Cell culture process for RSV F protein production

Abstract

To provide a method for producing an RSV F protein trimer by a fed-batch cell culture method.SOLUTION: Provided is a method comprising a temperature transition. The temperature is shifted to a lower temperature between about 30.0°C and about 32.0°C, preferably about 31.0°C.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for producing RSV F protein trimers in a fed-batch cell culture process. [Background technology]

[0002] Respiratory syncytial virus, or RSV, is a respiratory virus that infects the lungs and respiratory tract. RSV is the leading cause of serious viral lower respiratory tract illness in children worldwide and an important cause of respiratory disease in the elderly. However, there is no approved vaccine for the prevention of RSV infection.

[0003] RSV is a member of the Paramyxoviridae family. Its genome consists of a single-stranded, negative-sense RNA molecule encoding 11 proteins, including nine structural proteins (three glycoproteins and six internal proteins) and two nonstructural proteins. The structural proteins include three transmembrane surface glycoproteins: the attachment protein G, the fusion protein F, and a small hydrophobic SH protein. RSV exists in two subtypes, A and B. These subtypes differ primarily in the G glycoprotein, while the sequence of the F glycoprotein is more conserved between the two subtypes.

[0004] The mature F glycoprotein has three general domains: the ectodomain (ED), the transmembrane domain (TM), and the cytoplasmic tail (CT), which contains a single palmitoylated cysteine ​​residue.

[0005] The human RSV F glycoprotein is initially translated from mRNA as a single 574-amino acid polypeptide precursor (referred to as "F0" or "F0 precursor") containing a signal peptide sequence (amino acids 1-25) at its N-terminus. Upon translation, the signal peptide is removed by signal peptidase in the endoplasmic reticulum. The remaining portion of the F0 precursor (i.e., residues 26-574) is further cleaved by cellular proteases (specifically, furin) at two polybasic sites (aa 109 / 110 and 136 / 137), releasing a 27-amino acid intervening sequence (amino acids 110-136) called pep27, generating two linked fragments called F1 (C-terminal portion, amino acids 137-574) and F2 (N-terminal portion, amino acids 26-109). F1 contains a hydrophobic fusion peptide at its N-terminus and two heptad repeat regions (HRA and HRB). HRA is near the fusion peptide, and HRB is near the TM domain. The F1 and F2 fragments are linked by two disulfide bonds. Either the uncleaved F0 protein without the signal peptide sequence or the F1-F2 heterodimer can form the RSV F promoter. Three such promoters assemble to form the final RSV F protein complex, which is a homotrimer of three promoters.

[0006] The F proteins of subtypes A and B share approximately 90% amino acid sequence identity. The representative sequence of the F precursor polypeptide for subtype A is shown in SEQ ID NO:1 (strain A2, GenBank GI:138251, Swiss Prot P03420), and for subtype B is shown in SEQ ID NO:2 (strain 18537, GenBank GI:138250, Swiss Prot P13843). Both SEQ ID NO:1 and SEQ ID NO:2 are 574 amino acid sequences. The signal peptide sequences of SEQ ID NO:1 and SEQ ID NO:2 are reported to be amino acids 1-25 (GenBank and UniProt). In both sequences, the TM domain is approximately amino acids 530-550, although other reports have also reported it to be 525-548. The cytoplasmic tail region begins at either amino acid 548 or 550 and ends at amino acid 574, with a palmitoylated cysteine ​​residue located at amino acid 550.

[0007] One of the primary antigens under consideration for RSV subunit vaccines is the F protein. The RSV F protein trimer mediates virion membrane fusion with host cell membranes and also promotes syncytium formation. This largest population of F molecules forms a lollipop-shaped structure in the virion prior to fusion with the host cell membrane, with the TM domain anchored to the viral envelope [Dormitzer, PR, Grandi, G., Rappuoli, R., Nature Reviews Microbiol, 10, 807, 2012]. This conformation is called the prefusion conformation. Prefusion RSV F is recognized by the monoclonal antibodies (mAbs) D25, AM22, and MPE8, regardless of its oligomeric state. The prefusion F trimer is specifically recognized by the mAb AM14 [Gilman MS, Moin SM, Mas V, et al., Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein, PLoS Pathogens, 11(7), 2015]. When DRSV enters cells, the F protein rearranges from the prefusion state (sometimes referred to herein as "pre-F") through an unfolded intermediate structure to the postfusion state ("post-F"). During this rearrangement, the C-terminal coiled coil of the prefusion molecule dissociates into its three constituent chains, which then wrap around the globular head and, together with three additional helices, form a postfusion six-helix bundle. The prefusion RSV F trimer undergoes structural changes when exposed to increasingly harsh chemical or physical conditions, such as elevated temperatures. First, the trimeric structure is disrupted (at least locally within the molecule), then it rearranges into the post-fusion form, and then the domains denature.

[0008] To prevent viral entry, it is presumed that F-specific neutralizing antibodies must bind to the prefusion conformation of F on the virion, or possibly to an unfolded intermediate, before the viral envelope fuses with the cell membrane. Therefore, the prefusion form of the F protein is considered to be the preferred conformation for the desired vaccine antigen [Ngwuta, JO, Chen, M., Modjarrad, K., Joyce, MG, Kanekiyo, M., Kumar, A., Yassine, HM, Moin, SM, Killelikelly, AM, Chuang, GY, Druz, A., Georgiev, IS, Rundlet, EJ, Sastry, M., Stewart-Jones, GB, Yang, Y., Zhang, B., Nason, MC, Capella, C., Peeples, M., Ledgerwood, JE, McLellan, JS, Kwong, PD, Graham, BS, Science Translat. Med., 14, 7, 309 (2015)].The F glycoprotein is readily converted to its post-fusion form following membrane extraction with detergents such as Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, SDS, CHAPS, and CHAPSO, expression as an ectodomain, physical or chemical stress, or storage [McLellan JS, Chen M, Leung S, et al., Structure of RSV fusion glycoprotein trimer bound to a pre-fusion-specific neutralizing antibody, Science 340, 1113-1117 (2013); Chaiwatpongsakorn, S., Epand, RF, Collins, PL, Epand RM, Peeples, ME, J Virol. 85(8):3968-77 (2011); Yunus, AS, Jackson TP, Crisafi, K., Burimski, I., Kilgore, NR, Zoumplis, D., Allaway, GP, Wild, CT, Salzwedel, K., Virology. 2010 Jan 20;396(2):226-37]. Therefore, the preparation of pre-fusion F as a vaccine antigen remains a challenge. Because neutralizing and protective antibodies function by blocking virus entry, it is hypothesized that F antigens that elicit only post-fusion-specific antibodies are unlikely to be as effective as F antigens that elicit pre-fusion-specific antibodies. Therefore, it would be more desirable to utilize an F protein vaccine containing an F protein immunogen in a pre-fusion form (or potentially an unfolded intermediate form). Mutants of the RSV F protein have been proposed to improve the stability of the pre-fusion form of the protein (see, for example, PCT Application No. WO2017 / 109629), and are promising vaccine candidates. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] PCT Application No. WO2017 / 109629 [Patent Document 2] WO2009 / 079796 [Patent Document 3] WO2010 / 149745 [Patent Document 4] WO2011 / 008974 [Patent Document 5] WO2014 / 160463 [Patent Document 6] WO2014 / 174018 [Patent Document 7] WO2014 / 202570 [Patent Document 8] WO2015 / 013551 [Patent Document 9] WO2015 / 177312 [Patent Document 10] WO2017 / 005848 [Patent Document 11] WO2018 / 109220 [Patent Document 12] WO2004 / 104186 [Patent Document 13] WO2006 / 026445 [Patent Document 14] WO2008 / 109410 [Patent Document 15] WO2008 / 063892 [Patent Document 16] EP2243827 [Patent Document 17] WO2002 / 066603 [Patent Document 18] WO2015 / 140708 [Patent Document 19] WO2006 / 050050 [Patent Document 20] EP117,060 [Patent Document 21] EP117,058 [Patent Document 22] U.S. Patent No. 4,399,216 [Patent Document 23] U.S. Patent No. 5,166,320 [Patent Document 24] WO90 / 14837 [Patent Document 25] U.S. Patent No. 5,057,540 [Patent Document 26] WO90 / 03184 [Patent Document 27] WO96 / 11711 [Patent Document 28] WO2004 / 004762 [Patent Document 29] WO2005 / 002620 [Non-patent literature]

[0010] [Non-Patent Document 1] Dormitzer, P.R., Grandi, G., Rappuoli, R., Nature Reviews Microbiol, 10, 807, 2012. [Non-patent document 2] Gilman MS, Moin SM, Mas V et al., Characterization of a prefusion-specific antibody that recognizes a quaternary,cleavage-dependent epitope on the RSV fusion glycoprotein, PLoS Pathogens, 11(7), 2015 [Non-patent document 3] Ngwuta,JO,Chen,M.,Modjarrad,K.,Joyce,MG,Kanekiyo,M.,Kumar,A.,Yassine,HM,Moin,SM,Killikelly,AM,Chuang,GY,Druz,A.,Georgiev,IS,Rundlet ,EJ,Sastry,M.,Stewart-Jones,GB,Yang.Y.,Zhang,B.,Nason,MC,Capella,C.,Peeples,M.,Ledgerwood,JE,Mclellan,JS,Kwong,PD,Graham,BS,Science Translat.Med., 14, 7, 309(2015)

