at position 20 of the A chain and cysteine ​​at position 19 of the B chain.

[0030] The term "analog" or "derivative" with respect to a parent polypeptide refers to a modified polypeptide in which one or more amino acid residues of the parent polypeptide are replaced / deleted / added by other amino acid residues. Such addition, deletion or replacement of amino acid residues can occur at the N-terminus of the polypeptide or at the C-terminus of the polypeptide or within the polypeptide. Examples of insulin analogs are insulin aspart, insulin lispro, insulin glargine, oral insulin tregopir, etc. Other examples are porcine or bovine insulin, both of which are analogs of human insulin.

[0031] The term "glargine" specifically refers to a long-acting human insulin analogue that differs from human insulin in that the amino acid asparagine at position 21 on the insulin A chain is replaced by glycine and two arginine residues are added to the C-terminus of the B chain.

[0032] The term "lispro" specifically refers to a fast-acting human insulin analog that is chemically distinct from human insulin in that in insulin lispro the amino acid proline at position B28 is replaced by lysine and the lysine at position B29 is replaced by proline.

[0033] The term "CIEX chromatography" refers to "cation exchange liquid chromatography" where separation is performed due to the affinity of positively charged ions for negatively charged resins. Chromatography is used to capture and concentrate proteins during primary processing and clarification of fermentation supernatants.

[0034] The term "NTU" refers to "Nephelometric Turbidity Units", which is a measure of the cloudiness or haze of a liquid medium caused by finely suspended colloidal particles. The NTU of a solution is measured using a nephelometer.

[0035] The term "flocculation" refers to the process by which fine particles clump together to form a floc or flocs that can be separated by various methods such as sedimentation or filtration.

[0036] The term "cold" or "cold hold" refers to a temperature in the range of 2° C. to 8° C. The temperature may be, for example, from 4° C. to 6° C., including 5° C.

[0037] "Room temperature" or "RT" or "RT hold" refers to ambient temperature or temperatures in the range of 22°C to 25°C, including 23°C or 24°C.

[0038] The term "depth filtration" refers here to a type of filtration technique in which a porous filtration medium can retain particles when the fluid being filtered contains a high load of particulate matter. The filter is used throughout the medium, and as a result, larger particle masses can be retained before clogging occurs.

[0039] The term "DE diatomaceous earth (DE) filtration" refers to a process that uses diatoms or diatomaceous earth, the skeletal remains of small, single-celled organisms, as the filter medium.

[0040] The term "centrifugation" refers to a technique involving the application of centrifugal force to separate particles from a solution according to particle size, shape, density, viscosity of the medium, and rotor speed.

[0041] The term "v / v" refers to volume / volume. It indicates that the solute and solvent are liquid in nature. % v / v means that the solvent is in 100 mL. A % v / v solution is calculated using the following formula, using milliliters as the base measure of volume (v): %v / v = mL of solute / 100mL of solution It is calculated as follows.

[0042] Physical separation methods such as membrane filtration have been used initially to clarify fermentation supernatants. However, this filtration technique has challenges due to the variable nature of the particles (such as suspended particles, cells, cell debris, fine colloidal solids, protein aggregates, and other insoluble matter) present in the fermentation supernatant after the cell separation step. The filtration area required to handle this crude supernatant is 100 L / m2. 2The filtration efficiency was less than 1.2 u and the quality of the filtered feed was not suitable for direct loading onto capture chromatography. A commonly used filtration scheme involved the use of a 4-5 u filter / 1.2 u filter / 0.45 u (nominal) membrane filter in series. As a result, one is forced to conclude that the use of membrane filters is a rather impractical and uneconomical method for clarification of fermentation supernatants. Similar challenges were experienced with depth filters. Due to the wide distribution of particle sizes, frequent and rapid clogging of depth filters was commonly observed, resulting in extremely low filtrate production per unit filter area.

[0043] The challenges faced during membrane and depth filtration were primarily due to the fact that the supernatant contained particulate matter with a broad size distribution, such that the finer particles would clog the filter membrane at the start of filtration, thus greatly reducing the filter throughput.

