Patent Application
20240076320
The present disclosure relates generally to the field of purification of proteins. In particular, the present disclosure relates to methods of purifying viral proteins with a hydrophobic membrane domain for use in vaccine compositions.
In recent years, the development of viral protein subunit vaccines has been prioritized due to the appealing safety and stability attributes of such vaccines as compared to live attenuated or inactivated vaccines. Rather than injecting a whole pathogen to trigger an immune response, subunit vaccines use specific isolated proteins from viral pathogens.
Efforts to develop protein subunit vaccines for common viral pathogens, including influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 viruses, are continuing. For example, such vaccines may contain one or more proteins having a hydrophobic membrane domain. Examples of such proteins include recombinant influenza hemagglutinin (HA) proteins, recombinant SARS-CoV-2 spike (S) proteins, recombinant RSV fusion (F) proteins, or a combination thereof. These proteins can be generated through a baculovirus expression vector system (BEVS), in which the genetic instructions for the desired protein product are introduced into a baculovirus that is then used to infect insect cells. The insect cells produce the protein, which can then be harvested and purified for use in vaccine compositions.
To satisfy the requirements of use in pharmaceutical and therapeutic applications, isolated proteins that are used in protein subunit vaccines must undergo careful purification to remove any contaminants or impurities that may arise during protein production. Purification of the target proteins is often achieved by utilizing one or more chromatographic purification steps. However, because of similarities between the target proteins and baculovirus/host cell proteins, routine purification procedures based on particle size or charge are often inefficient or ineffective in removing host cell protein (HCP) impurities. In addition to impacting the purity of the target protein, HCP impurities can also negatively impact yield and process volume, making large scale manufacturing more complex, costly, and time consuming.
The present invention is directed to methods of purifying viral proteins for use in vaccine compositions, and specifically for purifying viral proteins having a hydrophobic membrane domain for use in protein subunit vaccines.
The present invention provides for a method of purifying a protein from a solution. The method includes a capture step (a) and a polish step (b). The capture step (a) includes: (i) passing the solution over a hydrophobic interaction chromatography column, and (ii) eluting a crude protein eluate from the hydrophobic interaction chromatography column. The polish step (b) includes: (i) passing the crude protein eluate obtained from the capture step over a ligand affinity chromatography column, (ii) recovering a first flow through intermediate from the ligand affinity chromatography column, (iii) passing the first flow through intermediate over an anion exchange chromatography column, (iv) recovering a second flow through intermediate from the anion exchange chromatography column, (v) passing the second flow through intermediate over a different ligand affinity chromatography column, and (vi) recovering a purified protein eluate from the ligand affinity chromatography column. The protein may be any protein having a hydrophobic membrane domain, including for example an HA protein derived from an influenza virus, an F protein derived from RSV, or an S protein derived from a SARS-CoV-2 virus.
The present invention also provides a purified protein having a hydrophobic membrane domain that is produced by a baculovirus expression system in cultured insect cells, wherein the purified protein has a purity of greater than 85%.
Protein purification methods in accordance with the present disclosure provide highly purified proteins for use in vaccine compositions while removing undesirable HCP impurities. The method provides highly purified protein substances while maintaining or increasing the yield of the purified target protein. The method reduces process volume, complexity, and cost, and can be scaled from laboratory to commercial settings.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The present invention can be more readily understood by reading the following detailed description of the invention and study of the included examples.
As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
The terms “comprise,” “comprising,” “include,” “including,” and “includes” when used in this specification and claims are intended to specify the presence of the stated materials, substances, features, integers, components, or steps, but they do not preclude the presence or addition of one or more other materials, substances, features, integers, components, steps, or combinations thereof.
The term “about” modifies the subject values, such that they are within an acceptable error range, as determined by one of ordinary skill in the art, which will depend in part on the limitations of the measurement system.