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[0011] Summary of the Invention [Problem to be solved by the invention]

[0012] Therefore, a process is needed to produce such antigens in the desired trimeric conformation and with suitable titers. Such a process should also be robust enough to be used on a large scale. In addition, the amount of host cell proteins (HCPs) or other impurities should be minimized to facilitate downstream processing of the produced trimers. [Means for solving the problem]

[0013] The present invention provides a method for producing RSV F protein trimers in a fed-batch cell culture process, comprising: (i) Initiating cell culture by providing mammalian cells containing a gene encoding a RSV F protein in a cell culture medium; (ii) culturing the cells at a temperature between about 33.0°C and 35.0°C; (iii) providing glucose to the cell culture in a limited manner by feeding glucose to the cell culture in response to an increase in pH above a predetermined pH value; The present invention relates to a method comprising:

[0014] In some embodiments, the method includes a temperature transition to a lower temperature between about 30.0°C and about 32.0°C, preferably about 31.0°C. [Brief explanation of the drawings]

[0015] [Figure 1A] 1 is a graph showing the effect of growth temperature on the percentage of HMMS, LMMS, and subtype A RSV F protein trimers as measured by size exclusion chromatography. [Figure 1B] 1 is a graph showing the effect of growth temperature on the percentage of HMMS, LMMS, and subtype B RSV F protein trimers as measured by size exclusion chromatography. [Figure 2A] 1 is a graph showing the effect of growth temperature on subtype A RSV F protein titer as measured by RP-HPLC. [Figure 2B] 1 is a graph showing the effect of growth temperature on subtype B RSV F protein titer as measured by RP-HPLC. [Figure 3A] 1 is a graph showing the effect of growth temperature on the amount of host cell protein (HCP) in material recovered from the production of subtype A RSV F protein, as measured by enzyme-linked immunoassay. [Figure 3B] 1 is a graph showing the effect of growth temperature on the amount of host cell protein (HCP) in material recovered from the production of subtype B RSV F protein, as measured by enzyme-linked immunoassay. [Figure 4A] 1 is a graph showing the effect of growth temperature on the amount of trimer titer in material recovered from the production of subtype A RSV F protein. [Figure 4B]1 is a graph showing the effect of growth temperature on the amount of trimer titer in material recovered from the production of subtype B RSV F protein. [Figure 5A] Graph showing the effect of production temperature on the percentage of HMMS, LMMS, and subtype A RSV F protein trimers as measured by size exclusion chromatography. [Figure 5B] 1 is a graph showing the effect of production temperature on the percentage of HMMS, LMMS, and subtype B RSV F protein trimers as measured by size exclusion chromatography. [Figure 6A] 1 is a graph showing the effect of production temperature on the titer of subtype A RSV F protein as measured by RP-HPLC. [Figure 6B] 1 is a graph showing the effect of production temperature on the titer of subtype B RSV F protein as measured by RP-HPLC. [Figure 7A] [Figure 7B] 1 is a graph showing the effect of production temperature on the amount of host cell protein (HCP) in material recovered from the production of subtype B RSV F protein, as measured by enzyme-linked immunoassay. [Figure 8A] 1 is a graph showing the effect of production temperature on the amount of trimer titer in material recovered from the production of subtype A RSV F protein. [Figure 8B] 1 is a graph showing the effect of production temperature on the amount of trimer titer in material recovered from the production of subtype B RSV F protein. [Figure 9A] Graph showing the effect of temperature shift timing on the percentage of HMMS, LMMS, and subtype A RSV F protein trimers as measured by size exclusion chromatography. [Figure 9B] Graph showing the effect of the timing of the temperature shift on the percentage of HMMS, LMMS, and subtype B RSV F protein trimers as measured by size exclusion chromatography. [Figure 10A] 1 is a graph showing the effect of the timing of the temperature shift on the titer of subtype A RSV F protein as measured by RP-HPLC. [Figure 10B] 1 is a graph showing the effect of the timing of the temperature shift on the titer of subtype B RSV F protein as measured by RP-HPLC. [Figure 11A] 1 is a graph showing the effect of the timing of the temperature shift on the amount of host cell protein (HCP) in material recovered from the production of subtype A RSV F protein, as measured by enzyme-linked immunoassay. [Figure 11B] 1 is a graph showing the effect of the timing of the temperature shift on the amount of host cell protein (HCP) in material recovered from the production of subtype B RSV F protein, as measured by enzyme-linked immunoassay. [Figure 12A] 1 is a graph showing the effect of temperature shift timing on the amount of trimer titer in material recovered from the production of subtype A RSV F protein. [Figure 12B] 1 is a graph showing the effect of the timing of the temperature shift on the amount of trimer titer in material recovered from the production of subtype B RSV F protein. [Figure 13A] Graph showing the effect of the presence of a temperature shift on the percentage of HMMS, LMMS, and subtype A RSV F protein trimers as measured by size exclusion chromatography. [Figure 13B] Graph showing the effect of the presence of a temperature shift on the percentage of HMMS, LMMS, and subtype B RSV F protein trimers as measured by size exclusion chromatography. [Figure 14A] 1 is a graph showing the effect of the presence of a temperature shift on the titer of subtype A RSV F protein as measured by RP-HPLC. [Figure 14B]1 is a graph showing the effect of the presence of a temperature shift on the titer of subtype B RSV F protein as measured by RP-HPLC. [Figure 15A] 1 is a graph showing the effect of the presence of a temperature shift on the amount of host cell protein (HCP) in material recovered from the production of subtype A RSV F protein, as measured by enzyme-linked immunoassay. [Figure 15B] 1 is a graph showing the effect of the presence of a temperature shift on the amount of host cell protein (HCP) in material recovered from the production of subtype B RSV F protein, as measured by enzyme-linked immunoassay. [Figure 16] Figure 1 shows a Western blot after hydrophobic interaction chromatography (HIC) of material recovered from the production of subtype A RSV F protein from nine bioreactors using various culture conditions. [Figure 17] Figure 1 shows a Western blot after hydrophobic interaction chromatography (HIC) of material recovered from the production of subtype B RSV F protein from nine bioreactors using various culture conditions. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides a method for producing RSV F protein trimers in a fed-batch cell culture process, comprising: (i) Initiating cell culture by providing mammalian cells containing a gene encoding a RSV F protein in a cell culture medium; (ii) culturing the cells at a temperature between about 33.0°C and about 35.0°C; (iii) providing glucose to the cell culture in a limited manner by feeding glucose to the cell culture in response to an increase in pH above a predetermined pH value; The present invention relates to a method comprising:

[0017] The method of the present invention is particularly useful for producing RSV F protein trimers used as antigens in immunogenic compositions. The method of the present invention can be used to produce RSV F protein trimers on a large scale, for example, in a cell culture medium volume of at least 500 L, or even at least 3000 L. The method of the present invention provides high titers and a high percentage of trimer-form RSV F protein, while minimizing the amount of HCP or other impurities, thereby facilitating further downstream processing. In addition, detailed conditions for optimizing protein processing have been identified and can be used in the method of the present invention.

[0018] In some embodiments, the RSV F protein is a subtype A RSV F protein. In some embodiments, the RSV F protein is a subtype B RSV F protein. In some embodiments, the RSV F protein is a mutant of a wild-type RSV F protein. In some embodiments, the RSV F protein is a mutant of a subtype A wild-type RSV F protein. In some embodiments, the RSV F protein is a mutant of a subtype B wild-type RSV F protein. In some embodiments, the mutants exhibit introduced mutations in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type RSV F protein and are immunogenic against viruses containing the wild-type RSV F protein or wild-type F protein. The amino acid mutations in the mutants include amino acid substitutions, deletions, or additions relative to the wild-type RSV F protein.

[0019] In some embodiments, the RSV F protein produced by the methods of the invention is a RSV protein mutant as disclosed in WO2017 / 109629, which is incorporated herein by reference.

[0020] In some embodiments, the RSV F protein is a mutant of the wild-type RSV F protein in which the introduced amino acid mutation is a mutation in which a pair of amino acid residues in the wild-type RSV F protein becomes a pair of cysteines ("engineered disulfide mutations"). The introduction of a pair of cysteine ​​residues allows for the formation of disulfide bonds between cysteine ​​residues, which stabilize the conformation or oligomeric state of the protein, for example, the pre-fusion conformation. Examples of specific pairs of such mutations include 55C and 188C, 155C and 290C, 103C and 148C, and 142C and 371C, such as S55C and L188C, S155C and S290C, T103C and I148C, and L142C and N371C.

[0021] In yet another embodiment, the RSV F protein mutant comprises one or more amino acid mutations that are gap-filling mutations. Examples of amino acids that can be replaced for gap-filling purposes include small aliphatic amino acids (e.g., Gly, Ala, Val) or small polar amino acids (e.g., Ser, Thr), and amino acids that are buried in the pre-fusion conformation but exposed to the solvent in the post-fusion conformation. Examples of supplemental amino acids include large aliphatic amino acids (Ile, Leu, Met) or large aromatic amino acids (His, Phe, Tyr, Trp). In some specific embodiments, the RSV F protein mutant comprises: (1) substitution of S at position 55, 62, 155, 190, or 290 with I, Y, L, H, or M; (2) substitution of T with I, Y, L, H, or M at position 54, 58, 189, 219, or 397; (3) substitution of G at position 151 with A or H; (4) substitution of A at position 147 or 298 with I, L, H, or M; (5) substitution of V with I, Y, or H at positions 164, 187, 192, 207, 220, 296, 300, or 495; and (6) Substitution of R by W at position 106 and a void-filling mutation selected from the group consisting of:

[0022] In some specific embodiments, the RSV F protein mutant comprises at least one void-filling mutation selected from the group consisting of T54H, S190I, and V296I.

