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  • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
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  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
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  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
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  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18611—Respirovirus, e.g. Bovine, human parainfluenza 1,3
  • C12N2760/18651—Methods of production or purification of viral material
  • C12N2760/18652—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

• the invention relates to methods for propagating viruses.
• the invention provides optimized conditions for propagating viruses. Optimization of the following parameters are provided: lipid concentrates as supplements to the medium, temperature shift from pre-infection to post-infection, multiplicity of infection and serum supplementation of pre-infection medium.
• the invention provides for the first time a method for propagating a virus by culturing cells that are infected with the virus in a medium comprising chemically defined lipid concentrate (CDLC).
• CDLC chemically defined lipid concentrate
• the CDLC is added to medium that is substantially free of serum for culture of virus-infected cells.
• the invention provides for propagating a viral cell culture by a direct bead-to-bead transfer method.
• Human parainfluenza virus types 1-3 hPIVl-3 and respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are non-segmented negative-strand RNA viruses of the paramyxovirus family.
• the Paramyxoviridae form a family within the order of Mononegavirales, consisting of the sub-families Paramyxovirinae and Pneumovirinae.
• Parainfluenza virus is a member of the Respirovirus genus (PIVl, PIV2 and PIV3) of the paramyxoviridae family.
• HRSV Human respiratory syncytial virus
• Parainfluenza viral infection results in serious respiratory tract disease in infants and children. (T ao et al, 1999, Vaccine 17: 1100-08). Infectious parainfluenza viral infections account for approximately 20% of all hospitalizations of pediatric patients suffering from respiratory tract infections worldwide. Id.
• PIV is made up of two structural modules: (1) an internal ribonucleoprotein core, or nucleocapsid, containing the viral genome, and (2) an outer, roughly spherical lipoprotein envelope.
• Its genome is a single strand of negative sense RNA, approximately 15,456 nucleotides in length, encoding at least eight polypeptides.
• proteins include, but are not limited to, the nucleocapsid structural protein (NP, NC, or N depending on the genera), the phosphoprotein (P), the matrix protein (M), the fusion glycoprotein (F), the hemagglutinin-neuraminidase glycoprotein (HN), the large polymerase protein (L), and the C and D proteins of unknown function. Id.
• the parainfluenza nucleocapsid protein (NP, NC, or N) consists of two domains within each protein unit including an amino-terminal domain, comprising about two-thirds of the molecule, which interacts directly with the RNA, and a carboxyl-terminal domain, which lies on the surface of the assembled nucleocapsid.
• a hinge is thought to exist at the junction of these two domains thereby imparting some flexibility to this protein ⁇ see Fields et al. (ed.), 1991, Fundamental Virology, Second Edition, Raven Press, New York, incorporated by reference herein in its entirety).
• the matrix protein (M) is apparently involved with viral assembly and interacts with both the viral membrane as well as the nucleocapsid proteins.
• the fusion glycoprotein (F) interacts with the viral membrane and is first produced as an inactive precursor, then cleaved post-translationally to produce two disulfide linked polypeptides.
• the active F protein is also involved in penetration of the parainfluenza virion into host cells by facilitating fusion of the viral envelope with the host cell plasma membrane.
• the glycoprotein, hemagglutinin-neuraminidase (FIN) protrudes from the envelope allowing the virus to contain both hemagglutinin and neuraminidase activities.
• FIN is strongly hydrophobic at its amino terminal which functions to anchor the HN protein into the lipid bilayer.
• the large polymerase protein (L) plays an important role in both transcription and replication. Id. 2.2 RSV INFECTIONS
• Respiratory syncytial virus is the leading cause of serious lower respiratory tract disease in infants and children (Feigen et al, eds., 1987, In: Textbook of Pediatric Infectious Diseases, WB Saunders, Philadelphia at pages 1653-1675; New Vaccine Development, Establishing Priorities, Vol. 1, 1985, National Academy Press, Washington DC at pages 397-409; and Ruuskanen et al, 1993, Curr Probl Pediatr 23:50-79). The yearly epidemic nature of RSV infection is evident worldwide, but the incidence and severity of RSV disease in a given season vary by region (Hall, 1993, Contemp Pediatr 10:92-110).
• RSV infects adults as well as infants and children. In healthy adults, RSV causes predominantly upper respiratory tract disease. It has recently become evident that some adults, especially the elderly, have symptomatic RSV infections more frequently than had been previously reported (Evans, A.S., eds., 1989, Viral Infections of Humans. Epidemiology and Control, 3rd ed., Plenum Medical Book, New York at pages 525-544). Several epidemics also have been reported among nursing home patients and institutionalized young adults (Falsey, A.R., 1991, Infect Control Hosp Epidemiol 12:602-608; and Garvie et al, 1980, 5rMedJ281 :1253-1254). Finally, RSV may cause serious disease in immunosuppressed persons, particularly bone marrow transplant patients (Hertz et al, 1989, Medicine 68:269-281).
• a humanized antibody directed to an epitope in the A antigenic site of the F protein of RSV is approved for intramuscular administration to pediatric patients for of serious lower respiratory tract disease caused by RSV at recommended monthly doses of 15 mg/kg of body weight throughout the RSV season (November through April in the northern hemisphere).
• SYNAGIS® is a composite of human (95%) and murine (5%) antibody sequences. See, Johnson et al., 1997, J. Infect. Diseases 176:1215-1224 and U.S. Patent No. 5,824,307, the entire contents of which are incorporated herein by reference.
• the human heavy chain sequence was derived from the constant domains of human IgGi and the variable framework regions of the VH genes or Cor (Press et al., 1970, Biochem. J. 117:641-660) and Cess (Takashi et al., 1984, Proc. Natl. Acad. Sci. USA 81 :194-198).
• the human light chain sequence was derived from the constant domain of CK and the variable framework regions of the VL gene K104 with J ⁇ -4 (Bentley et al., 1980, Nature 288:5194- 5198).
• the murine sequences derived from a murine monoclonal antibody, Mab 1129 (Beeler et al., 1989, J. Virology 63:2941-2950), in a process which involved the grafting of the murine complementarity determining regions into the human antibody frameworks.
• hRSV human respiratory syncytial virus
• the new virus was named human metapneumo virus (hMPV) based on sequence homology and gene constellation.
• hMPV human metapneumo virus
• hMPV can be isolated year-round, albeit at a lower rate (Robinson, 2005, J. Med. Virol. 76:98-105; Williams, 2004, New Engl. J. Med. 350:443-450). Risk factors for hMPV infection are also similar to those found for RSV. Highest incidence of infection with human metapneumovirus has been found in young children, in the elderly and immunocompromised humans.
• hMPV shares a similar genetic structure to RSV but lacks the non-structural genes found in RSV (van den Hoogen, 2002, Virology. 295:119-132). Both viruses code for similar surface proteins that are defined as the surface glycoprotein (G) protein and the fusion (F) protein. Based upon differences between the amino acid sequences of the G and F proteins, both RSV and hMPV have been subdivided into A and B groups. However, in hMPV there is a further bifurcation of A and B subgroups into Al, A2, Bl, and B2 groupings (Boivin, 2004, Emerg. Infect. Dis.10:1154-1157, 25).
• the sequences of the G proteins display a wide variance between subgroups; with hMPV the G protein has only 30% identity between A and B subgroups.
• the F protein is more conserved; across the known hMPV isolates the F protein amino acid sequence is 95% conserved (Biacchesi, 2003, Virology 315:1-9; Boivin, 2004, Emerg. Infect. Dis.lO:l 154-1157; van den Hoogen, 2004, Emerg. Infect. Dis. 10:658-666) .
• the F proteins of hMPV and RSV share only a 33% amino acid sequence identity and antisera generated against either RSV or hMPV do not neutralize across the pneumoviridae group (Wy de, 2003, Antiviral Research. 60:51-59). With RSV a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Similarly, because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies.
• Human metapneumovirus is related to avian metapneumovirus.
• the F protein of hMPV is highly homologous to the F protein of avian pneumonovirus ("APV").
• ADV avian pneumonovirus
• Alignment of the human metapneumoviral F protein with the F protein of an avian pneumovirus isolated from Mallard Duck shows 85.6% identity in the ectodomain.