[0044] Microfiltration is another technique that is widely used to clarify feeds with high solids. Extensive development has been done to develop a clarification process using microfiltration. A process was developed that uses a 0.1 micron microfiltration membrane. After microfiltration, the filtrate was further concentrated by ultrafiltration to overcome the dilution that occurred during microfiltration. This process was repeated for 100 ml. 2 The microfiltration was scaled up to a total area of ​​1000 μL, filtering 20–22 KL of supernatant. Filtration of the supernatant was achieved in 80–100 h, with a significant improvement in clarity. However, the clarified supernatant after microfiltration showed limited stability. Upon storage of the post-microfiltration supernatant at either room temperature or low temperature, microprecipitates reappeared, causing changes in sample clarity and turbidity, leading to subsequent clogging of the capture column.

[0045] In summary, the traditional centrifugation and / or filtration approaches relied upon to clarify supernatants pose the following challenges prior to application to chromatography: -Higher residual solids in the feed caused higher backpressure on the capture column; -NTU or suspended colloidal particles continued to increase during sample retention; This has had a significant impact on process time and column life, resulting in process fragmentation. As mentioned in EP 3070472, certain ionic polymers, especially cationic polymers, can be used to flocculate cells and / or cell debris and precipitate / coagulate proteins. Ionic polymers have also been used to modify fermentation media to enhance the removal of impurities from process streams in applications such as depth filtration and membrane absorbents. However, it is also known that as the fermentation media is processed, the pH and conductivity of the media continue to change. As a result, the effectiveness of these flocculants is typically reduced.

[0046] As mentioned in EP 1934242, conventional biopharmaceutical protein purification methods used to remove cells and cell debris are not always effective and sometimes result in significant binding of the desired product, increasing the overall process time which can be a challenge during scale-up of the operating procedure. Any improvement that allows for shorter recovery times and / or greater recovery is advantageous as it reduces the costs associated with protein production.

[0047] Another major approach commonly applied is flocculation using pH, anionic and cationic agents. In all such flocculation methods, the co-precipitation of product along with impurities is the main problem commonly faced. In such situations, when the flocculated impurities are physically separated by centrifugation or by filtration, the precipitated product is also removed along with the impurity precipitate, resulting in a high loss of product. This was mainly due to the phenomenon of physical adsorption or non-specific interaction of the protein of interest with the aggregates, or the precipitation of the actual product. This posed a serious challenge to the flocculation-based clarification approach due to the impact on process costs by causing loss of the protein of interest.

[0048] In the process disclosed herein, a fermentation broth is used. The fermentation broth may be the supernatant obtained by centrifugation of the recombinant host cells that produced the recombinant protein. In some embodiments, the process may include using the host cells to produce the recombinant protein.

[0049] Generally, any desired recombinant protein may be included in the fermentation broth. In some embodiments, the recombinant protein is insulin or its analogues / derivatives. Each protein may be expressed in any suitable host cell, such as eukaryotic systems. An example of a suitable eukaryotic host cell is yeast, such as Pichia pastoris. The process includes adding urea and non-ionic detergent to the fermentation broth.

[0050] A nonionic surfactant is a compound that does not have an ionic functional group. Thus, its hydrophilic head group is uncharged. Any nonionic surfactant can generally be used. For example, it may be an ether and / or may include a hydroxyl group. In some embodiments, the nonionic surfactant is a polyether. In some embodiments, the nonionic surfactant is an amine oxide or a phosphine oxide. In some embodiments, the nonionic surfactant is a sulfoxide.

[0051] In some embodiments, non-ionic surfactants, interchangeably referred to as non-ionic surfactants, are commercially available under the trade names Triton, Tween or Brij, such as Brij 35, C12E23, or polyoxyethylene (23) lauryl ether. Polyoxyethylene-based non-ionic surfactants are available under the trade names Brij35, Brij58, TritonX-100, IGEPALCA-630 (formerly NonidetP-40). In exemplary embodiments, non-ionic detergents are available under the trade name TritonX-100.

[0052] An illustrative example of a non-ionic surfactant with multiple hydroxy groups is ([N,N'-bis(3-D-gluconamidpropyl)deoxycholamide]) available under the trade name Deoxy Big CHAP, or N,N-bis-(3-D-gluconamidpropyl)cholamide available under the trade name Big CHAP. Further examples of non-ionic surfactants with multiple hydroxy groups are acyl-N-methylglucamide (MEGA) compounds, such as N-decanoyl-N-methylglucamine or N-octanoyl-N-methylglucamide.