[0023] In yet another embodiment, the RSV F protein mutant comprises an electrostatic mutation that reduces ionic repulsion or increases ionic attraction between residues in the protein that are adjacent to each other in the folded structure. In some embodiments, the RSV F protein mutant comprises an electrostatic substitution that reduces repulsive ionic interactions or enhances attractive ionic interactions with the acidic residues Glu487 and Asp489 from another promoter of the RSV F trimer. In some detailed embodiments, the RSV F protein mutant comprises (1) substitution of E at position 82, 92, or 487 with D, F, Q, T, S, L, or H; (2) substitution of K at position 315, 394, or 399 with F, M, R, S, L, I, Q, or T; (3) substitution of D at position 392, 486, or 489 with H, S, N, T, or P; and (4) Substitution of R at position 106 or 339 with F, Q, N, or W The electrostatic mutations are selected from the group consisting of:

[0024] In yet another embodiment, the RSV F protein mutant comprises a combination of two or more different types of mutations selected from engineered disulfide mutations, void-filling mutations, and electrostatic mutations. In some specific embodiments, the RSV F protein mutant comprises: (1) A combination of T103C, I148C, S190I, and D486S, (2) A combination of T54H, S55C, L188C, and D486S; (3) A combination of T54H, T103C, I148C, S190I, V296I, and D486S; (4) Combination of T54H, S55C, L142C, L188C, V296I, and N371C; (5) A combination of S55C, L188C, and D486S; (6) A combination of T54H, S55C, L188C, and S190I; (7) A combination of S55C, L188C, S190I, and D486S; (8) A combination of T54H, S55C, L188C, S190I, and D486S; (9) A combination of S155C, S190I, S290C, and D486S; (10) A combination of T54H, S55C, L142C, L188C, V296I, N371C, D486S, E487Q, and D489S, and (11) Combination of T54H, S155C, S190I, S290C, and V296I The combination of mutations relative to the corresponding wild-type RSV F protein is selected from the group consisting of:

[0025] In some embodiments, the RSV F protein is of subtype A and includes mutations T103C, I148C, S190I, and D486S.

[0026] In some embodiments, the RSV F protein is of subtype B and includes mutations T103C, I148C, S190I, and D486S.

[0027] Given that the RSV F sequence is substantially conserved, those skilled in the art can easily compare amino acid positions between different natural RSV F sequences to determine the corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, in almost all confirmed natural RSV F0 precursor proteins, the furin cleavage site is at the same amino acid position. Thus, the conservation of natural RSV F protein sequences across strains and subtypes allows for the comparison of amino acids at specific positions in the RSV F protein using a reference RSV F sequence. For purposes of this disclosure (unless otherwise indicated by context), the RSV F protein amino acid positions are indicated based on the amino acid sequence of the full-length natural F precursor polypeptide of the RSV A2 strain, which corresponds to the GenInfo identifier GI138251 and the SwissProt identifier P03420.

[0028] In some embodiments, the RSV F protein produced by the methods of the invention is an RSV protein mutant as disclosed in WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, and WO2018 / 109220. The RSV F proteins disclosed in these references are incorporated herein by reference.

[0029] As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture at the beginning of the culturing process or at various times thereafter. In some embodiments, these additional components are provided collectively as a feed medium. Such provided components typically include nutritional components for the cells that have become depleted during the culturing process. A fed-batch culture is typically interrupted at some point, and the cells and / or components in the medium are harvested and optionally purified. In some embodiments, a fed-batch culture comprises a basal medium supplemented with a feed medium.

[0030] In some embodiments, the cells are cultured at a temperature of 33.0°C, 33.1°C, 33.2°C, 33.3°C, 33.4°C, 33.5°C, 33.6°C, 33.7°C, 33.8°C, 33.9°C, 34.0°C, 34.1°C, 34.2°C, 34.3°C, 34.4°C, 34.5°C, 34.6°C, 34.7°C, 34.8°C, 34.9°C, or 35.0°C. In a preferred embodiment, the cells are cultured at a temperature between 34.0°C and 35.0°C. In a preferred embodiment, the cells are cultured at a temperature of 34.5°C.

[0031] The method of the present invention includes providing cells with limited glucose, wherein the glucose is supplied to the cells in response to an increase in pH above a predetermined pH value. Such a method of supplying glucose in response to a change in pH, also known as HiPDOG, is disclosed, for example, in WO 2004 / 104186 and Gagnon et al. ((2011) (Biotechnology and bioengineering 108:1328-1337), both of which are incorporated herein by reference.

[0032] In some embodiments, a pH sensor is used to monitor the pH of the cell culture.

[0033] In some embodiments, the predetermined pH value of the methods of the invention corresponds to an increase of 0.01 to 0.10 above the pH set point of the culture, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.10. In some embodiments, the predetermined pH value corresponds to an increase of 0.05 above the pH set point of the culture.

[0034] In some embodiments, the pH set point for the cell culture is between 6.70 and 7.30. In some embodiments, the pH set point for the cell culture is between 6.90 and 7.20. In some embodiments, the pH set point for the cell culture is between 7.00 and 7.10. In one preferred embodiment, the pH set point for the cell culture is 7.05.

[0035] In one preferred embodiment, the pH set point for the cell culture is 7.05 and the predetermined pH value corresponds to an increase of 0.05 above said set point.

[0036] In some embodiments, during a cell culture phase in which glucose is provided in a limiting manner, the pH of the cell culture is between 6.70 and 7.30. In some embodiments, during a cell culture phase in which glucose is provided in a limiting manner, the pH of the cell culture is between 6.90 and 7.20. In some embodiments, during a cell culture phase in which glucose is provided in a limiting manner, the pH set point is 6.95. In some embodiments, during a cell culture phase in which glucose is provided in a limiting manner, the pH set point is 7.07. In some embodiments, during a cell culture phase in which glucose is provided in a limiting manner, the pH set point is 7.01. In some embodiments, during a cell culture phase in which glucose is provided in a limiting manner, the pH set point is 7.20.

[0037] In some embodiments, after a cell culture stage in which glucose is provided in a limiting form, the pH set point is 7.20. In some embodiments, after a cell culture stage in which glucose is provided in a limiting form, the pH set point is 7.20 and the pH operating range is 7.05 to 7.35. In some embodiments, after a cell culture stage in which glucose is provided in a limiting form, the pH set point is 6.90. In some embodiments, after a cell culture stage in which glucose is provided in a limiting form, the pH set point is 6.90 and the pH operating range is 6.75 to 7.05.

[0038] In some embodiments, feeding glucose to the cell culture in response to an increase in pH above a predetermined pH value comprises feeding glucose until the pH drops to reach a pH set point for the culture.

[0039] In some embodiments, glucose is provided to the cell culture in a limiting manner during the growth phase of the culture, hi some embodiments, glucose is provided to the cell culture in a limiting manner for 1 to 6 days, preferably 3, 4, or 5 days, more preferably 4 or 5 days.

[0040] In some embodiments, providing the cell culture with glucose in a limiting manner begins on day 0, day 1, or day 2.

[0041] In some embodiments, when glucose is provided in a limiting form, it is provided as an independent feedstock, i.e., without other components of the feed medium.

[0042] In some embodiments, when glucose is provided in a limiting form, it is provided as part of the feed medium.

[0043] In some embodiments of the methods disclosed herein, the temperature is shifted to a lower temperature between about 30.0°C and about 32.0°C, preferably about 31.0°C. In some embodiments, the temperature is shifted to the lower temperature between days 3 and 7 (i.e., between day 3 and day 7 of culture). In a preferred embodiment, the temperature is shifted to the lower temperature on day 5 or 6. In a preferred embodiment, the temperature is shifted to the lower temperature after the limiting glucose supply is discontinued.

[0044] In some embodiments, the methods of the invention result in improved titer compared to other methods, such as methods performed at temperatures higher or lower than those or temperature ranges specified herein, and / or methods without a temperature shift, and / or methods using media containing glucocorticoids, and / or methods that do not include providing glucose to the cell culture in a limited manner by feeding the cell culture glucose in response to an increase in pH above a predetermined pH value. Titer may be determined by any method known in the art. In one embodiment, titer is measured by reverse-phase high performance liquid chromatography (RP-HPLC).

[0045] In some embodiments, the methods of the invention result in an increased percentage of trimers and a decreased percentage of high molecular mass species (HMMS) and / or low molecular mass species (LMMS) compared to other methods, such as methods performed at temperatures higher or lower than the temperatures or temperature ranges specified herein, and / or methods without a temperature shift, and / or methods using a medium containing glucocorticoids, and / or methods that do not include providing glucose to the cell culture in a limited manner by feeding glucose to the cell culture in response to an increase in pH above a predetermined pH value. The percentages of trimers, HMMS, and LMMS may be determined by any method known in the art. In some embodiments, the percentages of trimers, HMMS, and LMMS are measured by size exclusion chromatography (SEC-HPLC).