• Alignment of the human metapneumoviral F protein with the F protein of an avian pneumovirus isolated from Turkey (subgroup B) shows 75% identity in the ectodomain. See, e.g., co-owned and co-pending Provisional Application No.
• hMPV likewise appears to be a significant factor in human, particularly, juvenile respiratory disease.
• the present invention relates to methods for propagating viruses.
• the conditions for the propagation of virus are optimized in order to produce a robust and high-yielding cell culture which would be beneficial, e.g., for manufacture of virus vaccine candidates of the invention.
• the present invention relates to a method for propagating viruses.
• the invention provides a method for propagating negative strand RNA viruses.
• the invention provides a method for propagating non-segmented, negative strand RNA viruses, such as paramyxoviruses.
• the invention specifically provides a method for propagating parainfluenza virus (PIV) and respiratory syncytial virus (RSV) and metapneumo virus (MPV).
• PIV parainfluenza virus
• RSV respiratory syncytial virus
• MPV metapneumo virus
• the invention provides a method for propagating PIV with human and bovine sequences.
• chimeric human/bovine PIV expressing RSV nucleotide sequences is propagated using the methods of the invention.
• the invention provides a method for propagating a virus by culturing cells that are infected with the virus in a medium comprising chemically defined lipid concentrate (CDLC).
• CDLC-supplemented medium is substantially free of serum.
• the medium is a serum- free medium, such as OptiPROTM SFM or VP-SFMTM or SFM4MegaVirTM (SFM4MV) or Ex-Cell VeraTM or Williams' E medium.
• the cells used are Vera cells.
• the CDLC-supplemented medium contains a maximum serum concentration of 0.5% v/v.
• the CDLC comprises one or more of Pluronic F-68, Ethyl Alcohol, Cholesterol, Tween 80, DL-alpha-Tocopherol Acetate, Stearic Acid, Myristic Acid, Oleic Acid, Linoleic Acid, Palmitic Acid, Palmitoleic Acid, Arachidonic Acid, and Linolenic Acid.
• the invention provides a method for propagating a virus by culturing cells infected by a virus in a serum-free medium.
• Contemplated serum-free media include, for example, VP-SFMTM or SM4MegaVirTM, OptiPROTM SFM or Ex-Cell VeraTM or Williams' E medium.
• the cells used are Vera cells.
• the cells used are serum- free adapted Vera cells.
• the present invention provides further optimized conditions for propagating viruses.
• the cells are cultured at a first temperature before infection with the virus and at a second temperature after infection with the virus, wherein the second temperature is lower than the first temperature.
• the cells are cultured at about 37 0 C before infection with the virus, i.e., pre-infection, and from about 29 0 C to about 37 0 C after infection with the virus, i.e., post-infection.
• the cells are cultured at about 33 0 C after infection with the virus.
• the cells are cultured at about 3O 0 C after infection with the virus.
• the seeding density of the virus ranges from Ie5 to 2e5 cells/ cm 2 . In another embodiment, the seeding density is 2.1 e4 to 2.9 e4 cells/cm 2 . In another embodiment, the seeding density is 2.4 e4 cells/cm 2 . In yet another embodiment, the seeding density of the virus is 2e5 cells/cm 2 .
• the cells are cultured with a virus at a multiplicity of infection (MOI) ranging from about 0.0001 to about 0.1. In yet another embodiment, the cells are cultured at a MOI ranging from about 0.001 to about 0.01.
• MOI multiplicity of infection
• the cells are cultured in the presence of serum before infection with the virus.
• the pre-infection medium comprises fetal bovine serum.
• the point of infection is 3 or 4 days post-seeding (dps). In one embodiment, the post-infection time ranges from 4 to 11 days. In another embodiment, the post-infection time is about 5 days or about 6 days or about 8 days. In yet another embodiment, the point of infection is when the cells reach > Ie6 cells/cm 2 .
• the viral cell cultures of the invention are grown in suspension culture in a bioreactor either in a batch process or in a fed-batch process. In another embodiment, the viral cell cultures of the invention are grown on microcarrier beads in said bioreactors. In a specific embodiment, the viral cell cultures of the invention are grown in suspension culture in a single use bioreactor (SUB). In another embodiment, the viral cell cultures of the invention are grown on microcarrier beads in a SUB.
• SUV single use bioreactor
• the viral titer as a result of the methods and compositions of the invention is at least 5 log 10 TCID 50 /ml, at least 6 log 10 TCID 50 /ml, at least 7 log 10 TCIDso/ml, at least 8 log 10 TCID 50 /ml, at least 9 log 10 TCID 50 /ml, at least 10 log 10 TCID 50 /ml.
• the methods of the invention can also be used when rescuing virus from recombinant viral genomes.
• MEDI-559 is a live, attenuated RSV vaccine, termed rA2cp248/404/1030 ⁇ SH. (Karron et al., JID vol 191 p. 1093 (2005))
• Vero African green monkey kidney cell line v/v volume per volume ratio
• Figure 1 Virus production profiles at different MOIs. Duplicate T-75 flasks of serum- free Vero cells were infected three days post-seeding with MEDI-534 at MOI of 0.1, 0.01, 0.001, 0.0001 or 0.00001. Cultures were incubated at 37 0 C pre- and post-infection.
• Figure 2a Effects of time of infection and post-infection temperature on infectious virus titers. Serum-free Vero cultures were infected with MEDI-534 using MOI 0.001 at three days post-seeding (0.6 x 10 7 cells/flask). Duplicate T-75 flasks were incubated at either 33 0 C or 37 0 C post-infection.
• Figure 2b Effects of time of infection and post-infection temperature on infectious virus titers. Serum-free Vero cultures were infected with MEDI-534 using MOI 0.001 at five days post-seeding (0.6 x 10 7 cells/flask). Duplicate T-75 flasks were incubated at either 33 0 C or 37 0 C post-infection. [0037] Figure 3. Virus production profiles of RB cultures titrated with FBS pre- infection. Vera cells were seeded in one of the following media in triplicate RBs: OptiPROTM SFM, OptiPROTM SFM + 0.5 % (v/v) FBS, and OptiPROTM + 2% (v/v) FBS.
• OptiPROTM SFM (1.9 x 10 7 cells/flask), OptiPROTM SFM + 0.5% (v/v) FBS (9.3 x 10 7 cells/flask) and OptiPROTM + 2% (v/v) FBS (10.4 x 10 7 cells/flask).
• the remaining 2 x 3 RBs were infected with MEDI-534 at MOI 0.001.
• FIG. 4a Comparison of cell yield in different pre -infection media and supplements. Vera cells were seeded in one of the following five media at four RBs per condition: (1) OptiPROTM SFM, (2) OptiPROTM SFM + 1% (v/v) CDLC, (3) OptiPROTM + 0.5% (v/v) FBS, (4) VP-SFM and (5) VP-SFM + 1% (v/v) CDLC. Three days post-seeding, two RBs per condition were used for cell counts. The remaining duplicate sets of RBs were infected with MEDI-534 at MOI 0.001.
• FIG. 4b Comparison of virus production in different pre-infection media and supplements. Vera cells were seeded in one of the following five media at four RBs per condition: (1) OptiPROTM SFM, (2) OptiPROTM SFM + 1% (v/v) CDLC, (3) OptiPROTM + 0.5% (v/v) FBS, (4) VP-SFM and (5) VP-SFM + 1% (v/v) CDLC. Three days post-seeding, two RBs per condition were used for cell counts. The remaining duplicate sets of RBs were infected with MEDI-534 at MOI 0.001.
• Figure 5a Comparison of cell growth in RBs using different pre-infection media. Vera cells were seeded in either OptiPROTM + 0.5% (v/v) FBS or VP-SFM + 1% (v/v) CDLC. To generate the growth curves, duplicate RBs in each condition were counted daily.
• Figure 5b Comparison of virus production in RBs using different pre-infection media. Vera cells were seeded in either OptiPROTM + 0.5% (v/v) FBS or VP-SFM + 1% (v/v) CDLC. To generate the virus production profiles, duplicate RB cultures in the two different pre-infection media were infected with MEDI-534 three days post-seeding. The infected cultures were sampled daily from 2-7 days post-infection.