[0053] Another suitable non-ionic surfactant is dimethyldidecylphosphine oxide available under the trade name APO-12. Octyl beta glucoside is another example of a non-ionic surfactant. Another suitable non-ionic surfactant is n-dodecyl sucrose. Two more suitable non-ionic surfactants are n-dodecyl-β-D-glucopyranoside and n-dodecyl-β-D-maltoside. Two more suitable non-ionic surfactants are cyclohexyl-n-ethyl-β-D-maltoside and cyclohexyl-n-hexyl-β-D-maltoside. Cyclohexyl-n-methyl-β-D-maltoside and n-decanoyl sucrose are two more examples of a suitable non-ionic surfactant. Yet another suitable non-ionic surfactant is Digitonin.

[0054] Generally, the non-ionic surfactant is added to a final concentration ranging from greater than 0 to 0.5% v / v. In some embodiments, the final concentration of the non-ionic surfactant is greater than 0.05% v / v, for example, greater than 0.1% v / v. In some embodiments, the final concentration of the non-ionic surfactant is up to 0.6% v / v, including up to 0.4% v / v. As an illustrative example, the final concentration of the non-ionic surfactant may be 0.25% v / v.

[0055] Urea may be added to a final concentration ranging from greater than 0M to 0.5M. In some embodiments, the final concentration of urea may range from greater than 0.1 to 0.3M. In some embodiments, the final concentration of urea is greater than 0.02M, such as greater than 0.05M. In some embodiments, the final concentration of urea is greater than 0.1M, such as greater than 0.12M. In some embodiments, the final concentration of non-ionic surfactant is up to 0.35M, including up to 0.2M. In some embodiments, the final concentration of urea may be in the range of 0.15-0.25M. As an illustrative example, the final concentration of urea may be 0.1M.

[0056] The process further includes adjusting the pH of the fermentation broth to which the urea and non-ionic surfactant have been added. The pH may be adjusted, for example, to a pH value in the range of pH 2.5 to pH 4.0. The pH may also be adjusted to a pH value in the range of pH 3.0 to pH 3.8. The pH may illustratively be adjusted to pH 2.8 or pH 3.5. In some embodiments, the pH may be adjusted to a pH value in the range of pH 7.8 to pH 8.2. The pH may also be adjusted to a pH value in the range of pH 8.0 to pH 8.5.

[0057] Any acid or base can be used to adjust the pH value of the fermentation broth. If the pH needs to be increased, an organic or inorganic base may be added to the fermentation broth. Suitable bases for pH adjustment are, for example, sodium hydroxide and potassium hydroxide. The concentration of sodium hydroxide may be in the range of 1-4M, for example, 2.5M.

[0058] After pH adjustment, the fermentation broth with added urea and non-ionic surfactant is incubated for a time sufficient to allow flocculation to occur. In some embodiments, the fermentation broth is incubated for 30 minutes or more, including 1 hour or more. In some embodiments, the fermentation broth is incubated for 2 hours or more, including 4 hours or more.

[0059] After incubation, the fermentation broth is separated into soluble and insoluble matter. This removes the insoluble matter from the fermentation broth, giving a solution, which for ease of reference will be referred to as the supernatant below. The separation into soluble and insoluble matter is generally achieved using physical means. The fermentation broth may be centrifuged (apparatus: Beckman coulter) at a force of 8983 g, allowing sufficient removal of flocculation. After incubation, the fermentation broth may also be subjected to filtration (3M™ Zeta Plus™ capsules, 60SP nominal pore size 0.3 microns to 4 microns). The filter may be, for example, a membrane, allowing sufficient removal of flocculation.

[0060] After exposing the pH-treated fermentation broth to gravity and filtration enhanced by centrifugation or to centrifugation, the process may be completed. If desired, or if flocculation is still observed, a second adjustment of the pH of the supernatant may be made.

[0061] The pH of any second or further subsequent pH adjustments is selected independently from the pH used to adjust the pH of the fermentation broth to which urea and non-ionic surfactants were added. Illustratively, if the pH was increased in the first pH adjustment, the pH may be increased or decreased in the second or further subsequent pH adjustments. In some embodiments, the first pH adjustment may be, for example, at a pH value in the range of 2.0-4.5, and the subsequent pH adjustments may be at a pH value in the range of 7.5-8.5. In some embodiments, the first pH adjustment may be, for example, at a pH value in the range of 7.5-8.5, and the subsequent pH adjustments may be at a pH value in the range of 2-4.5. In some embodiments, both the first pH adjustment and the subsequent pH adjustments may be at a pH value in the range of 7.5-8.5, but at different pH values ​​within this range. Similarly, the first pH adjustment and the subsequent pH adjustments may be at a pH value in the range of 2.0-4.5, but at different pH values ​​within this range.