[0046] In some embodiments, the methods of the present invention result in an increased trimer titer compared to other methods, such as methods performed at temperatures higher or lower than the temperatures or temperature ranges specified herein, and / or methods without a temperature shift, and / or methods using a medium containing glucocorticoids, and / or methods that do not include providing glucose to the cell culture in a limited manner by feeding the cell culture glucose in response to an increase in pH above a predetermined pH value. Trimer titer is calculated by multiplying the percentage of trimer, preferably obtained by SEC-HPLC, by the titer, preferably obtained by RP-HPLC. Trimer titer estimates how much of the protein is produced in trimeric form.

[0047] In some embodiments, the methods of the present invention result in a reduction in the amount of host cell protein compared to other methods, such as, for example, methods performed at temperatures higher or lower than the temperatures or temperature ranges specified herein, and / or methods without a temperature shift, and / or methods using a medium containing glucocorticoids, and / or methods that do not include providing glucose to the cell culture in a limited manner by feeding the cell culture glucose in response to an increase in pH above a predetermined pH value. HCPs may be measured by any method known in the art. In some embodiments, HCPs were measured by enzyme-linked immunoassay (ELISA).

[0048] In some embodiments, the methods of the present invention result in an improved amount of processed RSV F(A) or RSV F(B) in a form suitable for the formation of trimers that can be used as an antigen in an immunogenic composition, compared to other methods, such as methods performed at temperatures higher or lower than the temperatures or temperature ranges defined herein, and / or methods without a temperature transition, and / or methods using a medium containing glucocorticoids, and / or methods that do not include providing glucose to a cell culture in a limited manner by feeding glucose to the cell culture in response to an increase in pH above a predetermined pH value. The amount of processed RSV F(A) or RSV F(B) in a suitable form can be determined by any method known in the art. In one embodiment, such an amount is measured, for example, by Western blot as shown in Example 3.

[0049] In some embodiments, the methods of the invention result in improved titers, and / or an increased percentage of trimers and a decreased percentage of high molecular mass species (HMMS) and / or low molecular mass species (LMMS), and / or a decreased amount of host cell protein, compared to other methods, such as, for example, methods performed at temperatures higher or lower than the temperatures or temperature ranges specified herein, and / or methods without a temperature shift, and / or methods using a medium containing glucocorticoids, and / or methods that do not include providing glucose to the cell culture in a limited manner by feeding glucose to the cell culture in response to an increase in pH above a predetermined pH value.

[0050] As used herein, the terms "culture medium," "cell culture medium," and "culture medium" refer to a solution containing nutrients that nourish growing mammalian cells. Typically, such a solution provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. In one embodiment, the medium may contain Ala, Arg, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, and cystine and / or Cys.

[0051] Such solutions may also contain supplements that promote growth and / or survival above minimal rates, including, but not limited to, hormones and / or other growth factors, specific ions (sodium, chloride, calcium, magnesium, phosphate, etc.), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), inorganic compounds (e.g., iron) present at high final concentrations, amino acids, lipids, and / or glucose or other energy sources. In some embodiments, it is advantageous to formulate the medium at a pH and salt concentration that is optimal for cell survival and proliferation. For example, the medium may be formulated to a pH of approximately 7.1-7.3 and a final osmolality of approximately 1000 mOsm-1300 mOsm.

[0052] Examples of known basal and / or feed cell culture media that can be used in the methods of the present invention include those disclosed in WO2006 / 026445, WO2008 / 109410, WO2008 / 063892, EP2243827, WO2002 / 066603, WO2015 / 140708, and WO2006 / 050050.

[0053] In a preferred embodiment, the feed medium used in the method of the present invention comprises 4-10 mM Ala, 30-60 mM Arg, 50-90 mM Asn, 10-30 mM Asp, 2-40 mM Glu, 2-15 mM Gly, 8-20 mM His, 25-32 mM Ile, 35-60 mM Leu, 28-60 mM Lys, 9-25 mM Met, 10-30 mM Phe, 15-40 mM Pro, 44-80 mM Ser, 20-45 mM Thr, 2-10 mM Trp, and 20-50 mM Val.

[0054] In some embodiments, the medium is a chemically defined medium, in which the components of the medium are known and controlled, hi some embodiments, the medium is a complex medium, in which not all components of the medium are known and / or controlled.

[0055] Chemically defined growth media for mammalian cell culture have been extensively developed and published over the past few decades. All components of defined media are well characterized, and therefore, defined media do not contain complex additives such as serum or hydrolysates. Early media formulations were developed to allow for cell growth and viability maintenance with little or no concern for protein production. More recently, media formulations have been developed with the express purpose of supporting highly productive cell cultures producing recombinant proteins. Such media are preferred for use in the methods of the present invention. Such media generally contain high amounts of nutrients, particularly amino acids, to support cell growth and / or maintenance at high densities. If necessary, one skilled in the art may modify such media for use in the methods of the present invention.

[0056] The components of complex media are not all well characterized, and therefore complex media may contain additives such as simple and / or complex carbon sources, simple and / or complex nitrogen sources, serum, etc. In some embodiments, complex media suitable for the present invention contain additives such as hydrolysates in addition to the other components of defined media as described herein.

[0057] In some embodiments, defined media typically contain approximately 50 chemical entities at known concentrations in water. Some defined media also contain one or more well-characterized proteins, e.g., insulin, IGF-1, transferrin, BSA, while others do not require a protein component and are therefore referred to as protein-free defined media. Typical chemical components of media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and a miscellaneous category that does not easily fit into a classification.

[0058] Cell culture media may optionally be supplemented with supplementary components. As used herein, the term "supplementary components" refers to components that promote growth and / or survival above a minimal rate, including, but not limited to, hormones and / or other growth factors, specific ions (sodium, chloride, calcium, magnesium, phosphate, etc.), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources. In some embodiments, supplementary components may be added to the initial cell culture. In some embodiments, supplementary components may be added after the cell culture has been initiated.

[0059] Typically, trace elements refer to various inorganic salts present at submicromolar levels. For example, commonly included trace elements include zinc, selenium, copper, etc. In some embodiments, iron (ferrous or ferric salts) can be included in the initial cell culture medium as a trace element at micromolar concentrations. Manganese is also often included among trace elements as a divalent cation (MnCl2 or MnSO4) at nanomolar to micromolar concentrations. Many less common trace elements are commonly added at nanomolar concentrations.

[0060] In some embodiments, the cell culture medium used in the methods of the invention does not contain glucocorticoid compounds.

[0061] Glucocorticoid compounds are known to modulate various cellular functions, such as cell growth, metabolism, glycosylation, and secretion of many proteins, and are therefore often included in cell culture media, especially those used in large-scale manufacturing processes.

[0062] Examples of glucocorticoid compounds used as cell culture media components include, but are not limited to, hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and fludrocortisone acetate.

[0063] As shown below in Example 3, the presence of glucocorticoids, such as hydrocortisone, in cell culture medium negatively impacts the amount of properly formed RSV F protein. Without being bound by any theory, this effect may be due to the glucocorticoid compounds interfering with the processing of the RSV F protein, resulting in an increase in the amount of unprocessed RSV protein in the harvested material.

[0064] In some embodiments, the cell culture medium used in the methods of the present invention does not contain glucocorticoid compounds. In some embodiments, the basal medium used in the methods of the present invention does not contain glucocorticoid compounds. In some embodiments, the feed medium used in the methods of the present invention does not contain glucocorticoid compounds. In some embodiments, the basal medium and feed medium used in the methods of the present invention do not contain glucocorticoid compounds.

[0065] In some embodiments, the cell culture medium used in the methods of the present invention does not contain hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, or fludrocortisone acetate. In some embodiments, the basal medium used in the methods of the present invention does not contain hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, or fludrocortisone acetate. In some embodiments, the feed medium used in the methods of the present invention does not contain hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, or fludrocortisone acetate. In some embodiments, the basal medium and feed medium used in the methods of the invention do not contain any of hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and fludrocortisone acetate.

[0066] In some embodiments, the cell culture medium used in the methods of the present invention does not contain hydrocortisone, prednisolone, betamethasone, or dexamethasone. In some embodiments, the basal medium used in the methods of the present invention does not contain hydrocortisone, prednisolone, betamethasone, or dexamethasone. In some embodiments, the feed medium used in the methods of the present invention does not contain hydrocortisone, prednisolone, betamethasone, or dexamethasone. In some embodiments, the basal medium and feed medium used in the methods of the present invention do not contain hydrocortisone, prednisolone, betamethasone, or dexamethasone.

[0067] In some embodiments, the cell culture medium used in the methods of the present invention does not contain hydrocortisone. In some embodiments, the basal medium used in the methods of the present invention does not contain hydrocortisone. In some embodiments, the feed medium used in the methods of the present invention does not contain hydrocortisone. In some embodiments, the basal medium and feed medium used in the methods of the present invention do not contain hydrocortisone.

[0068] In some embodiments, the medium used in the methods of the present invention can support cell cultures of, for example, 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL, 5×10 8 In some embodiments, the cell culture is a fed-batch CHO cell culture. In some embodiments, the cells are at a density of 1 x 10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL, or 5 x 10 8 Grow to a viable cell density higher than 1000 cells / mL.

[0069] As used herein, the term "viable cell density" refers to the number of cells present in a given volume of culture medium. Viable cell density may be measured by any method known to those skilled in the art. It is preferable to measure viable cell density using an automated cell counter, such as the Bioprofile Flex®. As used herein, the term "maximum cell density" refers to the maximum cell density achieved during cell culture. As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. Those skilled in the art will understand that the present invention encompasses many methods for determining cell viability. For example, to determine cell viability, a dye (e.g., trypan blue) may be used that does not pass through the membranes of living cells but can pass through the disrupted membranes of dead or dying cells.