• FIG. 6a Comparison of cell yield in RB cultures titrated with CDLC pre- infection. Vera cells were seeded in serum-free growth medium (VP-SFM) supplemented with CDLC at three different concentrations in duplicates. Four days post-seeding, the cultures were trypsinized for cell counting and passaged in the VP-SFM containing CDLC added at three different concentrations (in replicates of four). On the third day post-seeding, duplicate RBs per condition were counted, and the remaining duplicate sets of RBs were infected with MEDI-534.
• VP-SFM serum-free growth medium
• FIG. 6b Comparison of virus production in RB cultures titrated with CDLC pre-infection. Vera cells were seeded in serum- free growth medium (VP-SFM) supplemented with CDLC at three different concentrations in duplicates. Four days post- seeding, the cultures were trypsinized for cell counting and passaged in the VP-SFM containing CDLC added at three different concentrations (in replicates of four). On the third day post-seeding, duplicate RBs per condition were counted, and the remaining duplicate sets of RBs were infected with MEDI-534.
• VP-SFM serum- free growth medium
• FIG. 7 Comparison of MEDI-534 production in different post-infection media. RB cultures were inoculated in VP-SFM + 1% (v/v) CDLC. Three days post- seeding, duplicate RBs were infected at MOI 0.001 with MEDI-534 in one of the following SFM: VP-SFM + 1% CDLC, VP-SFM and WME.
• Figure 8a Comparison of cell growth in microcarrier cultures using different pre-infection media. Vera cells were seeded in duplicate spinner flasks containing 2 g/L CytodexTM 1 in either OptiPROTM + 0.5% (v/v) FBS or VP-SFM + 1% (v/v) CDLC. To generate the growth curves, samples were taken daily from the uninfected flasks for nuclei counts.
• FIG. 8b Comparison of virus production in microcarrier cultures using different pre-infection media. Vera cells were seeded in duplicate spinner flasks containing 2 g/L CytodexTM 1 in either OptiPROTM + 0.5% (v/v) FBS or VP-SFM + 1% (v/v) CDLC. To generate the virus production profiles, duplicate flasks in each pre-infection media were infected five days post-seeding with MEDI-534.
• FIG. 9a Comparison of pre-infection Vera cell growth in bioreactors controlled at different pHs. Vera cells were seeded in bioreactors containing 2 g/L CytodexTM 1 in VP-SFM + 1% (v/v)CDLC maintained at pH 7.0, ph 7.2 or pH 7.4. Samples were taken daily from the bioreactors for nuclei counts pre-infection.
• FIG. 9b Comparison of virus production in bioreactors controlled at different pHs. Vera cells were seeded in bioreactors containing 2 g/L CytodexTM 1 in VP- SFM + 1% (v/v)CDLC maintained at pH 7.0, ph 7.2 or pH 7.4. Four days post-seeding, the bioreactor cultures were infected with MEDI-534.
• Figure 10 Effects of agitation rate on Vera cell growth in 3L bioreactors using DO at 50% air saturation, pH at 7.1, temperature at 37°C. High agitation rate is at 125 rpm and low agitation rate is at 65 rpm. An agitation rate of 125 rpm improved cell growth and cells grew to a higher density.
• FIG. 11 Effect of CytodexTM 1 density on Vero cell growth in 3L bioreactors. DO was at 50% air saturation, pH was set at 7.1 and temperature was at 37°C. Agitation rate of 125 rpm was used. Two bioreactors contained CytodexTM 1 at 2 g/L and two at 4 g/L. Cultures with 4 g/L of CytodexTM 1 had higher cell density than the culture with 2 g/L of CytodexTM 1.
• FIG. 12 Effects of CytodexTM 1 density on RSV ⁇ M2-2 Production.
• Cells were infected with RSV ⁇ M2-2 virus at MOI of 0.01 and cultured in a shake incubator at 33°C and 5% CO2 with shaking at 100 rpm.
• Cultures with 4 g/L of CytodexTM 1 produced higher virus titer than the culture with 2 g/L of the microcarrier beads.
• Figure 13 Vero cell growth curve in bioreactors which were cultured with 4 g/L of CytodexTM 1, 125 rpm agitation rate, 50% of DO, pH at 7.1 and 37°C for 3 days.
• FIG. 14 Production of MEDI-559 in a bioreactor.
• Duplicate bioreactor cultures were inoculated with Vero cells at 2e5 cells/mL in the serum- free growth medium and cultured with 4 g/L of CytodexTM 1, 125 rpm agitation rate, 50% of DO, pH at 7.1 and 37°C for 3 days.
• Cell growth was monitored by taking samples daily from each bioreactor and counting nuclei number in the culture samples. On day 3 of culturing, agitation was stopped to allow the microcarrier beads to settle to the bottom of the bioreactor.
• Spent growth medium was then removed from the bioreactor while leaving the cells on the microcarrier beads behind and was replaced with equal volume of fresh post-infection medium (SFM4MegaVirTM + 4 mM L-GIn). Agitation was resumed at 125 rpm. Temperature of the culture was reduced to 30 0 C and pH was set at 7.0. Cells were then infected with MEDI-559 at the MOI of 0.01 and continued to be cultured at 30 0 C for 10 days. Samples were taken from the cultures daily from day 7 to day 10. Virus titers were determined by TCID50 assay. Using a straight batch process, a productivity of approximately 8 logio TCID 50 /ml was achieved.
• Figure 15 Infectious MEDI-560 titer in the spent culture medium from the following infection conditions: (0) SFM4MegaVir medium and 30 0 C, ( ⁇ ) in William's medium E at 30 0 C, ( ⁇ ) in SFM4MegaVir medium at 32°C, (D) in William's medium E at 32°C, and ( ⁇ ) in Ex-Cell Vero medium at 32°C.
• FIG. 16 Cell growth profiles of the three bioreactor cultures. ( ⁇ ) 3L260307- R9; (A) 3L120407-R10; and ( ⁇ ) SUB120407. Cell density was measured in cells per milliliter (cells/mL). [0056] Figure 17. Cell growth profiles of the three bioreactor cultures. ( ⁇ ) 3L260307- R9; (A) 3L120407-R10; and ( ⁇ ) SUB120407. Cell density was measured in cells per microcarrier.
• FIG. 18A-B Glucose and lactate profiles of the three bioreactor cultures pre- infection. ( ⁇ ) 3L260307-R9; (A) 3L120407-R10; and ( ⁇ ) SUB120407.
• FIG 19A-B Glutamine and ammonium ion profiles of the three bioreactor cultures pre-infection. ( ⁇ ) 3L260307-R9; (A) 3L120407-R10; and ( ⁇ ) SUB120407.
• FIG. 20 A-B Glucose and lactate profiles of the three bioreactor cultures during the infection phase. ( ⁇ ) 3L260307-R9; (A) 3L120407-R10; and ( ⁇ ) SUB120407.
• FIG. 21 A-B Glutamine and ammonium ion profiles of the three bioreactor cultures during virus infection phase. ( ⁇ ) 3L260307-R9; (A) 3L120407-R10; and ( ⁇ ) SUB120407.
• Figure 22 Cell growth profiles in bioreactors expanded using four different intermittent agitation regimes for bead-to-bead transfer over time after being split at a 1 :5 ratio.
• FIG. 23 Cell growth profile in 3L bioreactor seeded with cells from roller bottle (freshly seeded), bioreactor culture after IX expansion at 1 :5 split ratio (IX 1 :5 transfer) and cultures derived from two consecutive expansion at 1 :5 split ratio in bioreactors (2X 1 :5 transfer).
• Figure 25 Comparative MEDI-560 productions in bioreactor cultures: seeded with cells from a roller bottle (freshly seeded); after IX expansion at 1 :5 split ratio (IX 1 :5 transfer); and cultures derived from two consecutive expansion at 1 :5 split ratio in bioreactors (2X 1 :5 transfer).
• the present invention relates to a method for propagating viruses.
• conditions for the propagation of virus are optimized in order to produce a robust, scalable and high-yielding cell culture which would be beneficial, e.g., for manufacture of virus vaccine candidates of the invention.
• a chemically defined medium is utilized to avoid or reduce substances of animal origin and increase virus production.
• Critical parameters can be identified, and the production process can be first optimized in small-scale experiments to determine the scalability, robustness, and reproducibility and subsequently adapted to large scale production of virus.
• the virus that is propagated using the methods of the invention is a negative strand RNA viruses.
• the virus that is propagated is a non-segmented, negative strand RNA viruses, such as a paramyxovirus.