[0062] Generally, in any second or further subsequent pH adjustment, the pH may be adjusted, for example, to a pH value in the range of pH 7.8 to pH 8.2. The pH may also be adjusted to a pH value in the range of pH 8.0 to pH 8.5. In some embodiments, the pH may be adjusted to a pH value in the range of pH 2.5 to pH 4.0. The pH may also be adjusted to a pH value in the range of pH 3.0 to pH 3.8. The pH may illustratively be adjusted to pH 2.8 to pH 3.5.

[0063] After the second or further subsequent pH adjustment, the supernatant is incubated for a time sufficient to allow further aggregation to occur. In some embodiments, the supernatant is incubated for 30 minutes or more, including 1 hour or more. In some embodiments, the supernatant is incubated for 2 hours or more, including 4 hours or more.

[0064] After the second or further pH adjustment, the supernatant is again incubated for a time sufficient to allow aggregation to occur. This second or further incubation may last for 30 minutes or more, including 1 hour or more. In some embodiments, the second or further incubation may last for 2 hours or more, including 4 hours or more.

[0065] After the second or further incubation, the supernatant is centrifuged under gravity, allowing for sufficient removal of flocculation. Gravity may be used similar to that detailed above. After incubation, the supernatant may be subjected to filtration. Filters as detailed above may be employed. The filter may be, for example, a membrane, allowing for sufficient removal of flocculation.

[0066] In some embodiments, a method for harvesting a yeast cell culture is provided, comprising culturing Pichia pastoris cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and / or packed cell volume is achieved, removing the cells by centrifugation, adding urea and a non-ionic detergent such as TritonX-100 to the cell-free fermentation supernatant to obtain a cell-free supernatant, and initiating pH-based flocculation, mixing the cell-free supernatant during flocculation, allowing the flocs to settle, and recovering the clarified supernatant.

[0067] Following this, the recombinant protein may be subjected to further downstream processing steps, which will typically involve chromatography.

[0068] example In the following, embodiments of the process disclosed herein are illustrated by way of examples. A protein of interest, such as insulin or its analogs / derivatives, was expressed in a yeast expression system of choice, with the yeast Pichia pastoris. Protein expression was carried out in fermentation reactors with volumes ranging from 20-22 KL for large-scale production. The protein was secreted from the cells into the medium in the form of a precursor.

[0069] At the end of the fermentation, the broth was harvested and centrifuged at 8983 g for 10 min. The supernatant collected after centrifugation still contained soluble as well as insoluble materials along with the protein of interest. Adding an aggregation initiator at this step could cause the protein to aggregate with other medium components. Therefore, to further reduce the process time, an improved process was devised in which the supernatant was treated with a solution of urea and non-ionic detergent, followed by pH adjustment to a range of 2-4.5 or 7.5-8.5 using a suitable base, incubated for a specified time, and centrifuged to remove aggregates, as shown in Figures 1 and 3. Depending on the clarity of the harvested broth, additional pH adjustment and centrifugation steps may be performed.

[0070] The addition of urea and non-ionic detergents at a specific pH initiated flocculation, causing media components, cells, cell debris, colloids and other materials to cluster and form flocs / aggregates of various sizes, which also prevented the proteins to be harvested from binding to the flocs. The base used to adjust the pH was sodium hydroxide or potassium hydroxide, mainly sodium hydroxide. The concentrations of sodium hydroxide are shown below.

[0071] material and method The table details the materials and material grades used in the experiments performed below. The table details the reagents used and their preparation in the experiments performed. [Table 1]

[0072] For efficient separation of the agglomerated particles from the solution, the centrifugation method was repeated 2-3 times in the process. -A wide pH range was scanned to study the solubility pattern of the protein. An effective pH of either pH 2-4.5 or pH 7.5-8.5 was identified as suitable for selectively precipitating impurities and colloidal particles. -Optimized individual concentrations of urea and TritonX-100, as well as a mixture of urea and TritonX-100, were selected as co-solubilizing agents. -Urea and TritonX-100 were used to induce selective aggregation of impurities and other colloidal particles, keeping the main product in solution with minimal loss.