[0070] Cell culture method As used herein, the terms "culture" and "cell culture" refer to a population of cells suspended in a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of skill in the art, in some embodiments, these terms as used herein refer to the combination of a cell population and the medium in which the cell population is suspended.

[0071] As used herein, the term "fed-batch culture" or "fed-batch cell culture" refers to a method of culturing cells in which additional components are provided to the culture at the beginning of the culturing process or at various times thereafter. Such provided components typically include nutritional components for the cells that have become depleted during the culturing process. A fed-batch culture is typically interrupted at some point, and the cells and / or components in the medium are harvested and optionally purified. In some embodiments, a fed-batch culture comprises a basal medium supplemented with a feed medium.

[0072] Cells may be grown in any convenient volume selected by the practitioner. For example, cells may be grown in small-scale reactors ranging in volume from a few milliliters to several liters. Alternatively, cells may be grown in large-scale industrial bioreactors ranging in volume from at least 500 to 1000, 2500, 5000, 8000, 10,000, 12,000, 15,000, 20,000, 25,000 liters or larger, or any volume in between. In some embodiments, the cell culture volume is at least 500 L. In some embodiments, the cell culture volume is at least 3000 L.

[0073] In some embodiments, cells may be grown for a longer or shorter period of time during the initial growth stage (or growth stages), depending on the desires of the practitioner and the requirements of the cells themselves. In some embodiments, cells are grown for a period of time sufficient to achieve a predetermined cell density. In some embodiments, cells may grow for a period of time sufficient to achieve a predetermined cell density of about 1×10 6 cells / mL, approximately 5×10 6 cells / mL, approximately 1×10 7 cells / mL, approximately 5×10 7 cells / mL, approximately 1×10 8 cells / mL, or approximately 5 x 10 8The cells are grown for a sufficient time to achieve a predetermined cell density in cells / mL. In some embodiments, the cells are grown for a sufficient time to achieve a cell density that is a given percentage of the maximum cell density that the cells would ultimately reach if allowed to grow unimpeded. For example, the cells may be grown for a sufficient time to achieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent of the maximum cell density. In some embodiments, the cells are grown until the cell density does not increase by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% per culture day. In some embodiments, the cells are grown until the cell density does not increase by more than 5% per culture day.

[0074] In some embodiments, the cells are allowed to grow for a defined period of time. For example, depending on the starting concentration of the cell culture and the intrinsic growth rate of the cells, the cells may be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days, preferably 4-10 days. The practitioner of the present invention can select the duration of the initial growth phase depending on the protein production requirements and the needs of the cells themselves.

[0075] To enhance oxygenation and nutrient distribution to the cells, the cell culture may be agitated or shaken during the initial cultivation stage. According to the present invention, it will be understood by those skilled in the art that it may be beneficial to control or regulate certain internal conditions of the bioreactor during the initial growth stage, including but not limited to pH, temperature, oxygenation, etc.

[0076] It will be understood by those skilled in the art that, according to the present invention, the temperature at which cells are cultured is a temperature set point that is controlled to limit temperature fluctuations around the set point during cell culture.

[0077] The methods of the present invention can employ a temperature transition to a lower temperature. In such cases, those skilled in the art will understand that a lower temperature set point is established and that once the temperature reaches the lower set point, the temperature is controlled to limit temperature fluctuations around the lower set point. When transitioning the temperature of a culture, the temperature transition can be relatively gradual. For example, it may take several hours or days to complete the temperature change. Alternatively, the temperature transition can be relatively abrupt. For example, the temperature change may be completed in less than a few hours. Given appropriate production and control equipment, such as is standard in large-scale industrial production of polypeptides or proteins, the temperature change may even be completed in less than an hour.

[0078] In some embodiments, once the cell culture conditions have been transitioned as discussed above, the cell culture is maintained during the subsequent production phase under conditions conducive to cell culture survival and viability and suitable for expression of the desired polypeptide or protein at commercially relevant levels. In some embodiments, the cells may be maintained in the subsequent production phase until a desired cell density or product titer is reached. In some embodiments, the duration of the production phase is between 2 and 10 days, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably between 4 and 8 days, preferably 6 days.

[0079] In some embodiments, the duration of the growth phase is about 6 days and the duration of the production phase is about 6 days.

[0080] To enhance oxygenation and nutrient distribution to the cells, the cell culture may be agitated or shaken during the subsequent production stage. In accordance with the present invention, those skilled in the art will appreciate that it may be beneficial to control or adjust certain internal conditions of the bioreactor during the subsequent production stage, including, but not limited to, pH, temperature, oxygenation, etc.

[0081] cell Any mammalian cell that can be cultured can be utilized in accordance with the present invention. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include the BALB / c mouse myeloma line (NSO / 1, ECACC number: 85110503), human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands), SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651), human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980), mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1 587), human cervical carcinoma cells (HeLa, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (HepG2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad. Sci., 383:44-68, 1982), MRC5 cells, FS4 cells, and human hepatoma line (HepG2). In some preferred embodiments, the cells are CHO cells. In some preferred embodiments, the cells are GS-CHO cells.

[0082] Protein expression As noted above, in many instances, cells are selected or engineered to produce high levels of a desired product. Often, cells are engineered by the hand of man to produce high levels of a recombinant protein, for example, by introducing a gene encoding the protein of interest and / or introducing genetic control elements that regulate expression of that gene (whether endogenous or introduced).

[0083] Even within a population of a specific type of cell engineered to express a specific protein, there is variability within the cell population, so that individual specific cells grow better and produce more of the target protein. In certain embodiments, the practitioner empirically selects cell lines that grow robustly under the specific conditions selected for culturing the cells. In some embodiments, individual cells engineered to express a specific protein are selected for large-scale production based on cell growth, final cell density, percent cell viability, titer of expressed protein, or any combination thereof, or any other criteria that the practitioner considers important.

[0084] As used herein, the term "host cell" refers to a cell that has been engineered to produce a protein of interest as described herein. The protein may be expressed from a gene that is endogenous to the cell or from a heterologous gene that has been introduced into the cell. The protein may be naturally occurring or, alternatively, may contain a sequence that has been manipulated or selected by the hand of man.

[0085] Isolation of expressed proteins In general, it is usually desirable to isolate and / or purify proteins expressed according to the present invention. In certain embodiments, the expressed protein is secreted into the culture medium and therefore may be purified by removal of cells and other solids, for example, by centrifugation or filtration, as an initial step in the purification process.

[0086] The expressed protein can be isolated and purified by standard methods, including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation, and / or any other available technique for protein purification (see, e.g., Scopes, Protein Purification Principles and Practice, 2nd ed., Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (co-eds.), Protein Expression: A Practical Approach, Oxford University Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN (co-eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, each of which is incorporated herein by reference). In particular, for immunoaffinity chromatography, proteins can be isolated by binding to an affinity column containing antibodies activated against the protein and attached to a solid support. Alternatively, affinity tags, such as influenza envelope sequences, polyhistidine, or glutathione S-transferase, can be attached to the protein using standard recombinant techniques, allowing for easy purification by passage through an appropriate affinity column. To reduce or eliminate protein degradation during the purification process, protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, or aprotinin, can be added at any or all stages. Protease inhibitors are particularly useful when cells must be lysed to isolate and purify the expressed protein.

[0087] It will be appreciated by those skilled in the art that the exact purification techniques will vary depending on the characteristics of the protein being purified, the characteristics of the cells from which the protein is expressed, and / or the composition of the medium in which the cells are grown.

[0088] Introduction of genes into host cells for protein expression Generally, the nucleic acid molecule introduced into the cell encodes a protein that it is desired to express in accordance with the present disclosure.

[0089] Suitable methods for introducing nucleic acids sufficient to achieve expression of a protein of interest into mammalian host cells are known in the art. See, for example, Gething et al., Nature, 293:620-625, 1981; Mantei et al., Nature, 281:40-46, 1979; Levinson et al., EP 117,060 and EP 117,058, each of which is incorporated herein by reference. For mammalian cells, common methods for introducing genetic material into mammalian cells include the calcium phosphate precipitation method of Graham and van der Erb (Virology, 52:456-457, 1978) or the lipofectamine™ (Gibco BRL) method of Hawley-Nelson (Focus 15:73, 1993). General aspects of mammalian cell host system transformation are described in U.S. Patent No. 4,399,216, issued August 16, 1983, by Axel. For various techniques for introducing genetic material into mammalian cells, see Keown et al., Methods in Enzymology, 1989; Keown et al., Methods in Enzymology, 185:527-537, 1990; and Mansour et al., Nature, 336:348-352, 1988.

[0090] In some embodiments, the introduced nucleic acid is in the form of a naked nucleic acid molecule. For example, the nucleic acid molecule introduced into a cell may consist only of the nucleic acid encoding the protein and the necessary genetic control elements. Alternatively, the nucleic acid encoding the protein (including the necessary regulatory elements) may be contained within a plasmid vector. Non-limiting representative examples of vectors suitable for expressing proteins in mammalian cells include pCDNA1, pCD (see Okayama et al., Mol. Cell Biol. 5:1136-1142, 1985), pMClneo Poly-A (see Thomas et al., Cell 51:503-512, 1987), baculovirus vectors such as pAC373 and pAC610, CDM8 (see Seed, B., Nature 329:840, 1987), and pMT2PC (see Kaufman et al., EMBO J. 6:187-195, 1987), each of which is incorporated by reference in its entirety. In some embodiments, the nucleic acid molecule introduced into the cell is contained within a viral vector. For example, a nucleic acid encoding a protein may be inserted into a viral genome (or a partial viral genome). The regulatory elements that direct protein expression may be included with the nucleic acid inserted into the viral genome (i.e., linked to the gene inserted into the viral genome) or may be provided by the viral genome itself.