• the invention specifically provides a method for propagating parainfluenza virus (PIV) and respiratory syncytial virus (RSV) and metapneumo virus (MPV). Even more specifically, the invention provides a method for propagating PIV with human and bovine sequences.
• chimeric human/bovine PIV expresses RSV nucleotide sequences.
• the virus that is propagated is MEDI-534. In another specific embodiment, the virus that is propagated is MEDI-559.
• the virus that is propagated is one which has a deletion of an open reading frame, such as, for example M2-2 or NS-I .
• the virus that is propagated is one which is rcp45 hPIV3 or MEDI-560.
• the virus that is propagated using the methods of the invention is an enveloped viruses.
• the virus that is propagated is a virus that infects and replicates in attached cells. (See also Section 5.5).
• the pre-infection medium may contain serum such as FBS. Subsequently, the cells are infected with the virus and cultured in medium.
• the post-infection medium may be substantially free of serum. The virus is subsequently harvested.
• the invention provides for the first time a method for propagating a virus by culturing cells in chemically defined lipid concentrate (CDLC)-supplemented medium.
• CDLC-supplemented medium is substantially free of serum.
• the medium is serum-free.
• a chemically defined medium is utilized to avoid variability and increase virus production.
• the CDLC-supplemented medium contains a maximum serum concentration of 0.5% v/v.
• the CDLC comprises one or more of Pluronic F-68, Ethyl Alcohol, Cholesterol, Tween 80, DL-alpha-Tocopherol Acetate, Stearic Acid, Myristic Acid, Oleic Acid, Linoleic Acid, Palmitic Acid, Palmitoleic Acid, Arachidonic Acid, and Linolenic Acid.
• the CDLC comprises 100,000 mg/L of Pluronic F-68, 100,00 mg/L of Ethyl Alcohol, 220 mg/L of Cholesterol, 2,200 mg/L of Tween 80, 70 mg/L of DL-alpha-Tocopherol Acetate, 10 mg/L of Stearic Acid, 10 mg/L of Myristic Acid, 10 mg/L of Oleic Acid, 10 mg/L of Linoleic Acid, 10 mg/L of Palmitic Acid, 10 mg/L of Palmitoleic Acid, 2 mg/L of Arachidonic Acid, and 10 mg/L of Linolenic Acid. (See also Section 5.1.1).
• the CDLC-supplemented medium is substantially free of serum.
• the medium is a serum-free medium, such as OptiPROTM SFM or VP-SFMTM or SFM4MegaVirTM (SFM4MV) or Ex-Cell VeraTM or Williams' E medium.
• the cells used are Vera cells.
• cell titer is maximized prior to culturing cells with the virus.
• the cells are cultured in a medium containing serum before infection with a virus or a viral construct of interest and the cells are cultured in a medium without serum after infection with the virus or viral construct.
• the serum is fetal bovine serum and is present a concentration of 10% of culture volume, 5% of culture volume, 2% of culture volume, or 0.5% of culture volume.
• the virus is cultured with cells that have reduced tumorigenicity and are conducive to virus propagation.
• the cells used for virus propagation are Vera cells.
• the cell culture used for virus propagation is a perfusion culture. (See also Section 5.1.2).
• virus titer is increased by modifying the parameters for the post-infection culture conditions.
• the virus can be propagated in serum- free media.
• Serum-free media may be any serum-free media including but not limited to OptiPROTM SFM (Gibco Cat 12309-019, 2005) and virus-production serum-free medium (VP-SFM) (Gibco Cat 11681-020, 2005) or SFM4MegaVirTM (Hyclone) or Ex-Cell VeraTM (SAFC Biosciences) or William's E media (Hyclone). (See Section also 5.1.1).
• the present invention relates to a method of optimal virus production by modifying virus titers used to infect cells.
• the average number of viruses used to infect a cell is referred to as a multiplicity of infection (MOI).
• MOI used to infect Vera cells is in a range of about 0.0001 to about 0.1.
• MOI is in a range of about 0.001 to about 0.01.
• MOI is 0.001.
• the MOI is 0.01. (See also Section 5.3).
• the present invention relates to a method of increased virus production by modifying the parameters of the process and culture conditions.
• the invention relates to an improved method of a shifting to a lower postinfection cultivation temperature.
• the cells are cultured at about 37 0 C, i.e., at 37 0 C ⁇ 1, before infection with the virus and from about 29 0 C, i.e., at 29 0 C ⁇ 1 to about 35 0 C, i.e., at 35 0 C ⁇ 1 after infection with the virus.
• the cells are cultured at about 33 0 C, i.e., at 33 0 C ⁇ 1 after infection with the virus.
• the cells are cultured at about 3O 0 C, i.e., at 3O 0 C ⁇ 1 after infection with the virus. (See also Section 5.2).
• Routine assays may be used to optimize individual parameters for a particular host cell-type and/or virus.
• Small-scale experiments are conducted to identify and optimize critical process parameters and culture conditions for virus production.
• T-flasks such as, for example, T-25 or T-75 flasks
• the virus production processes are increased to mid-scale production, e.g., using roller bottles or spinner flasks.
• spinner flasks use microcarriers for mid-scale production of the virus. (See Section 5.4).
• Viruses can be propagated using a microcarrier for cell culture.
• the advantage to using microcarriers is to increase the surface area for the cells grown in culture, especially for adherent cell lines, such as, for example, Vero cells, in order to improve cell growth.
• Microcarriers used in connection with the present invention may be any microcarrier including but not limited to Pronactin F, CytodexTM 1 and CytodexTM 3.
• the microcarrier is CytodexTM 1.
• the amount of microcarrier beads used are range from about 2 g/L to about 20g/L.
• the amount of microcarrier beads used are about 2 g/L, about 4 g/L, or about 5g/L for batch processes.
• the amount of microcarrier beads used are about 20 g/L for fed-batch processes.
• trypsin is used to detach cultured cells from the microcarrier beads to allow the cells to subsequently adhere to new microcarrier beads added to the culture in order to expand and grow.
• expansion of the viral cell culture may occur in the absence of trypsin.
• a direct bead-to-bead transfer to expand viral cell cultures in bioreactors is used instead.
• Viral cell culture cells are allowed to directly migrate from the microcarrier beads they are attached to, to adhere to freshly added beads in order to expand and grow.
• One embodiment of the invention for a direct bead-to-bead transfer involves various intermittent agitation of the viral cell culture for the duration of the culture time.
• the intermittent agitation is performed in 1, 2, 3 or more cycles.
• each cycle may have a duration of up to 5 hours, up to 8 hours, up to 24 hours or for the entire duration of the culture time.
• the intermittent agitation is performed at 125 rpm for 5 minutes and then stopped at 0 rpm for 30 minutes in the first cycle. In another embodiment, the intermittent agitation is performed at 125 rpm for 10 minutes and then stopped at 0 rpm for 50 minutes in the first cycle. In another embodiment, the intermittent agitation is performed at 125 rpm for 1 hour and then stopped at 0 rpm for 1 hour in the second cycle. In yet another embodiment, agitation may be constant at 125 rpm in the second cycle. In yet another embodiment, the agitation may be constant at 125 rpm in the third cycle. See Table VIII.
• direct bead-to-bead transfer can also involve expansion of the viral cell culture by splitting the culture by a ratio of 1 : 1 or 1:5 into fresh growth medium containing microcarrier beads.
• the culture is split when the viral cell culture density is >le6 cells/mL.
• the viral constructs and methods of the present invention can be used for commercial production of viruses, e.g., for vaccine production.
• the vaccine contains only the live attenuated viruses that have been propagated. Contamination of vaccines with adventitious agents introduced during production should also be avoided. Methods known in the art for large scale production of viruses or viral proteins can be used for commercial production of a vaccine of the invention.
• cells are cultured in a bioreactor or fermenter.
• Bioreactors are available in volumes from under 1 liter to in excess of 100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, MN); NBS bioreactors (New Brunswick Scientific, Edison, N. J.); and laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsungen, Germany).
• the viral cell cultures of the invention are grown in a 3L bioreactor. In one embodiment, the viral cell cultures of the invention are grown in a 15L bioreactor.
• the viral cell cultures of the invention are grown in a 30L bioreactor.
• Such bioreactors can be, for example, a stirred-tank Applikon bioreactor.