[0073] Flocculation process of insulin glargine-containing fermentation broth Figure 1 shows a step-by-step flow chart of the flocculation process of fermentation broth containing insulin glargine. The insulin glargine supernatant from the fermentation broth was further clarified with different strengths and different pH of primary treatment stocks (urea and 30X stocks of TritonX-100 (as shown in Table 1)).

[0074] At the end of the fermentation, the fermentation broth was harvested and first centrifuged. Upon centrifugation, a mixture of urea and TritonX-100 from a 30X stock was added to the cell-free fermentation supernatant. The quantities of the reagents were added on a volume / volume basis. After adjusting the pH to 3.5±0.1 with 2.5 M sodium hydroxide solution, the mixture was left for incubation for 2 hours. The pH depends on the insulin or insulin analogue present in the fermentation broth.

[0075] After incubation, the mixture was centrifuged to remove solids in the form of aggregates formed at pH 3.5. After this centrifugation, the pH of the resulting supernatant was further adjusted to 8.5 ± 0.1 using 2.5 M sodium hydroxide solution. The second pH adjustment was made to remove additional solids from the solution that inherently aggregate only in the pH range of 7.5-8.5. In this way, the sample goes through two types of aggregation processes, each followed by a centrifugation step. While this two-step aggregation step was followed for insulin glargine, for most of the other analogs a single aggregation step is sufficient to remove the solids from the cell-free supernatant.

[0076] The pH of the flocculated supernatant obtained after the final centrifugation step was readjusted to 2.5±0.1. The mixture was further clarified using depth and terminal filtration followed by cation exchange liquid chromatography. The methods provided herein have been further refined with the aid of experimentation, however, these experiments should not be construed as limiting the scope of the invention. In the following experiments, the efficiency of flocculation is expressed as product recovery and NTU in the primary recovery step.

[0077] Experiment 1 - Primary processing of glargine supernatant for better clarification and optimal recovery In this study, primary processing of glargine was carried out at different pH values ​​and stock concentrations. Primary processing was also carried out in two different batches. The turbidity of the solutions was measured with a nephelometer and the resulting turbidity was reported in Nephelometric Turbidity Units (NTU). Process recovery was calculated for each value from both the batches to evaluate the optimized pH value.

[0078] At the end of the fermentation, the broth was harvested and centrifuged at 8983g for 10 minutes (to remove cells and cell debris from the broth), followed by the addition of a solution of urea and TritonX-100 from a 30X stock to the supernatant. After this addition, the pH was adjusted to 3.5±0.1 using 2.5M sodium hydroxide solution. Once pH adjusted, the broth was incubated for 2 hours, followed by centrifugation of the broth at 8983g for 10 minutes. After the end of the centrifugation after incubation at pH 3.5, the pH was increased to 8.5 using 2.5M sodium hydroxide solution and incubated for an additional 2 hours to allow flocculation. These flocculate formed after incubation at pH 8.5 were removed by another centrifugation. This process followed test number 5 in the table below. All other tests were incubated at a single pH with or without the primary treatment stock. Following this, the pH was readjusted to 2.5±0.1 as shown in Figure 1. Ten trials were conducted in two batches. Trials 1, 2, and 3 did not contain any added primary treatment stock.

[0079] Table 2 details the results observed after conducting the tests as per experiment 1. [Table 2]

[0080] As can be seen in Table 2, there was a striking difference in primary recovery at pH 4.5 between the cases with (Tests Nos. 5, 7, and 10) and without (Tests Nos. 3 and 7) the addition of co-solubilizer. It was observed that the addition of co-solubilizer (primary processing stock) during the primary treatment process significantly reduced product losses. It was also observed that pH 3.5 - pH 8.5, pH 3.5 (alone) were the most optimal process conditions for primary clarification, and these tests (Tests Nos. 5 and 6) offered an excellent balance between NTU and process recovery.

[0081] Experiment 2 - Stability of glargine clarified supernatants by primary processing approach (NTU stability) In order to determine the stability of the clarified supernatant after primary treatment, independent experiments were carried out for NTU. The experiments were carried out in triplicate at different pH values, different urea and TritonX concentrations. The experiments were carried out at two different holding conditions, i.e. low temperature, i.e. 5±3°C, and room temperature, i.e. 22±3°C.