The articles “a” and “an” as used herein refer to “one or more” or “at least one,” unless otherwise indicated. That is, reference to any element or component of an embodiment by the indefinite article “a” or “an” does not exclude the possibility that more than one element or component is present.
The term “purified” as used herein refers to a target substance, such as a target protein, that has been isolated from a complex mixture that includes unwanted components. Where reference is made to a purified substance, such as a purified protein, the reference is intended to include both target substances that have been entirely isolated from a complex mixture and target substances that are substantially isolated, even where a pharmaceutically acceptable residual amount of impurities remain.
The term “purity” is used herein to refer to the percentage of a particular sample that is comprised of the target substance, such as a target protein. Unless otherwise specified the percentage purity of a substance is calculated using known densitometry image analysis techniques.
A “hydrophobic membrane domain” refers to a hydrophobic region of a protein's polypeptide chain that spans the viral membrane.
An “influenza hemagglutinin protein” or “HA protein” is an envelope glycoprotein present in the influenza virion that is responsible for viral attachment and penetration into the host cell. The influenza virus initiates infection by attachment of the virion surface HA protein to a sialic acid-containing cellular receptor. Following attachment, the HA protein undergoes conformational changes that lead to fusion of viral and host cell membranes followed by virus uncoating and M2-mediated release of M1 proteins from nucleocapsid-associated ribonucleoproteins (RNPs), which migrate into the cell nucleus for viral RNA synthesis. The membrane-distal “head” portion of the HA protein is a primary target of host cell antibodies that confer protective immunity to influenza viruses, and as a result the HA protein is a desirable antigen for use in commercial influenza vaccines. When reference is made to an “influenza hemagglutinin protein” or “HA protein” in the present disclosure, it should be understood that such reference may include wild type and/or modified HA proteins.
An “RSV fusion protein” or “F protein” is glycoprotein present on the surface of the RSV virion that controls the initial phase of RSV infection, and in particular causes the virion membrane to fuse with the target cell membrane. RSV initiates infection by attachment via interaction between the attachment glycoprotein (“G protein”) and a receptor surface of the host cell. Following attachment, the F protein undergoes conformational changes that lead to fusion of the viral and host cell membranes and expression of viral proteins. The RSV F protein is a primary target of host cell antibodies that confer protective immunity to RSV, and as a result the F protein is a desirable antigen for use in commercial RSV vaccines. When reference is made to an “RSV fusion protein” or “F protein” in the present disclosure, it should be understood that such reference may include wild type and/or modified F proteins.
A “SARS-CoV-2 spike protein” or “S protein” is a large structural protein present in the SARS-CoV-2 (COVID-19) virus that is responsible for viral attachment and penetration into the host cell. The SARS-CoV-2 virus initiates infection by attachment via interaction between the receptor-binding domain (S1 region) of the S protein and ACE2 receptor surface of the host cell. Following attachment, the S protein undergoes conformational changes that lead to fusion of viral and host cell membranes and expression of viral proteins. The SARS-CoV-2 S protein is a primary target of host cell antibodies that confer protective immunity to coronaviruses, and as a result the S protein is a desirable antigen for use in commercial COVID-19 vaccines. When reference is made to a “SARS-CoV-2 spike protein” or “S protein” in the present disclosure, it should be understood that such reference may include wild type and/or modified S proteins.
The term “baculovirus expression system” as used herein refers to a known platform for expressing recombinant proteins. To initiate baculovirus expression, a recombinant baculovirus is constructed by cloning the genes of interest into a transfer plasmid behind a strong promotor and surrounded by DNA homologous to the parent baculovirus. To generate the target protein, the recombinant baculovirus is scaled up and used to infect insect cells, which generate large quantities of the target protein for subsequent harvesting and purification. Insect cell lines for use in baculovirus expression systems are well known in the art.
The terms “host cell protein” or “HCP” as used herein refer to any non-target protein present in the baculovirus expression system. For example, HCPs may include recombinant baculovirus proteins, insect cells proteins, or combinations thereof.