[0091] Naked DNA can be introduced into cells by forming a precipitate containing DNA and calcium phosphate. Alternatively, naked DNA can be introduced into cells by forming a mixture of DNA and DEAE-dextran and incubating the mixture with cells, or by incubating cells and DNA together in an appropriate buffer and exposing the cells to a high-voltage electric pulse (for example, by electroporation). Another method for introducing naked DNA into cells is by mixing DNA with a liposome suspension containing cationic lipids. The DNA / liposome complex is then incubated with cells. Naked DNA can also be directly injected into cells, for example, by microinjection.

[0092] Alternatively, naked DNA can be introduced into cells by complexing it with a cation such as polylysine that has been conjugated to a ligand for a cell surface receptor (see, e.g., Wu, G. and Wu, C.H., J. Biol. Chem. 263:14621, 1988; Wilson et al., J. Biol. Chem. 267:963-967, 1992; and U.S. Pat. No. 5,166,320, each of which is incorporated by reference in its entirety). Binding of the DNA-ligand complex to the receptor promotes uptake of the DNA by receptor-mediated endocytosis.

[0093] The use of viral vectors containing specific nucleic acid sequences, e.g., cDNAs encoding proteins, is a common method for introducing nucleic acid sequences into cells. Infection of cells with viral vectors has the advantage that the majority of cells receive the nucleic acid, thereby eliminating the need for selection of cells that have received the nucleic acid. In addition, molecules encoded within the viral vector, e.g., by cDNAs housed within the viral vector, are generally expressed efficiently in cells that have been loaded with viral vector nucleic acid.

[0094] Defective retroviruses have been well characterized for use in gene transfer for gene therapy purposes (for a review, see Miller, AD, Blood 76:271, 1990). Recombinant retroviruses can be constructed in which a nucleic acid encoding a protein of interest has been inserted into the retroviral genome. In addition, portions of the retroviral genome can be removed to render retroviral replication defective. Such replication-defective retroviruses are then packaged into virions that can be used to infect target cells using helper viruses by standard techniques.

[0095] The adenoviral genome can be engineered to encode and express a protein of interest but be inactivated with respect to its replicative potential during the normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616, 1988; Rosenfeld et al., Science 252:431-434, 1991; and Rosenfeld et al., Cell 68:143-155, 1992. Suitable adenoviral vectors derived from adenovirus Ad strain type 5 dl324 or other adenovirus strains (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including respiratory epithelia (Rosenfeld et al., 1992, supra), endothelial cells (Lemarchand et al., Proc. Natl. Acad. Sci. USA 89:6482-6486, 1992), hepatocytes (Herz and Gerard, Proc. Natl. Acad. Sci. USA 90:2812-2816, 1993), and muscle cells (Quantin et al., Proc. Natl. Acad. Sci. USA 89:2581-2584, 1992). In addition, the introduced adenoviral DNA (and the foreign DNA contained therein) does not integrate into the host cell genome but remains episomal, thereby avoiding potential problems that can arise as a result of insertional mutagenesis in situations where the introduced DNA is integrated into the host genome (e.g., retroviral DNA). Moreover, the adenoviral genome has a large capacity (up to 8 kilobases) to carry foreign DNA compared to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267, 1986). Most replication-defective adenoviral vectors currently in use have all or part of the viral E1 and E3 genes deleted, but retain approximately 80% of the adenoviral genetic material.

[0096] Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpesvirus, as a helper virus for efficient replication and a productive life cycle (for a review, see Muzyczka et al., Curr. Topics in Micro. and Immunol., 158:97-129, 1992). This virus is also one of the few viruses that can integrate its DNA into non-dividing cells and exhibits high-frequency, stable integration (see, e.g., Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356, 1992; Samulski et al., J. Virol. 63:3822-3828, 1989; and McLaughlin et al., J. Virol. 62:1963-1973, 1989). Vectors containing as little as 300 base pairs of AAV can be packaged and integrated. Space for exogenous DNA is limited to approximately 4.5 kb. AAV vectors such as those described by Tratschin et al. (Mol. Cell. Biol. 5:3251-3260, 1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see, e.g., Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470, 1984; Tratschin et al., Mol. Cell. Biol. 4:2072-2081, 1985; Wondisford et al., Mol. Endocrinol. 2:32-39, 1988; Tratschin et al., J. Virol. 51:611-619, 1984; and Flotte et al., J. Biol. Chem. 268:3781-3790, 1993).

[0097] When the method used to introduce the nucleic acid molecule into the cell population results in a majority of cells being modified and the protein being efficiently expressed by the cells, the modified cell population can be used without further isolation or subcloning of individual cells within the population. That is, because the cell population can efficiently produce the protein, no further isolation of the cells is necessary and the population can be immediately used to seed cell cultures for protein production. Alternatively, it may be desirable to isolate and grow a homogenous population from a few cells or single cells that efficiently produce the protein.

[0098] In some cases, the gene encoding the protein of interest may be linked to one or more regulatory gene control elements. In certain embodiments, the gene control element directs constitutive expression of the protein. In certain embodiments, a gene control element can be used that leads to inducible expression of the gene encoding the protein of interest. The use of an inducible gene control element (e.g., an inducible promoter) allows modulation of the production of the protein in cells. Non-limiting examples of potentially useful inducible gene control elements for use in eukaryotic cells include elements regulated by hormones (see, e.g., Mader, S. and White, JH, Proc. Natl. Acad. Sci. USA 90:5603-5607, 1993), elements regulated by synthetic ligands (see, e.g., Spencer, DM et al., Science 262:1019-1024, 1993), and elements regulated by ionizing radiation (see, e.g., Manome, Y. et al., Biochemistry 32:10607-10613, 1993; Datta, R. et al., Proc. Natl. Acad. Sci. USA 89:10149-10153, 1992). Other cell-specific or other regulatory systems known in the art can also be used in accordance with the present invention.

[0099] Those skilled in the art can select and, if necessary, appropriately improve the method for introducing a gene that causes expression of a protein of interest into a cell, following the teachings of the present invention.

[0100] immunogenic composition The subtype A and B RSV F proteins produced by the methods disclosed herein can be included in immunogenic compositions used as vaccines.

[0101] In addition to the immunogenic component, the vaccine may further comprise an immunomodulatory agent, such as an adjuvant. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and / or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions), including squalene-water emulsions such as MF59 (see, e.g., WO 90 / 14837); saponin preparations, such as QS21 and immunostimulating complexes (ISCOMS) (see, e.g., U.S. Pat. Nos. 5,057,540, WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2005 / 002620); bacterial or microbial derivatives, for example, monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), oligonucleotides containing CpG motifs, ADP-ribosylating bacterial toxins or mutants thereof, e.g., E. coli heat-labile enterotoxin LT, cholera toxin CT, and the like. It is also possible to use a vector-encoded adjuvant by using a heterologous nucleic acid encoding a fusion of the oligomerization domain of C4 binding protein (C4bp) with an antigen of interest (e.g., Solabomi et al., 2008, Infect Immun 76:3817-23). ​​In certain embodiments, the compositions herein include aluminum as an adjuvant, e.g., in the form of aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate, or combinations thereof, at a concentration of 0.05-5 mg, e.g., 0.075-1.0 mg, of aluminum per dose. [Example]

[0102] GS-CHO clones recombinantly expressing RSV F proteins of subtype A (hereinafter RSV F(A)) or subtype B (hereinafter RSV F(B)) were maintained at 36.5°C and 5% CO2 in a 120 or 140 rpm shaking incubator. Cultures were maintained at 0.35 x 10 6 cells / mL or 0.20 x 10 6 N-1 seed cultures for all experiments were performed in 2 L Applikon® bioreactors with a working volume of 1 L and were cultured at 0.70 x 10 cells / mL in nutrient-rich medium. 6 cells / mL and passaged for 4 days.

[0103] Production experiments were performed in a 2 L Applikon® bioreactor equipped with a BioNet® controller using a glucose-limited fed-batch process, hereafter referred to as the HiPDOG process (Gagnon et al. (2011), Biotechnology and bioengineering 108:1328-1337). Detailed methods and parameters are provided in the experimental section that follows.

[0104] On the day of harvest, the cell culture broth is clarified by centrifugation and depth filtration. Downstream processing includes ultrafiltration and diafiltration 1 (UF / DF1) to concentrate and buffer the exchange material before the capture chromatography step, which is an anion exchange chromatography (AEX) column operated in bind-and-elute mode. Polishing columns include ceramic hydroxyapatite chromatography (CHA) in flow-through mode and a hydrophobic interaction chromatography (HIC) column in bind-and-elute mode. Downstream processing concludes with a virus-retaining filtration step, ultrafiltration and diafiltration 2 (UF / DF2), and a final filtration step.

[0105] In the following experiments, titer, trimers, high molecular mass species (HMMS), low molecular mass species (LMMS), and host cell proteins (HCPs) are reported.