• the viral cell cultures of the invention are grown in suspension culture in a single use bioreactor (SUB).
• the viral cell cultures of the invention are grown on microcarrier beads in a SUB.
• small-scale process optimization studies are performed before the commercial production of the virus, and the optimized conditions are selected and used for the commercial production of the virus.
• a virus is propagated as follows: cells in which the virus is known to grow well are grown under their optimal growth conditions, e.g., with serum and at 37 0 C; cells are placed into the CDLC enriched medium, e.g., medium without serum or substantially free of serum, and the cells are infected with the virus; the infected cells are cultured at a lower temperature than the pre-infection culture, e.g., at 33 0 C or 3O 0 C.
• Viral cell cultures grown according to the invention may achieve a virus titer obtained of at least 7 log 10 TCID 50 /mL.
• the virus titer achieved is at least 7.5 log 10 TCID 50 /mL.
• the virus titer achieved is at least 8 log 10 TCID 50 /mL.
• the virus titer achieved is at least 8.5 log 10 TCID 50 /mL.
• the virus titer achieved is at least 9 log 10 TCID 50 /mL.
• Viral cell cultures grown according to the invention may yield a certain number of vaccine doses per virus harvest batch.
• the viral cell cultures grown according to the invention may yield at least 1 million, at least 2 million, at least 5 million, at least 9 million, at least 10 million, at least 11 million, at least 12 million, at least 15 million, at least 20 million, at least 25 million, at least 30 million, at least 35 million, at least 40 million, at least 45 million, at least 50 million, at least 55 million, at least 60 million, at least 65 million, at least 70 million, at least 75 million, at least 80 million, at least 85 million, at least 90 million, at least 100 million, at least 105 million, at least 110 million, at least 115 million, at least 120 million, at least 125 million, at least 130 million, or at least 135 million vaccine doses per 3OL of virus harvest batch.
• the cells used for viral propagation are cells that can be grown and/or maintained without the addition of components derived from animals or humans.
• the cells used for viral propagation are grown in substantially serum-free medium.
• the cells for virus propagation are cells that are adapted to growth without serum.
• Vero cells are used for virus propagation.
• serum- free media may be any serum-free media including but not limited to OptiPROTM SFM (Gibco Cat 12309-019) and virus-production serum- free medium (VP SFM) (Gibco Cat 11681-020).
• the medium used for culturing of cells infected with the virus that is to be propagated is substantially free of serum.
• OptiPROTM SFM is a serum-free, ultra-low protein (7.5 ⁇ g/ml) medium containing no proteins, peptides, or other components of animal or human origin. Stock solutions of the OptiPROTM SFM are stored at the temperature between 2 0 C to 8 0 C in the dark. OptiPROTM SFM is unique in that it supports the growth of numerous attachment- dependent cell lines without the need for addition of attachment proteins to the medium or pre-treatment of the attached surface by inducing the cells to manufacture their own attachment proteins.
• VP-SFM is a serum-free, ultra-low protein (5 ⁇ g/ml) medium containing no proteins, peptides, or other components of animal or human origin. VP- SFM is available in a ready to use liquid format and is stored at the temperature between 2 0 C to 8 0 C in the dark.
• EX-CELLTM Vero (SAFC BioSciences, JRH Catalog No. 14585) is serum-free and free of animal-derived components.
• the medium contains a plant-derived hydrolysate and low levels of recombinant proteins, but does not contain phenol red or Pluronic® F68.
• the serum-free medium is chemically defined, such as FNC Coating Mix® (Athena Environmental Sciences), UltaMEMTM (Cambrex Corporation), HL- 1TM (Cambrex Corporation), NeurobasalTM A Medium (Invitrogen), MAM -PF- 1,-2-3 (Promocell), RenCyteTM BHK (Medicult), Williams' Medium E (Sigma- Aldrich), and Nutridoma-NS Supplement (Roche).
• the serum-free medium is chemically undefined, such as OptiPROTM SFM and VP-SFM or SFM4MegaVirTM (Hy clone).
• the present invention relates to a method of increased virus production using a medium that is substantially serum-free.
• Substantially serum-free medium can be medium that contains less than 5% v/v of serum, less than 2.5% v/v of serum, less than 1% v/v of serum, less than 0.1% v/v of serum, less than 0.01% v/v of serum, or less than 0.001% v/v of serum.
• virus is propagated by incubating cells that are infected with the virus in a culture medium containing less than 5% v/v of serum, less than 2.5% v/v of serum, less than 1% v/v of serum, less than 0.1% v/v of serum, less than 0.01% v/v of serum, or less than 0.001% v/v of serum.
• virus is propagated by incubating cells that are infected with the virus in the complete absence of serum.
• virus is propagated in a serum-free medium comprising chemically defined lipid concentrate (CDLC).
• CDLC chemically defined lipid concentrate
• the cells are first cultured in medium containing serum and then transferred into medium without serum, by removing the serum-containing medium from the cells and adding the medium without serum.
• the cells are washed with medium without serum to ensure that cells once infected with the virus are incubated in the absence of serum.
• the cells are washed with medium without serum at least one time, two times, three times, four times, five times, or at least ten times.
• the cells once the cells are in serum-free medium, they are infected with the virus.
• CDLC can be added before or after the infection with the virus.
• the present invention provides for the first time a method for propagating a virus by culturing cells in a medium comprising chemically defined lipid concentrate (CDLC).
• CDLC chemically defined lipid concentrate
• the present invention contemplates culturing cells in a medium comprising chemically defined lipid concentrate (CDLC) prior to infection with virus.
• defined medium that is substantially free of serum is the avoidance of risks of contamination and immunogenic stimuli. Contaminants include, but are not limited to, viruses, prions, and mycoplasma.
• the addition of CDLC to serum- free medium is effective in facilitating cell attachment thereby increasing titers of enveloped viruses in mammalian culture systems.
• the CDLC-supplemented medium is substantially free of serum. In another embodiment, the CDLC-supplemented medium contains a maximum serum concentration of 0.5% v/v. In certain embodiments, the CDLC-supplemented medium contains one or more of Pluronic F-68, Ethyl Alcohol, Cholesterol, Tween 80, DL-alpha- Tocopherol Acetate, Stearic Acid, Myristic Acid, Oleic Acid, Linoleic Acid, Palmitic Acid, Palmitoleic Acid, Arachidonic Acid, and Linolenic Acid.
• the CDLC comprises 100,000 mg/L of Pluronic F-68, 100,00 mg/L of Ethyl Alcohol, 220 mg/L of Cholesterol, 2,200 mg/L of Tween 80, 70 mg/L of DL-alpha- Tocopherol Acetate, 10 mg/L of Stearic Acid, 10 mg/L of Myristic Acid, 10 mg/L of Oleic Acid, 10 mg/L of Linoleic Acid, 10 mg/L of Palmitic Acid, 10 mg/L of Palmitoleic Acid, 2 mg/L of Arachidonic Acid, and 10 mg/L of Linolenic Acid.
• the CDLC comprises 50,000 to 250,000 mg/L of Pluronic F-68, 50,000 to 250,000 mg/L of Ethyl Alcohol, 100 to 300 mg/L of Cholesterol, 1,000 to 4,000 mg/L of Tween 80, 50 to 100 mg/L of DL-alpha-Tocopherol Acetate, 5 to 20 mg/L of Stearic Acid, 5 to 20 mg/L of Myristic Acid, 5 to 20 mg/L of Oleic Acid, 5 to 20 mg/L of Linoleic Acid, 5 to 20 mg/L of Palmitic Acid, 5 to 20 mg/L of Palmitoleic Acid, 1 to 5 mg/L of Arachidonic Acid, and 5 to 20 mg/L of Linolenic Acid.
• the CDLC-supplemented medium contains 0.1% v/v to 5% v/v CDLC. In certain embodiments, the CDLC-supplemented medium contains 0.1% v/v, 0.5% v/v, 1% v/v, 2% v/v, 3% v/v, 4% v/v, or 5% v/v. In another embodiment, the CDLC-supplemented medium contains 1% v/v of CDLC.
• the CDLC is added to the cell culture medium pre- infection. In other embodiments, at least one lipid is exogenously added to the post-infection medium.
• the cells are cultured in a medium containing serum before infection with a virus or a viral construct of interest and the cells are cultured in a medium without serum after infection with the virus or viral construct.