[0082] Table 3 details the steps taken in the study. [Table 3]

[0083] Table 4 details the testing that was performed on the process detailed in Table 3. The results are better illustrated when viewed in conjunction with Figures 2A and 2B. [Table 4]

[0084] As detailed in Table 4, Test 1 was subjected to primary treatment after adjusting the pH to 3.5. In Tests 2 and 3, no primary treatment was used and the pH was adjusted to 3.5 and 2.5, respectively. All studies included a second pass of centrifugation at 8983 g for 10 min, after which the pH was adjusted to 2.5±0.2. As per the study run, NTU was stable for 7 days. As is evident from the stability data above (Table 4), NTU was almost stable (insignificant increase) for 7 days at low temperature in Test 1 (pH 3.5, with primary treatment approach) and Test 2 (pH 3.5, without primary treatment approach), while it was found to have increased in Test 3 (pH 2.5, without primary treatment approach) at the same time point. During the RT hold, test 1 at pH 3.5 with the primary treatment approach had higher stability compared to the other tests (pH 3.5 without the primary treatment approach and pH 2.5 without the primary treatment approach), clearly indicating that primary treatment offers a significant advantage in terms of NTU stability.

[0085] Study 3: Depth filtration study of clarified supernatants of glargine-A comparative evaluation High turbidity in NTU of the supernatant can pose a significant challenge at the depth filtration stage, thereby requiring larger filter areas and significantly longer processing times. Filtration capacity was studied in various trials (with and without primary treatment), as shown in Table 5 below. The tests were run using depth filtration (3M™ Zeta Plus™ capsules, 60SP nominal pore size rate 0.3 microns to 4 microns) and filtration throughput data was observed at a cut-off pressure limit of 2.0 bar, as detailed in Table 5. The depth filtration data for Test 1 was generated in the lab, while the remaining two data sets (pH 3.5-8.5 and pH 3.5, both with primary treatment) were referenced from scale batches (past manufacturing runs). [Table 5] Results - Trial 1 at pH 2.5 without primary treatment showed the lowest filtration throughput compared to the other arms as shown in the table above. The volumetric throughput for Trial 1 (clarification approach without primary treatment at pH 2.5) was 252 L / m for batches 1 and 2, respectively. 2 and 176 L / m 2 On the other hand, the other arm, which used a first-line treatment approach (pH 3.5 and 3.5 followed by 8.5), had a higher volumetric throughput in both cases (1000 L / m 2 was larger). This means that there are four benefits to using a primary treatment process: 1. Increased processing recovery; 2.Provides better clarity; 3. Greatly increase filtration capacity; and 4. Reduction of batch running costs, It was shown that there is.

[0086] Flocculation process of cell-free fermentation supernatant containing insulin lispro Figure 3 shows a stepwise flow chart of the aggregation process of cell-free fermentation supernatant containing insulin lispro. Cell-free fermentation supernatant of insulin lispro was obtained by centrifugation of the broth at 8983 g for 15-30 min. The pH of the cell-free fermentation supernatant obtained after centrifugation of the first broth was adjusted from 6-6.5 (fermentation pH) to 2.0 ± 0.1 using orthophosphoric acid / 2.5 M sodium hydroxide solution. After pH adjustment, this supernatant mixture was left to incubate (settle) for 8-12 hours to allow flocculation of certain types of fermentation impurities or medium components or salts. Unique to this first stage flocculation, which is performed at pH 2, is the absence of the need to add a primary treatment agent (urea / TritonX-100) since the product is highly soluble under these pH conditions and therefore does not co-precipitate with impurities or salts. After approximately 12 hours of incubation, the flocculated solids are removed by centrifugation. The pH 2 supernatant obtained after centrifugation is further adjusted to different pH conditions, namely pH 3.5, 4, and 4.5, to explore the second step of flocculation. Before the pH adjustment, a primary treatment agent is added to the base to avoid product precipitation in the samples. The pH 3.5, pH 4, or pH 4.5 samples with this primary treatment agent are incubated for 2-4 hours until flocculation occurs, and then centrifuged at 8983 g for 15-30 minutes. The samples obtained after the final centrifugation are readjusted to pH 2.5 and further filtered through depth and terminal filters, followed by loading onto a cation exchange chromatography column for capture.