[0106] The titer may be determined by any method known in the art. In the following experiments, the titer was measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Reverse-phase chromatography separates molecules based on polarity. Relatively non-polar molecules, including subtype A or B RSV F proteins, bind to the column, while polar molecules pass through the column unbound. Bound molecules are eluted from the column by applying a mobile phase gradient that progresses from polar to less polar conditions. Molecules are eluted in order of most to least polar. Detection is performed using ultraviolet (UV) absorption at 220 nm. Titer determination is achieved by comparing sample peak areas with those of calibration standards.

[0107] [Table 1]

[0108] [Table 2]

[0109] Trimers, HMMS, and LMMS were measured by size exclusion chromatography (SEC-HPLC). SEC-HPLC is an analytical method known to those skilled in the art and is used to determine the relative content of high molecular mass species (HMMS), trimers, and low molecular mass species (LMMS) in subtype A or B RSV F protein samples obtained by the method of the present invention. SEC-HPLC separates molecules based on their hydrodynamic volume. When the analyte is applied to the top of the column bed, molecules smaller than the pores of the packing material can diffuse into and out of the pores, while larger molecules do not enter the pores. As a result, larger molecules pass through the column more quickly and smaller molecules pass more slowly. Upon elution, molecular species are detected by UV absorption at 280 nm. Low molecular mass species (LMMS) is the term used for all molecular species whose apparent molecular mass is smaller than that of the trimer when measured by SEC-HPLC. These molecular species are eluted after the trimer peak. High molecular mass species (HMMS) is the term used for all peaks with apparent molecular masses greater than the trimer as measured by SEC-HPLC. These species elute before the trimer peak and may contain aggregates.

[0110] HCP was measured by enzyme-linked immunoassay (ELISA), a quantitative assay that measures residual Chinese hamster ovary (CHO) host cell protein (HCP) using a sandwich-type ELISA analysis. The major steps in the HCP assay are outlined below.

[0111] A set of standard samples is prepared from the highly enriched CHO HCP material. The standard samples have CHO HCP concentrations ranging from 2 ng / mL to 256 ng / mL. Test samples are diluted to four concentrations of subtype A or B RSV protein F. Finally, a control sample is tested in each assay plate. The assay plate is coated with polyclonal antibodies (anti-CHO HCPP pAbs) activated against the highly enriched CHO HCP preparation. After coating, the plate is blocked to minimize nonspecific binding of analytes and reagents. After blocking, the standards, test samples, and control samples are added to the assay plate and incubated to allow HCPs in these samples to be captured by the anti-CHO HCP antibody. The plate is then washed to remove unbound protein, leaving behind HCP-antibody complexes. To quantify the amount of bound HCP in each well, a preparation of anti-CHO HCP antibody conjugated to biotin is added to the assay plate and allowed to bind to the captured HCP. The plate is washed to remove unbound biotin-labeled antibody, and a streptavidin-horseradish peroxidase (HRP) conjugate is added, which binds to the biotin-anti-CHO HCP conjugate. The plate is washed to remove unbound streptavidin-HRP, and a solution of 3,3',5,5'-tetramethylbenzidine (TMB) is added to the assay plate. TMB is a substrate that produces a blue color in the presence of HRP. The assay plate is incubated with the TMB reagent for a period of time to generate an appropriate signal in each well, and the peroxidase reaction is quenched by adding sulfuric acid. Finally, the absorbance in each well is measured and recorded at 450 nm using an appropriate plate reader. The resulting signal is proportional to the amount of HCP captured in the assay plate. The signal in the standard sample wells is plotted against the standard HCP concentration. The plot is fitted with a four-parameter logistic (4PL) fit to generate an HCP standard curve.The signals in the test and external control samples are then used to determine the HCP content in these samples by interpolating the absorbance signal into the assumed linear portion of the standard 4PL function.

[0112] In terms of overall productivity and downstream filterability, the process is most optimal when titer and trimers are increased to the limit and HMMS, LMMS, and HCP are minimized. The RP-HPLC titer provides an estimate of the total amount of RSV protein present in the sample, including aggregates and non-trimeric RSV proteins. Trimers measured by SEC provide an estimate of the approximate percentage of trimeric RSV molecules present as a percentage of the total protein present (including any process impurities). Manipulation of process parameters such as growth temperature can increase trimers while negatively impacting titer (or vice versa). To show the overall impact on both titer and trimer, we report the "trimer titer," calculated by multiplying trimer by titer. Trimer titer estimates how much protein is produced in trimeric form.

[0113] Example 1 Effect of temperature on RSV F protein production in CHO cells This set of experiments was designed to evaluate the effect of pre- and post-transfer temperatures and transfer timing on titer and trimer formation during production of subtype A and B RSV F protein in CHO cells.

[0114] Production experiments were conducted in a 2 L Applikon® bioreactor equipped with a BioNet® controller using the conditions detailed in Table 1. All conditions included a stage in which the amount of glucose provided to the cells was limiting (HipDOG days 0-5 for RSV F(A) and days 0-4 for RSV F(B)) and were imposed in a fed-batch process using cell culture medium without hydrocortisone.

[0115] [Table 3]

[0116] 1.1 Effect of growth temperature In this experiment, cells were grown at temperatures of 33° C., 34.5° C., or 36° C. to assess the effect of growth temperature on titer, percent trimer, HMMS, LMMS, trimer titer, and amount of host cell protein (HCP). The results are shown in Table 2 and Figures 1A, 1B, 2A, 2B, 3A, 3B, 4A, and 4B.

[0117] [Table 4]

[0118] For both antigens, growth temperature was negatively correlated with the percentage of trimers and positively correlated with the percentage of HMMS and LMMS (see Figures 1A and 1B). The highest titers were consistently obtained at a temperature of 34.5°C (see Figures 2A and 2B). Growth temperatures between 34°C and 35°C, preferably 34.5°C, are suitable for limiting trimer titers and minimizing impurities. For both antigens, HCP levels were positively correlated with temperature (see Figures 3A and 3B). For subtype B, the highest trimer titers were obtained at a temperature of 34.5°C, and for subtype A, trimer titers at 33°C and 34.5°C were higher than at 36°C (see Figures 4A and 4B).

[0119] 1.2 Effect of production temperature In this experiment, the growth temperature was 34.5° C. and the production temperature was varied (28.5° C., 31° C., or 34° C.) to evaluate the effect of production temperature on titer, percent trimer, HMMS, LMMS, trimer titer, and amount of HCP. The results are shown in Table 3 and Figures 5A, 5B, 6A, 6B, 7A, 7B, 8A, and 8B.

[0120] [Table 5]

[0121] The production temperature (after the temperature shift) showed a negative linear correlation with trimer and a positive linear correlation with LMMS and HMMS for both antigens (see Figures 5A and 5B). The lowest HCP levels and highest trimer titer levels were obtained at a production temperature of 31°C (see Figures 7A and 7B, 8A and 8B).

[0122] 1.3 Effect of the timing of temperature transitions In this experiment, the timing of the temperature shift was varied to assess its effect on titer, percent trimer, HMMS, LMMS, trimer titer, and amount of HCP. The results are shown in Table 4 and Figures 9A, 9B, 10A, 10B, 11A, 11B, 12A, and 12B.

[0123] [Table 6]

[0124] Temperature shifting at 144 hours after the start of culture improved trimer quantity, titer, and HCP levels compared to shifts at different culture durations. This was true for both antigens and all attributes, except for trimers for RSV F(B), which were highest at the temperature shift at 185.5 hours after the start of culture. For RSV F(A), the highest trimer titer was obtained at the 144-hour temperature shift. Trimer titer levels for RSV F(B) were similar at 144 and 114 hours, both lower than at 185.5 hours.

[0125] Example 2 Effect of temperature shift on RSV F protein production in CHO cells This experiment was designed to evaluate the effect of a temperature shift on process performance, titer, and trimer formation during the production of subtype A and B RSV F protein in CHO cells.

[0126] Production experiments were conducted in a 2 L Applikon® bioreactor equipped with a BioNet® controller using the conditions detailed in Table 5. All conditions included a stage in which the amount of glucose provided to the cells was limiting (HipDOG days 0-5 for RSV F(A) and days 0-4 for RSV F(B)) and were imposed in a fed-batch process using cell culture medium without hydrocortisone.

[0127] [Table 7]

[0128] The results are shown in Tables 6 and 7 and Figures 13A, 13B, 14A, 14B, 15A, and 15C.

[0129] [Table 8]

[0130] [Table 9]

[0131] The presence of a temperature shift increased trimer levels, decreased HCP, and increased titers for both antigens (see Figures 13A, 13B, 14A, 14B, 15A, and 15B).

[0132] Example 3 Effect of glucocorticoid compounds on RSV F protein production in CHO cells This experiment was designed to understand the effect of glucocorticoid compounds, such as hydrocortisone, on the titer and product quality of subtype A and B RSV F proteins produced in CHO cells.

[0133] Production experiments were conducted in a 2 L Applikon® bioreactor equipped with a BioNet® controller using the steps detailed in Table 8 in cell culture medium with and without hydrocortisone.

[0134] All bioreactors were operated at 34.5°C, with a temperature shift to 31°C performed in bioreactor (B08) on day 6. Glucose was provided in a limiting manner (Hipdog) from days 0 to 4 for RSV F(B) and from days 0 to 5 for RSV F(A).