• the serum is fetal bovine serum and is present a concentration of 10% of culture volume, 5% of culture volume, 2% of culture volume, or 0.5% of culture volume.
• the serum can be, but is not limited to, bovine calf serum, human serum, newborn bovine serum, newborn calf serum, donor bovine serum, donor horse serum.
• the cells are incubated before infection with the virus in medium containing serum. In certain embodiments, subsequent to infection of the cells with the virus, the cells are incubated in the absence of serum. In other embodiments, the cells are first incubated in medium containing serum; the cells are then transferred into medium without serum; and subsequently, the cells are infected with the virus and further incubated in the absence of virus.
• the cells are transferred from medium containing serum into medium in the absence of serum, by removing the serum-containing medium from the cells and adding the medium without serum.
• the cells are centrifuged and the medium containing serum is removed and medium without serum is added.
• the cells are washed with medium without serum to ensure that cells once infected with the virus are incubated in the absence of serum.
• the cells are washed with medium without serum at least one time, two times, three times, four times, five times, or at least ten times.
• cells are cultured in a medium containing serum and at a temperature that is optimal for the growth of the cells before infection with a virus or transfection with a viral construct of interest, and the cell culture is incubated at a lower temperature (relative to the standard incubation temperature for the corresponding virus or viral vector) after infection with the virus or transfection with the viral construct of interest.
• cells are cultured in a medium containing serum before infection with a virus or transfection with a viral construct of interest at 37°C, or about 37°C (i.e., 37 ⁇ PC) and the cell culture is incubated at 33°C or about 33°C (i.e., 33 ⁇ PC) after infection with the virus or transfection with the viral construct of interest.
• cells are cultured in a medium containing serum before infection with a virus or transfection with a viral construct of interest at 37°C, or about 37°C (i.e., 37 ⁇ PC) and the cell culture is incubated at 30 0 C or about 30 0 C (i.e., 30 ⁇ PC) after infection with the virus or transfection with the viral construct of interest.
• cells are cultured in a medium containing serum and at a temperature that is optimal for the growth of the cells before infection with a virus or transfection with the viral construct of interest, and the cell culture is incubated without serum at a lower temperature (relative to the standard incubation temperature for the corresponding virus or viral vector) after infection with the virus or transfection with the viral construct of interest.
• cells are cultured in a medium containing serum before infection with a virus or transfection with a viral construct of interest at 37°C, and the cell culture is incubated without serum at 33°C or about 33°C (e.g., 33 ⁇ 1 C) after infection with the virus or transfection with the viral construct of interest.
• cells are cultured in a medium containing serum before infection with a virus or transfection with a viral construct of interest at 37°C, and the cell culture is incubated without serum at 30 0 C or about 30 0 C (e.g., 30 ⁇ 1 C) after infection with the virus or transfection with the viral construct of interest.
• a cell culture infected with a virus or transfected with a viral construct of interest and is incubated at a lower post-infection incubation temperature as compared to the standard incubation temperature for the cells in culture.
• a cell culture infected with a virus or transfected with a viral construct of interest is incubated at 33°C or about 33°C (e.g., 33 ⁇ 1°C).
• the postinfection incubation temperature is about 25°C, 26°C, 27°C, 28°C, 29°C, 30 0 C, 31°C, 32°C, 33°C, 34°C, 35°C, 36 ° C or 37 ° C.
• virus is propagated by incubating a cells before infection with the virus at a temperature optimized for the growth of the cells and subsequent to infection of the cells with the virus, i.e., post-infection, the temperature is shifted to a lower temperature.
• the shift is at least PC, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10 0 C, 11 0 C, or at least 12°C.
• the shift is at most 1 0 C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10 0 C, I PC, or at most 12°C.
• the shift is 4°C.
• the present invention relates to a method of optimal virus production by modifying virus titers used to infect cells.
• the average number of viruses used to infect a single cell is referred to as a multiplicity of infection (MOI).
• MOI multiplicity of infection
• an optimal balance of MOI is preferred. Less virus inoculums per infection can extend the lifespan of master virus banks, but an increased virus inoculums can yield higher virus titer.
• MOI used to infect Vera cells is in a range of about 0.0001 to about 0.1. In another embodiment, MOI is in a range of about 0.001 to about 0.01. In a specific embodiment of the invention, a cell culture is infected with the virus having the MOI of about 0.1. In another specific embodiment, a cell culture is infected with the virus having the MOI of about 0.01. In yet another specific embodiment, a cell culture is infected with the virus having the MOI of about 0.001.
• a cell culture is infected with a virus having the MOI of about 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.0011, 0.0012, 0.0013, 0.0014, or 0.0015. In another embodiment, a cell culture is infected with the virus having the MOI of 0.001.
• small-scale experiments are conducted to identify and optimize critical process parameters and culture conditions for virus production.
• T-flasks are utilized in small-scale experiments to identify and optimize critical process parameters and culture conditions for virus production.
• T- flask experiments are conducted to investigate the combined effects of time of infection and post-infection cultivation temperature on MEDI-534 or MEDI-559 or other viral construct production as disclosed herein, such as, but not limited to a ⁇ M2-2, ⁇ NS-1, or rcp45 hPIV3 (MEDI-560).
• T-flask experiments can assess the impact of pre- infection culture media on virus production.
• the virus production processes is increased to mid-scale production.
• roller bottles are used for mid- scale production of the virus.
• spinner flasks are used for mid-scale production of the virus.
• spinner flasks use microcarriers for mid-scale production of the virus.
• roller bottles use microcarriers for mid-scale production of the virus.
• the virus production is performed in microcarrier cultures.
• Microcarrier culture is a technique which makes possible the practical high yield culture of anchorage-dependent cells.
• the microcarriers provide convenient surfaces for the growth of animal cells and can be used in suspension culture systems or to increase the yield from monolayer culture vessels and perfusion chambers.
• Microcarriers' application include production of large quantities of cells, viruses and recombinant cell products, studies on cell adhesion, differentiation and cell function, perfusion column culture systems, harvesting cells, etc.
• the microcarriers used are CytodexTM 1 and CytodexTM 3 (Amersham Biosciences).
• the microcarrier used is Pronectin® F (Sayno Chemical Industries).
• CytodexTM 1 and CytodexTM 3 are commonly used microcarriers. They were specifically developed for the culture of a wide range of animal cells in culture volumes ranging from a few milliliters to thousand liters. Using CytodexTM in simple suspension culture systems provides yields of several million cells per milliliter.
• CytodexTM is designed to meet the special requirements of a microcarrier technique, which are: the size and density are optimized to give good growth and high yields for a wide variety of cells; the matrix is biologically inert and provides a strong but non-rigid substrate for stirred microcarrier cultures; the microcarriers are transparent allowing easy microscopic examination of the attached cells.
• CytodexTM 1 is based on a cross-linked dextran matrix which is substituted with positively charged N, N-diethylaminoethyl groups. The charged groups are distributed throughout the microcarrier matrix. CytodexTM 1 is suitable for general purpose microcarrier culture, particularly for most established cell lines. This microcarrier can also be used for production from cultures of primary cells and normal diploid cell strains when maximum recovery of culture products is not essential.
• CytodexTM 3 consists of a thin layer of denaturated collagen chemically coupled to a matrix of cross-linked dextran.
• the denaturated collagen layer on CytodexTM 3 is susceptible to digestion by a variety of proteases, including trypsin and collagenase, and provides unique opportunities for removing cells from microcarriers while maintaining maximum cell viability, function and integrity.
• CytodexTM 3 is the microcarrier of choice for cells known to be difficult to grow in culture, for differentiated cell culture systems and particularly for cells with an epithelial-like morphology. It can be used as a general purpose collagen-coated culture surface.
• the virus production is performed in microcarrier cultures.
• Vera cells are cultured in OptiPROTM SFM supplemented with FBS and containing CytodexTM 1.
• a concentration of FBS is about 0.5% (v/v) to about 2.0% (v/v).
• a concentration of FBS is about 0.5% (v/v) to about 1.0% (v/v).
• a concentration of FBS is about 0.5% (v/v).
• a concentration of CytodexTM 1 is about 1 g/L to about 5 g/L.
• a concentration of CytodexTM 1 is about 1 g/L to about 3 g/L. In another embodiment, a concentration of CytodexTM 1 is about 2 g/L. In yet another embodiment, a concentration of CytodexTM 1 is about 4 g/L.