[0087] Experiment 4: Primary processing of lispro fermentation supernatant for better clarification and optimal recovery Lispro fermentation supernatant was clarified with the primary treatment stock (30X stock of urea and TritonX-100, i.e., 3M urea and 4.5% TritonX-100) at different strengths and different pH according to the process flow described in Figure 3. Table 6 shows the results of these tests. [Table 6] Results: Considering NTU and process recovery as the two main evaluation criteria, clarification trials (3 and 6) with primary treatment approach at pH values ​​of 2.0 and 4.5 (1x strength of urea and TritonX-100), respectively, showed better process recovery and clarification compared to other combinations.

[0088] Experiment 5: Stability of lispro clarified supernatants by primary processing approach (NTU-based stability monitoring) An independent set of experiments was performed to determine the stability of the clarified supernatant with respect to NTU. The stability of the clarified Lispro supernatant was performed with respect to NTU at two different holding conditions: low temperature (5±3° C.) and RT (24±2° C.). The experiments were performed in two sets of tests: - Test 1 and Test 3 with primary treatment were carried out / performed at pH 2.0±0.2 and pH 4.0±0.2, respectively. - Test 2 and Test 4 without primary treatment were performed / conducted at pH 2.0±0.2 and pH 4.0±0.2, respectively. Tests 1 and 3 were performed with primary treatment, and tests 2 and 4 were performed without primary treatment. The process steps for conducting these experiments are detailed in Tables 7 and 8. [Table 7] [Table 8]

[0089] NTU stability was performed at RT (22±3° C.) as shown in Table 9, and at low temperature (5±3° C.) as shown in Table 10. a) NTU stability of clarified supernatants kept at RT (22±3° C.) - Results are shown in FIG. 4A. [Table 9] b) NTU stability of clarified supernatants during cold storage (5±3° C.)—results are shown in FIG. 4B. [Table 10]

[0090] A similar set of experiments was performed at RT using Tests 3 and 4. NTU stability was performed at RT (22±3° C.) as shown in Table 11, and at low temperature (5±3° C.) as shown in Table 12. c) NTU stability of clarified supernatants kept at room temperature (22±3° C.)—Results are shown in FIG. 5A. [Table 11] d) NTU stability of clarified supernatants during cold storage (5±3° C.) - Results are shown in FIG. 5B. [Table 12] Results: In the absence of the addition of urea and TritonX-100 stock solution, NTU was observed to increase from day 5 onwards at room temperature (RT). This observation was found to be similar in both tests without primary treatment, i.e., at pH 2.0±0.2 and pH 4.0±0.2. No clear differences were observed in NTU during cold holding for either arm (with or without primary treatment) at either pH stage (i.e., pH 2.0 ± 0.2 and pH 4.0 ± 0.2), although the study with primary treatment performed better in terms of NTU compared to the study without primary treatment.

[0091] Conclusion: The process disclosed herein provides a simple approach to the flocculation of soluble impurities from complex fermentation supernatants. It uses simple chemical agents and pH parameters to cause flocculation of soluble impurities. The agents used in the flocculation process, unlike commercially available flocculation agents, do not interfere with subsequent chromatographic purification by ion exchange chromatography. The disclosed process avoids product loss during the flocculation process by using an appropriate mixture of urea and detergents, thus keeping the desired product in solution. The flocculation process increases the size of the aggregates so that they can be removed by simple physical separation methods such as centrifugation. The complete removal of impurities from solution by this flocculation method is indicated by the stability of the clarified solution for extended periods of time, almost 5 days at room temperature and more than 7 days under low temperature conditions. This new approach allows for negative purification by taking impurities from the solution and clarifying it, making it suitable for chromatographic loading. After capture chromatography, it is ensured that more than 99% of the flocculation agent is removed and does not appear as residual in the final product. The proposed method offers the major advantage of reducing the process time and cost burden of the primary recovery step and avoiding clogging of the trapping column. The filtration process employed prior to chromatography was positively affected by the clarification provided by this primary recovery approach, with the filtration capacity of the depth filters increasing by 5-20 fold. The flocculation process was scaled up 1000-fold and performed similarly to the small-scale observations.