The term “impurity” as used herein refers to any component that is not desired in the final product, including host cell DNA and HCPs.
The term “solution” as used herein refers to any homogenous mixture of two or more substances.
The term “capture step” as used herein refers to quickly isolating, concentrating, and stabilizing the target protein after harvest. During the capture step, clarified lysate containing the target protein is passed through a chromatography column, and the target protein is eluted into a significantly smaller volume for further downstream processing. In general, capture steps are designed to accommodate larger and more viscous process volumes, and therefore employ larger columns and resin particle sizes compared to other purification steps. The resin selected for the capture step should maximize retention and recovery of the target protein.
The term “polish step” as used herein refers to removing trace impurities from the target protein to provide a purified protein. During the polish step, the target protein-containing solution obtained from the capture step (referred to herein as a crude protein eluate) is passed through a series of chromatography columns designed to remove specific impurities. Polishing is achieved via the use of different separation principles over a series of chromatography columns to maximize removal of impurities with different characteristics.
The terms “hydrophobic interaction column” or “hydrophobic interaction chromatography column” as used herein are intended to refer to a chromatography column designed to separate molecules based on their hydrophobicity. The ligand and resin used in the HIC column should be selected to maximize the specificity of binding to the target protein and step yield.
The terms “ligand affinity column” or “ligand affinity chromatography column” as used herein are intended to refer to a chromatography column designed to separate molecules based on highly specific biological interactions between the ligand and the molecule of interest. The ligand and resin used in the ligand affinity column should be selected based on its impurity clearance and step yield.
The terms “anion exchange column” or “anion exchange chromatography column” as used herein are intended to refer to a type of chromatography column designed to separate molecules based on their net surface charge. An anion exchange column uses a positively charged ion exchange resin with an affinity for molecules having a net negative surface charge. The ligand and resin used in the anion exchange column should be selected based on its impurity clearance and step yield.
The term “protein eluate” as used herein is intended to refer to the target protein-containing product obtained from a chromatography step via elution. The term “crude protein eluate” is intended to refer to a protein eluate obtained through the capture step, whereas the term “purified protein eluate” is intended to refer to the purified protein product obtained after the final polish step.
The term “flow through intermediate” as used herein is intended to refer to the target protein-containing product that flows through a chromatography step while impurities remain bound to the column resin. The term “first flow through intermediate” is intended to refer to the flow through protein product obtained from the second chromatography step and the term “second flow through intermediate” is intended to refer to the flow through protein product obtained from the third chromatography step.
The term “filtration” as used herein is intended to refer to the separation process by which certain particles are removed from a solution based on particle size.
The terms “tangential flow filtration” or “TFF” as used herein are intended to refer to a unit operation used for clarifying, concentrating, and/or purifying proteins via a pressure driven filtration process. During TFF, the feed solution is passed tangentially along the surface of a membrane while an applied pressure forces a portion of the fluid through the membrane. Particles that are too large to pass through the membrane pores are retained on the upstream side of the membrane (referred to as the retentate), and can be recycled through the TFF process as desired. Particles that are small enough to pass through the membrane pores (the filtrate) are collected and either disposed of or passed on to the next unit operation.
The same TFF unit operation can be used for micro- or ultrafiltration and diafiltration, which decreases process time and avoids product loss. When both micro- or ultrafiltration and diafiltration are preformed in the same unit operation, the diafiltration step is initiated by feeding the diafiltration solution (i.e. water or a buffer) into the recycled retentate stream and recirculating the solution across the membrane filter. In this way, the TFF process can be used to first concentrate the target protein (via microfiltration or ultrafiltration) and then diafilter the protein by exchanging the buffer solution, decreasing salt concentration, and/or removing solvents or other additives.