[0135] [Table 10]

[0136] Hydrocortisone negatively affected furin processing of the RSV F protein, as suggested by the Western blot results shown in Figures 16 and 17. Western blot analysis allows for the monitoring of processed RSV F(A) or RSV F(B) monomers and related species. The pre-fusion F trimer is specifically recognized by the mAb AM14 (Gilman MS et al., PLoS Pathogens, 11(7), 2015). The term "AM14" refers to the antibody described in WO2008 / 147196A2, which has a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 4. Results are collected to monitor process performance and the levels of processed RSV F(A) or RSV F(B) monomers, partially processed or unprocessed F+p27 or other size variants. The lanes containing hydrocortisone (B-07, B-04, B-03, and A-01, and A-04, A-05, B-03, and B-07 in Figure 16) show a smear just above the RSV band (approximately 60 kDa) identified by AM-14 antibody binding. The presence of a smear indicates a partially processed RSV variant.

[0137] Therefore, to improve the amount of processed material suitable for use in vaccine compositions, particularly in trimeric form, it is advantageous to exclude hydrocortisone or other related glucocorticoid compounds from the cell culture medium used in the methods of the invention.

[0138] Example 4 Effect of HiPDOG on RSV F production in CHO cells Stabilization of the pre-fusion conformation is important for RSV proteins because the post-fusion conformation is energetically preferred, less immunogenic, and the transition from pre-fusion to post-fusion is irreversible. Subtype A and B RSV F proteins can be engineered to stabilize the protein in the pre-fusion conformation, and disulfide bonds contribute to this stability. Therefore, the integrity of disulfide bonds can affect the stability of the desired conformation. The intrasubunit disulfide bonds in RSV were found to be slightly unpaired during the initial fed-batch process. The corresponding two unpaired cysteines were found to be modified with cysteinyl moieties. This modification was measured and reported as "cysteinylation," measured by amino acid analysis coupled to a QDa mass detector.

[0139] This experiment was designed to understand the effect of HiPDOG on the level of cysteinylation in subtypes A and B of RSV F produced in CHO cells. Bioreactor parameters are shown in Table 9.

[0140] [Table 11]

[0141] Cysteinylation levels were reduced for both RSV F(A) and RSV F(B) antigens when the HiPDOG control was used (Table 10).

[0142] [Table 12]

[0143] In addition, when HiPDOG was used, titers were improved for both RSV F(A) and RSV F(B) antigens (Table 11). The titers reported were after the first purification step (ultrafiltration).

[0144] [Table 13]

[0145] Example 5 Large scale manufacturing process To test whether the method of the present invention is suitable for large-scale use, CHO cells expressing subtype A or subtype B RSV protein F were cultured in a 12-day fed-batch process using HiPDOG, a growth temperature of 34.5°C, and a production temperature of 31°C, with a temperature shift on day 6. As shown in Table 12 below, the method of the present invention produced favorable trimer titer values, even when performed in 2500 or 12500 L bioreactors.

[0146] [Table 14] [Sequence List Free Text]

[0147] SEQ ID NO: 1. Amino acid sequence of full-length F0 of natural RSV A2 (GenBank GI:138251, Swiss Prot P03420) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIE LSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLS LIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN Amino acid sequence of the full-length F0 of SEQ ID NO: 2. Natural RSV B (strain 18537, GenBank GI: 138250, Swiss Prot P13843) MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIE LSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVN AGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV VQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSN GCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK SEQ ID NO: 3: Amino acid sequence of the heavy chain variable domain of antibody AM14: EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS SEQ ID NO: 4: Amino acid sequence of the light chain variable domain of antibody AM14: DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTV

Claims

1. A method for producing an RSV F protein trimer in a fed-batch cell culture process, comprising: (i) initiating a cell culture by providing Chinese hamster ovary (CHO) cells containing a gene encoding an RSV F protein in a cell culture medium; (ii) culturing the cells at a temperature between about 33.0°C and 35.0°C; (iii) providing glucose to the cell culture in a limited manner by feeding glucose to the cell culture in response to an increase in pH above a predetermined pH value; A method comprising:

2. 10. The method of claim 1, wherein the temperature is about 34.5°C.

3. The method according to claim 1 or 2, wherein after step (ii) of claim 1, the temperature is shifted to a lower temperature, preferably between about 30.0°C and about 32.0°C.

4. 4. The method of claim 3, wherein the lower temperature is about 31.0°C.

5. 5. The method of claim 3 or 4, wherein the temperature is shifted to a lower temperature between the third and seventh days.

6. 6. The method of any one of claims 3 to 5, wherein the temperature is shifted to a lower temperature on the fifth or sixth day.

7. 7. The method of claim 1, wherein the pH of the cell culture is monitored using a pH sensor.

8. 8. The method of any one of claims 1 to 7, wherein the predetermined pH value corresponds to an increase of 0.01 to 0.10 above the pH set point of the culture.

9. 8. The method of any one of claims 1 to 7, wherein the predetermined pH value corresponds to an increase of 0.05 above the pH set point of the culture.

10. 10. The method of claim 8 or 9, wherein the pH set point of the cell culture is between 6.70 and 7.

30.

11. 10. The method of claim 8 or 9, wherein the pH set point of the cell culture is between 6.90 and 7.

20.

12. 10. The method of claim 8 or 9, wherein the pH set point of the cell culture is between 7.00 and 7.

10.

13. 10. The method of claim 8 or 9, wherein the pH set point of the cell culture is 7.

05.

14. During the cell culture phase in which glucose is provided in a limited manner, the pH set point is 6.95, 7.01, 7.05, 7.07, or 7.20; - After the cell culture stage where glucose is provided in a limited form, the pH set point is 6.90 or 7.20; 10. The method according to any one of claims 1 to 9.

15. 15. The method of any one of claims 1 to 14, wherein feeding glucose to the cell culture in response to an increase in pH above a predetermined pH value comprises feeding glucose until the pH drops to reach a pH set point for the culture.

16. 16. The method of any one of claims 1 to 15, wherein glucose is provided in a limiting manner on days 1 to 6.

17. 17. The method of any one of claims 1 to 16, wherein the cell culture medium does not contain glucocorticoid compounds.

18. 18. The method of any one of claims 1 to 17, wherein the cell culture medium does not contain any of hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and fludrocortisone acetate.

19. 18. The method of any one of claims 1 to 17, wherein the cell culture medium does not contain hydrocortisone.

20. 20. The method of any one of claims 1 to 19, wherein the cell culture medium is serum-free.

21. 21. The method of any one of claims 1 to 20, wherein the cell culture medium is protein-free.

22. 22. The method of any one of claims 1 to 21, wherein the cell culture medium is a defined medium.

23. 23. The method of any one of claims 1 to 22, wherein the cell culture is further provided with a feed medium.

24. 24. The method of claim 23, wherein the feed medium is provided continuously or at multiple intervals.

25. 25. The method of claim 23 or 24, wherein the feed medium does not contain glucocorticoid compounds.

26. 26. The method of any of claims 23 to 25, wherein the feed medium does not contain any of hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and fludrocortisone acetate.

27. 27. The method of any of claims 23 to 26, wherein the feed medium does not contain hydrocortisone.

28. 28. The method of any of claims 23 to 27, wherein the feed medium is serum-free.

29. 29. The method of any of claims 23 to 28, wherein the feed medium is protein-free.

30. 30. The method of any of claims 23 to 29, wherein the feed medium is a defined medium.

31. The maximum viable cell density during cell culture was 1 x 10 ６ 31. The method of any one of claims 1 to 30, wherein the concentration exceeds cells / mL.

32. The maximum viable cell density is 5 x 10 ６ cells / mL, 1 x 10 ７ cells / mL, 5 x 10 ７ cells / mL, 1 x 10 ８ cells / mL, or 5 x 10 ８ 31. The method of any one of claims 1 to 30, wherein the concentration exceeds cells / mL.

33. 33. The method of any one of claims 1 to 32, wherein the volume of the cell culture medium is at least 500 L.

34. 34. The method of claim 33, wherein the volume of the cell culture medium is at least 3000 L.

35. A method described in any one of claims 1 to 34, wherein the Chinese hamster ovary cells (CHO) are GS-CHO cells.

36. 36. The method of any one of claims 1 to 35, wherein the RSV F protein is of subtype A.

37. 36. The method of any one of claims 1 to 35, wherein the RSV F protein is of subtype B.

38. 38. The method of any one of claims 1 to 37, wherein the RSV F protein comprises a mutation that stabilizes the trimer in the prefusion conformation.

39. The RSV F protein is (1) a combination of T103C, I148C, S190I, and D486S; (2) a combination of T54H, S55C, L188C, and D486S; (3) a combination of T54H, T103C, I148C, S190I, V296I, and D486S; (4) a combination of T54H, S55C, L142C, L188C, V296I, and N371C; (5) a combination of S55C, L188C, and D486S; (6) a combination of T54H, S55C, L188C, and S190I; (7) a combination of S55C, L188C, S190I, and D486S; (8) a combination of T54H, S55C, L188C, S190I, and D486S; (9) A combination of S155C, S190I, S290C, and D486S; (10) A combination of T54H, S55C, L142C, L188C, V296I, N371C, D486S, E487Q, and D489S; and (11) A combination of T54H, S155C, S190I, S290C, and V296I 39. The method of claim 38, comprising a combination of mutations selected from the group consisting of:

40. 40. The method of any one of claims 1 to 39, further comprising obtaining the RSV F protein trimer produced by the cell.

41. 41. The method of claim 40, further comprising purifying the RSV F protein trimer.

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