• Vera cells are cultured in OptiPROTM SFM supplemented with FBS and containing CytodexTM 3.
• a concentration of FBS is about 0.5% (v/v) to about 2.0% (v/v).
• a concentration of FBS is about 0.5% (v/v) to about 1.0% (v/v).
• a concentration of FBS is about 0.5% (v/v).
• a concentration of CytodexTM 3 is about 1 g/L to about 5 g/L.
• a concentration of CytodexTM 3 is about 1 g/L to about 3 g/L.
• a concentration of CytodexTM 3 is about 2 g/L.
• Vera cells are cultured in VP- SFM supplemented with CDLC and containing CytodexTM 1.
• a concentration of CDLC is about 0.5% (v/v) to about 2.0% (v/v).
• a concentration of CDLC is about 0.5% (v/v) to about 1.0% (v/v).
• a concentration of CDLC is about 1.0% (v/v).
• a concentration of CytodexTM 1 is about 1 g/L to about 5 g/L.
• a concentration of CytodexTM 1 is about 1 g/L to about 3 g/L.
• a concentration of CytodexTM 1 is about 2 g/L.
• a concentration of CytodexTM 1 is about 4 g/L.
• Vera cells are cultured in VP-SFM supplemented with CDLC and containing CytodexTM 3.
• a concentration of CDLC is about 0.5% (v/v) to about 2.0% (v/v).
• a concentration of CDLC is about 0.5% (v/v) to about 1.0% (v/v).
• a concentration of CDLC is about 1.0% (v/v).
• a concentration of CytodexTM 3 is about 1 g/L to about 5 g/L.
• a concentration of CytodexTM 3 is about 1 g/L to about 3 g/L.
• a concentration of CytodexTM 3 is about 2 g/L.
• bead to bead transfer methods may be utilized, wherein fresh beads and media are mixed with the confluent beads and the culture is incubated under conditions which facilitate the transfer of cells to the new beads in order to expand and grow the cell culture.
• Such bead to bead transfer may utilize an intermittent agitation scheme, such as, for example, agitation of the culture containing microcarrier beads at 100-130 rpm for 5 to 10 minute intervals, cease agitation for up to 40 minutes, for up to 50 minutes, for up to 60 minutes. Then, resume this pattern of agitation for a period of 4 hours, for 5 hours, for 6 hours, for 7 hours, for 8 hours up to overnight (12-24 hours).
• Bioreactors are available in volumes from under 1 liter to in excess of 100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, MN); NBS bioreactors (New Brunswick Scientific, Edison, N.J.); and laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsungen, Germany).
• Cyto3 Bioreactor Osmonics, Minnetonka, MN
• NBS bioreactors New Brunswick Scientific, Edison, N.J.
• laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsoder, Germany).
• small-scale process optimization studies are performed before the commercial production of the virus, and the optimized conditions are selected and used for the commercial production of the virus.
• a reactor system comprising disposable elements such as a flexible plastic bag for culturing cells.
• Such reactor systems are known in the art and are available commercially. See for example International Patent Publications WO 05/108546; WO 05/104706; and WO 05/10849 and Section 6.14 infra.
• Reactor systems comprising disposable elements also referred herein as "single use bioreactor(s)" or by the abbreviation "SUB(s)" may be pre-sterilized and do not require a steam-in-place (SIP) or clean-in-place (CIP) environment for changing from batch to batch or product to product in a culture or production system.
• SIP steam-in-place
• CIP clean-in-place
• a disposable reactor system is a stirred-tank reactor system which allows for a hydrodynamic environment for mixing the cell culture which allows for more efficient nutrient, O 2 and pH control.
• the present invention provides methods for the production of virus in a stirred-tank SUB wherein one or more parameters selected from the group consisting of temperature, agitation rate, pH, dissolved oxygen (DO), O 2 and CO 2 flowrate, are monitored and/or controlled.
• the host cells e.g., Vera cells
• DO dissolved oxygen
• cultivation of the cells and/or the virus production is performed in a bioreactor (e.g., a SUB) at a CO 2 concentration of at least 1%, or of at least 2%, or of at least 3%, or of at least 4%, or of at least 5%, or of at least 6%, or of at least 7%, or of at least 8%, or of at least 9%, or of at least 10%, or of at least 20%.
• a bioreactor e.g., a SUB
• the CO 2 flowrate is maintained at between about 0.1 L/min to about 1 L/min.
• the dissolved oxygen (DO) concentration (p ⁇ 2 value) is advantageously regulated during the cultivation of the cells and/or the production of virus and is in the range from 5% and 95% (based on the air saturation).
• the DO is maintained between about 10% to about 80%, or between about 20% to about 70%, or at about 50%.
• the dissolved oxygen (DO) concentration (p ⁇ 2 value) is at least 10%, or at least 20%, or at least 30%, or at least 50%, or at least 60%.
• the acceptable range for the DO is between about 100 to about 35%.
• the DO is maintained at between about 35% to about 50%, or at about 50%.
• the DO should not drop below about 35%.
• the initial DO may be 100% and that the DO may be allowed to drop down to a predetermined level (e.g., 50%) where it is maintained.
• the DO is maintained used any method known in the art, such as, for example, by sparging O 2 .
• the O 2 flowrate is maintained at less then about 2.0 L/min.
• the pH of the culture medium used for the cultivation of the cells and/or the production of virus is regulated and is in the range from pH 6.4 to pH
• the pH of the culture medium is maintained at about 6.4, or at about 6.6, or at about 6.8, or at about 7.0, or at about
• the initial pH may be lower or higher then the desired range and that the pH may be allowed to increase or decrease to the desired level (e.g., 7.1) where it is maintained.
• the pH is maintained by any method known in the art.
• the pH may be controlled by sparging CO 2 and/or by adding acid (e.g., HCL) or base (e.g., NaOH) as needed.
• the pH is regulated by the addition of NaOH and/or the sparging of CO 2 .
• Vera cells are cultured in a SUB system to a cell density of at least 5xlO 5 cells/mL, a least 7.5xlO 5 cells/mL, at least IxIO 6 cells/mL, at least 2.5xlO 6 cells/mL, at least 5xlO 6 cells/mL, at least 7.5xlO 6 cells/mL, at least 1OxIO 6 , at least 15x10 6 cells/mL, at least 2OxIO 6 cells/mL, or at least 25x10 6 cells/mL prior to infection.
• Vera cells are cultured in a SUB in a serum-free medium such as those described supra (see, for e.g., Section 5.1).
• the media is supplemented with additional glucose.
• Vera cells are cultured in a SUB as adherent cells on a microcarrier such as those described supra (see, for e.g., paragraphs [00109]-[00111]).
• the microcarrier is used at a concentration of between about 1 to about 4 g/L.
• the microcarrier is used at a concentration of between about 2 to about 3 g/L.
• the cells are cultured without the supplementation of any media component.
• the cells are cultured with the supplementation of glucose and glutamine.
• the cells are cultured with the supplementation of CDLC.
• the viral cell culture post-infection or cell culture supernatant has a lactate concentration of about 1.0 to 2.0 g/L, more particularly from about 1.25 to 1.5 g/L. In certain embodiments, the viral cell culture post-infection or cell culture supernatant, has a glutamine concentration of about 2.0 to about 4.0 g/L, more particularly from about 2.0 to about 3.0 g/L.
• the viral cell culture post-infection or cell culture supernatant has a glucose concentration of about 0.5 to 2.5 g/L, more particularly from about 1.5 to 1.75 g/L. In certain embodiments, the viral cell culture post-infection or cell culture supernatant, has an ammonium ion concentration of about 1.25 to about 2.5 mM, more particularly from about 2.0 to about 2.25 mM.
• the virus is recovered (i.e., harvested) from the viral cell culture 2 to 12 days post-infection. In another embodiment, the virus is recovered from the viral cell culture 3 to 4 days post-infection.
• the SUB is seeded with the Vera cells to be cultured at a seeding density of about IxIO 4 cells/mL to about 5 ⁇ 10 5 cells/mL.