The term “viral filtration” as used herein refers to a filtration process designed to remove endogenous or adventitious viruses from the target protein solution. Viral filtration is a direct flow filtration process that employs a membrane filter with a size exclusion mechanism that allows target proteins to pass through the membrane without adsorption or denaturation while efficiently capturing viruses.
The target proteins that are subjected to the purification method of the present invention are typically produced by recombinant expression in host cells, and in particular via a baculovirus expression system. Target proteins may be expressed in any suitable host cell, for example in insect cells. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells (e.g Sf9, Sf21, Sf22a), Trichoplusiani cells, and Drosophila S2 cells.
Following host cell expression and expansion, the host cells are harvested via lysis in the bioreactor and clarified via centrifugation. The resulting clarified lysate solution is then subjected to the method for protein purification provided in the present invention. Referring to
It should be understood that certain steps may be added or modified to the method of the present invention so long as the primary steps described in
As shown in
As further shown in
As shown in
Given the high specificity on binding to the target protein, it is possible to pass large volumes of clarified lysate solution through the hydrophobic interaction column used in first chromatography step a while achieving very high target protein recovery. The high purity and recovery of the crude protein eluate versus the load solution is shown in
Returning to
As is also shown in
As is further shown in
As is further shown in
In certain embodiments, the fourth chromatography step f may include a detergent exchange during elution. By exchanging detergents, it is possible to encourage protein attributes that are desirable for use in the vaccine composition. For example, an elution buffer containing a non-ionic detergent may be applied to the column during fourth chromatography step f to facilitate the formation of the target protein into nanoparticles. In certain embodiments, the non-ionic detergent may be selected from the group consisting of polysorbate-20 (PS-20), polysorbate-40 (PS-40), polysorbate-60 (PS-60), polysorbate-65 (PS-65), and polysorbate-80 (PS-80).
As would be understood by a person of ordinary skill in the art, following execution of each chromatography step the column can be flushed, stripped and/or cleaned, and stored for repeat use.
Returning to
During the first filtration step b, the crude protein eluate is concentrated in the filtrate through use of a filtration membrane sized to allow removal of the salts from the first chromatography step a while retaining the target protein in the retentate. The crude protein eluate is concentrated, diafiltered to remove salts, and chased with a buffer to recover the crude protein eluate for loading into the second chromatography step c. Because the isoelectric points for viral proteins having hydrophobic membrane domains may vary depending on the strain or variant of the particular virus, it may be necessary to vary the pH and/or salt concentration of the solution during first filtration step b so as to prevent the target protein from binding to the anion exchange resin.
During the third filtration step g, the purified protein eluate is concentrated in the retentate while smaller impurities are passed through the membrane filter into the filtrate and removed from the process. Following concentration, the purified protein eluate is diafiltered, diluted to a target concentration, and spiked with a non-ionic detergent to facilitate the nanoparticle formation desired in the vaccine composition. It should be noted that where a detergent exchange is employed during the fourth chromatography step to introduce the non-ionic detergent, the non-ionic detergent will also be concentrated in the retentate during third filtration step g.
A process flow diagram depicting one embodiment of the method of purifying proteins described above is provided herein as
It should be understood that certain steps may be added or modified to the method previously described so long as the primary steps described in
An aspect of the present invention relates to a method of purifying a protein from a solution. The method includes (a) a capture step and (b) a polish step. The capture step (a) includes: (i) passing the solution over a hydrophobic interaction chromatography column, and (ii) eluting a crude protein eluate from the hydrophobic interaction chromatography column. The polish step (b) includes: (i) a second chromatography step wherein the crude protein eluate obtained from the capture step is passed over a ligand affinity chromatography column, (ii) recovering a first flow through intermediate from the ligand affinity chromatography column, (iii) a third chromatography step wherein the first flow through intermediate is passed over an anion exchange chromatography column, (iv) recovering a second flow through intermediate from the anion exchange chromatography column, (v) a fourth chromatography step wherein the second flow through intermediate is passed over a second ligand affinity chromatography column, and (vi) recovering a purified protein eluate from the ligand affinity chromatography column. The protein may be an HA protein derived from an influenza virus, an S protein derived from a SARS-CoV-2 virus, or an F protein derived from RSV.