Claims

1. 1. A process for purifying a recombinant protein in a fermentation broth, comprising: a) adding urea and a non-ionic detergent to the fermentation broth; b) adjusting the pH of the fermentation broth to a value in the range of pH 2 to 4.5, or to a value in the range of pH 7.5 to 8.5; c) incubating the fermentation broth for at least 30 minutes; and d) separating the insoluble matter from the fermentation broth, thereby obtaining a supernatant; The process comprising:

2. below: a) adding urea and a non-ionic detergent to the supernatant; b) adjusting the pH of the supernatant to a value in the range of pH 2 to 4.5, or to a value in the range of pH 7.5 to 8.5; c) incubating the supernatant for at least 30 minutes; and d) separating the insoluble matter from the supernatant; 10. The process of claim 1 further comprising:

3. 3. The process of claim 1, wherein urea is added at a concentration of 0.1 to 0.3 M.

4. 4. The process of claim 3, wherein urea is added at a concentration of 0.15 to 0.25M.

5. 3. The process of claim 1 or 2, wherein the non-ionic detergent is added at a concentration in the range of 0.1 to 1% (v / v).

6. 3. The process of claim 1 or 2, wherein separating the insoluble matter from the fermentation broth and / or supernatant is performed by centrifugation or filtration.

7. 3. The process of claim 1 or 2, wherein the recombinant protein is insulin or an insulin analogue or derivative.

8. 8. The process of claim 7, wherein the insulin analogue is insulin glargine, insulin lispro, insulin aspart or oral insulin tregopir.

9. 3. The process of claim 1 or 2, wherein the recombinant protein is produced by yeast.

10. 10. The process of claim 9, wherein the yeast is Pichia pastoris.

11. 3. The process of claim 1 or 2, wherein the non-ionic detergent is selected from the group of Triton, Tween, or Brij series.

12. 12. The process of claim 11, wherein the non-ionic detergent is Triton X-100.

13. 13. The process of claim 12, wherein the concentration of Triton X-100 is 0.1 to 0.4%.

14. 14. The process of claim 13, wherein the concentration of Triton X-100 is 0.15%.

15. 3. The process according to claim 1 or 2, wherein the pH adjustment is carried out using a suitable base selected from the group of sodium hydroxide and potassium hydroxide.

16. 16. The process of claim 15, wherein the concentration of sodium hydroxide is 2.5M.

17. 3. The process of claim 1 or 2, wherein separating the insoluble matter from the fermentation broth and / or supernatant comprises subjecting the insoluble matter to depth filtration.

18. 3. The process of claim 1 or 2, wherein the final capture of the protein is by cation exchange chromatography.

19. 20. The process of claim 18, wherein greater than 99% of the urea and non-ionic detergent is removed after cation exchange chromatography.

20. 20. The method of claim 19, wherein greater than 95% of the final protein is recovered.

21. 1. A method for purifying a recombinant protein, wherein the recombinant protein is insulin glargine, comprising: a) harvesting the fermentation broth obtained after producing insulin glargine using the host Pichia pastoris; b) adding 0.25 M urea and 0.25% (v / v) Triton X-100 to the fermentation broth; c) adjusting the pH of the fermentation broth to a value in the range of pH 3.0 to 3.5; d) incubating the fermentation broth for at least 2 hours; e) separating the insoluble matter from the fermentation broth by centrifugation, thereby obtaining a supernatant; f) adjusting the pH of the supernatant to 8.5; g) incubating the supernatant for at least 2 hours; and h) separating the insoluble matter from the supernatant by centrifugation; 3. The process of claim 1 or 2, comprising:

22. 22. The process of claim 21, further comprising readjusting the pH to a value of 2.5 and comprising purification by cation exchange chromatography as a final capture of insulin glargine.

23. A method for purifying a recombinant protein, wherein the recombinant protein is insulin lispro, comprising: a) harvesting the fermentation broth obtained after producing insulin lispro using the host Pichia pastoris; b) adjusting the pH of the fermentation broth to a value in the range of 2.0 to 2.5; c) incubating the fermentation broth for at least 30 minutes; d) separating the insoluble matter from the fermentation broth by centrifugation, thereby obtaining a supernatant; e) adding 0.1 M urea and 0.15% Triton X-100 to the supernatant; f) adjusting the pH to a value in the range of pH 3.8 to 4.2; g) incubating the supernatant for at least 30 minutes; and h) separating the insoluble matter from the supernatant by centrifugation; 3. The process of claim 1 or 2, comprising:

24. 24. The process of claim 23, further comprising readjusting the pH to a value of 2.5 and purification by cation exchange chromatography as a final capture of insulin lispro.

25. 13. The process of claim 12, wherein the Triton X-100 is 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

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