The present invention also provides a purified protein having a hydrophobic membrane domain that is produced by a baculovirus expression system in cultured insect cells, wherein the purified protein has a purity of greater than 85%.
The present invention also provides a purified HA protein produced by a baculovirus expression system in cultured insect cells, wherein the purified influenza HA protein has a purity of greater than 85%.
The present invention also provides a purified F protein produced by a baculovirus expression system in cultured insect cells, wherein the purified RSV F protein has a purity of greater than 85%.
The specific embodiments describing the components, materials, ranges, values, and steps provided below are for illustration purposes only, and do not otherwise limit the scope of the disclosed subject matter, as defined by the claims.
In various embodiments, the hydrophobic interaction chromatography column employs a resin with high specificity for the target protein. For example, the hydrophobic interaction chromatography column may comprise a Butyl-S resin.
In various embodiments, the ligand affinity column of the second chromatography step employs a resin with high specificity for GP64 proteins. For example, the ligand affinity column of the second chromatography step may comprise a GP64 affinity resin.
In various embodiments, the anion exchange column employs a resin for effectively attracting negatively-charged impurities. For example, the anion exchange column may comprise a TMAE resin.
In various embodiments, the ligand affinity column of the fourth chromatography step employs a resin with high specificity for the target protein. For example, the ligand affinity column of the fourth chromatography step may comprise a lentil lectin resin.
In various embodiments, the target protein in the purified protein product is an HA protein derived from an influenza virus. The influenza virus may be one or more influenza virus subtypes selected from the group consisting of: Influenza A Group 1, Influenza A Group 2, and Influenza B.
In various embodiments, the target protein in the purified protein product is an F protein derived from a RSV. The RSV may be one or more RSV subtypes selected from the group consisting of: respiratory syncytial virus subtype A (RSV-A) and respiratory syncytial virus subtype B (RSV-B).
In various embodiments, the method for purifying the protein from the solution further comprises one or more filtration steps. For example, the capture step (a) may further include a first filtration step after the hydrophobic interaction chromatography column. In certain embodiments the first filtration step may include tangential flow filtration. As another example, polish step (b) may further include a second filtration step after the anion exchange chromatography step and a third filtration step after the final ligand affinity chromatography step. In certain embodiments the second filtration step may include viral filtration and the third filtration step may include tangential flow filtration.
Specific enumerated Embodiments 1 to 18 provided below are for illustration purposes only, and do not otherwise limit the scope of the disclosed subject matter, as defined by the claims. These enumerated embodiments encompass all combinations, sub-combinations, and multiply referenced (e.g., multiply dependent) combinations described therein.
Embodiment 1: A method including a capture step (a) that includes (i) passing the solution over a hydrophobic interaction chromatography column, and (ii) eluting a crude protein eluate from the hydrophobic interaction chromatography column, and a polish step (b) that includes (i) passing the crude protein eluate obtained from the capture step over a second chromatography step including a ligand affinity chromatography column, (ii) recovering a first flow through intermediate from the ligand affinity chromatography column, (iii) passing the first flow through intermediate over an anion exchange chromatography column, (iv) recovering a second flow through intermediate from the anion exchange chromatography column, (v) passing the second flow through intermediate over a fourth chromatography step including a ligand affinity chromatography column, and (vi) recovering a purified protein eluate from the ligand affinity chromatography column.
Embodiment 2: The method of embodiment 1, wherein the protein is a protein having a hydrophobic membrane domain.
Embodiment 3. The method of any one of embodiments 1-2, wherein the protein is derived from an influenza virus, RSV, or a SARS-CoV-2 virus.
Embodiment 4: The method of any one of embodiments 1-3, wherein the protein is an HA protein derived from an influenza virus.
Embodiment 5: The method of embodiment 4, wherein the influenza virus may be one or more influenza virus subtypes selected from the group consisting of: Influenza A Group 1, Influenza A Group 2, and Influenza B.
Embodiment 6: The method of any one of embodiments 1-3, wherein the protein is an F protein derived from an RSV.
Embodiment 7: The method of embodiment 6, wherein the RSV may be one or more RSV subtypes derived from the group consisting of: respiratory syncytial virus subtype A (RSV-A) and respiratory syncytial virus subtype B (RSV-B).
Embodiment 8: The method of any one of embodiments 1-7, wherein the hydrophobic interaction chromatography column includes a Butyl-S resin.
Embodiment 9: The method of any one of embodiments 1-8, wherein the ligand affinity column of the second chromatography step includes a GP64 affinity resin.
Embodiment 10: The method of any one of embodiments 1-9, wherein the anion exchange column includes a trimethylaminoethyl (TMAE) resin.
Embodiment 11: The method of any one of embodiments 1-10, wherein the ligand affinity column of the fourth chromatography step includes a lentil lectin resin.
Embodiment 12: The method of any one of embodiments 1-11, wherein the method for purifying the protein includes one or more filtration steps.
Embodiment 13: The method of embodiment 12, wherein the method includes a first filtration step after the hydrophobic interaction chromatography column.
Embodiment 14: The method of embodiment 13, wherein the first filtration step is a tangential flow filtration step.
Embodiment 15: The method of any one of embodiments 13-14, wherein the method includes a second filtration step after the anion exchange chromatography column.
Embodiment 16: The method of embodiment 15, wherein the second filtration step is a viral filtration step.
Embodiment 17: The method of any one of embodiments 15-16, wherein the method includes a third filtration step after the lentil lectin chromatography column of the fourth chromatography step.
Embodiment 18: The method of embodiment 17, wherein the third filtration step is a tangential flow filtration step.
Embodiment 19: A purified protein having a hydrophobic membrane domain that is produced by a baculovirus expression system in cultured insect cells, wherein the purified protein has a purity of greater than 85%.
Embodiment 20: A purified HA protein produced by a baculovirus expression system in cultured insect cells, wherein the purified influenza HA protein has a purity of greater than 85%.
Embodiment 21: A purified F protein produced by a baculovirus expression system in cultured insect cells, wherein the purified RSV F protein has a purity of greater than 85%.
Example 1: The following example provides process parameters for a capture step employing hydrophobic interaction chromatography with Butyl-S Sepharose 6 Fast Flow resin according to one embodiment of the present invention.
Example 2: The following example provides process parameters for a capture step employing tangential flow filtration with a Repligen TangenX® SIUS® 50 kDa cassette membrane according to one embodiment of the present invention.
Example 3: The following example provides process parameters for an intermediate filtration step that employees viral filtration with a Asahi Kasei Planova 20N filter according to one embodiment of the present invention.
Example 4: The following example provides process parameters for a polish step employing GP64 affinity chromatography with Repligen/Navigo GP64 affinity resin according to one embodiment of the present invention.
Example 5: The following example provides process parameters for a polish step employing anion exchange chromatography with Fractogel EMD TMAE (M) resin according to one embodiment of the present invention.
Example 6: The following example provides process parameters for a polish step employing viral filtration with a Asahi Kasei Planova 20N filter according to one embodiment of the present invention.
Example 7: The following example provides process parameters for a polish step employing lentil lectin chromatography with Capto Lentil Lectin resin according to one embodiment of the present invention.
Example 8: The following example provides process parameters for a polish step employing tangential flow filtration with a Repligen TangenX® SIUS® 50 kDa cassette membrane according to one embodiment of the present invention.
This application claims priority to U.S. Provisional Patent Application No. 63/374,278, filed Sep. 1, 2022, the contents of which are hereby incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63374278 | Sep 2022 | US |