• the seeding density is between about 3 ⁇ 10 4 cells/mL to about 3 ⁇ 10 5 cells/mL, or between about 7 ⁇ 10 4 cells/mL to about 2 ⁇ 10 5 cells/mL, or between about 8 ⁇ 10 4 cells/mL to about 2 ⁇ 10 5 cells/mL, or between about 9 ⁇ 10 4 cells/mL to about 1 ⁇ 10 5 cells/mL, or between about l ⁇ 10 5 cells/mL to about 2 ⁇ 10 5 cells/mL.
• the seeding density is between about l ⁇ 10 5 cells/mL to about 2 ⁇ 10 5 cells/mL.
• the agitation rate of the SUB is maintained at between about 50 to 150 rpm. In a specific embodiment the rate of agitation is maintained at between about 80 to about 120 rpm, or between about 90 to about 100 rpm. In another specific embodiment, the rate of agitation is maintained at between about 100 to about 125 rpm. In yet another embodiment, agitation rates may be maintained at one rate during the cell culturing, but then altered to another rate at another point during the cell culturing (i.e., intermittent agitation). Agitation rates are controlled by means well known in the art.
• a medium exchange may be performed after cultivation of the cells and prior to infection.
• the portion of the medium to be exchanged is between about 20% to about 100%, or between about 30% to about 80%, or between about 30% to about 60%, or between about 66% to about 90%.
• the medium is exchange with an equal volume of medium.
• the medium is exchange with a reduced volume of medium, effectively concentrating the cells.
• the medium may be exchanged for a medium having the same or different composition.
• a growth medium i.e., a medium used for proliferation of cells
• an infection medium i.e., a medium used during infection and viral growth.
• the growth medium is supplemented with and/or comprises additional components (e.g., glucose, trace mineral, amino acids, etc) such that media exchange is not required.
• the virus that is propagated using the methods of the invention is a negative strand RNA virus.
• the virus is a non- segmented, negative strand RNA virus, such as paramyxovirus.
• the viruses that are propagated using the methods of the inventions are parainfluenza virus (PIV) and respiratory syncytial virus (RSV) and metapneumo virus (MPV).
• the invention provides a method for propagating PIV with human and bovine sequences.
• the virus is a chimeric human/bovine PIV expresses RSV nucleotide sequences.
• the virus that is propagated is MEDI-534.
• the virus that is propagated is MEDI-559. In another specific embodiment, the virus that is propagated is one which has a deletion of an open reading frame, such as, for example M2-2 or NS-I . In another specific embodiment, the virus that is propagated is one which is rcp45 hPIV3 or MEDI-560. In certain embodiments, the virus that is propagated using the methods of the invention is an enveloped viruses. In other embodiments the virus that is propagated is a virus that infects and replicates in attached cells.
• the virus that is propagated is MEDI-534.
• MEDI- 534 was constructed by first constructing a recombinant bovine PIV3 vector by replacing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes in bovine PIV3 with the human PIV3 and NH genes, respectively (Haller et al, 2000, J Virol 74:11626-11635). Subsequently, the RSV F gene was inserted into the bovine-human PIV3 vector backbone to generate a chimeric virus that expresses the RSV F protein (Tang et al., 2003, J Virol 77:10819-10828).
• the chimeric virus was named MEDI-534 and it functions as a live, attenuated, bivalent vaccine in animal studies: hamsters and non-human primates immunized with MEDI-554 demonstrated protection from challenge with RSV and hPIV3, and the animals produced RSV -neutralizing and hPIV3 hemagglutination-inhibiting serum antibodies (Tang et al., 2003, supra; Tang et al., 2004, supra).
• the virus that is propagated is MEDI-559.
• MEDI- 559 is a live, attenuated RSV vaccine, termed rA2cp248/404/1030 ⁇ SH that is temperature sensitive, contains point mutations and a gene deletion of the SH gene. It was found to be well-tolerated and safe when administered to infants in Phase I clinical trials (Karron et al., JID vol 191 p. 1093 (2005)) and was able to elicit an immune response in such patients.
• the virus that is propagated is MEDI-560.
• MEDI-560 is a live, attenuated HPIV3.
• a derivative called cp45 was produced by 45 cycles of passage of wt HPIV3 at progressively suboptimal temperatures by Dr. Robert Belshe, now at St. Louis University.
• This biologically-derived vaccine candidate has been evaluated in adults, seropositive and seronegative children and young infants in Phase I and II trials and appears to be satisfactorily attenuated and immunogenic (Karron et al. Pediatr. Inf. Dis. J. 2003, 22: 394-405).
• cp45 The significant point mutations in cp45 were identified by sequence analysis and placed in wild type recombinant HPIV3 individually or in various combinations to assess their associated phenotypes. This identified three major ts and attenuating point mutations in the L protein as well as several non-ts attenuating point mutations in the C and F proteins. This virus has now been recovered from cDNA (rcpPIV3), which provides a virus with a known passage history using acceptable substrates.
• the viruses that are propagated using the methods of the invention are enveloped viruses.
• Enveloped viruses include, but are not limited to Paramyxovirus, Herpesvirus, Togavirus, Rhabdovirus, Coronavirus.
• the viruses that are propagated are viruses that infect and replicate in anchorage-dependent cells.
• Viruses that infect and replicate in anchorage-dependent cells include, but are not limited to Sindbis virus, Vesicular Stomatitis virus, Oncornavirus, Herpes simplex virus, Hepatitis A virus, RSV virus, Parainfluenza virus, Corona virus, FMDV virus, Rabies virus, Polio virus, and Reo virus.
• the viruses are propagated using the methods of the invention in a mammalian cell line.
• the viruses are propagated using the methods of the invention in cells that are anchorage dependent.
• Anchorage-dependent cells used with the methods of the invention can be cell lines derived from anchorage- dependent type cells including, but not limited to human adipose stem cells, human proximal tubule cells, mouse smooth muscle cells, human endothelial cells, human kidney cells, human large intestine cells, dog kidney cells, hamster ovary cell, green monkey kidney cells, rat small intestine cells, human bladder cells, and human prostate cells.
• the viruses are propagated in kidney-derived cell lines, that include, but are not limited to MDBK cells, MDCK cells, Vera cells, PK- 15 cells, and BHK-21 cells. In other embodiments, the viruses are propagated in BHK-21 cells or Vera cells. In another embodiment, the viruses are propagated in Vera cells.
• Vera cells originating from a continuous African green monkey kidney cell line, are the most commonly used cell line for vaccine production and they have demonstrated an absence of tumorigenicity (Vincent-Falquet et al, 1989, Dev Biol Stand 70: 153-156). Human polio and rabies vaccines are currently manufactured commercially in Vera cells (Montagnon, 1989, Dev Biol Stand 93: 119-123), following specified guidelines provided by regulatory authorities on the use of Vera cells for viral vaccine production (WHO, 1987a,b).
• Vera cells are usually considered to be anchorage-dependent and are typically propagated in static culture or on microcarriers (Yokomizo et al., 2004, Biotechnol Bioeng 85: 506-515; Wu et al., 2004, Vaccine 22: 3858-3864; Berry et al., 1999, Biotechnol Bioeng 62: 12-19), although they can grow in suspension as cell aggregates (Litwin 1992, Cytotechnology 10: 169-1974). Adherent cultures of Vera cells are well-characterized and have an excellent safety record (Montagnon and Vincent-Falquet, 1998, Dev Biol Stand 93: 119-123).
• the cells used for viral propagation are cells that can be grown and/or maintained without the addition of components derived from animals or humans. In certain embodiments, the cells used for viral propagation are cells that can be grown and/or maintained in substantially serum- free medium, or in medium without serum.
• the cells to be used with the methods of the invention are capable of attachment to fibronectin.
• the cells are grown post-infection while attached to a fibronectin substrate.
• the viral titer can be measured by any method well-known in the art, for example, but not limited to Tissue Culture Infectious Dose at 50% (TCID50) Assay.
• the assay measures the potency/infectivity of the virus and uses cells that can be infected with the virus, such as Vera cells.
• the TCID50 Assay is performed as follows: Vera cells are seeded two days prior to the addition of virus-containing samples in a 96 well plate. The cell plate lot number can be recorded and used as a tool for verifying that the cell passage number is greater than or equal to 126 and less than or equal to 148. The plates are 100% confluent; the cells in the plate are distributed in a smooth, continuous monolayer throughout the well.
• the cell plates are washed using the SkatronTM cell washer.
• the washed plates are transferred to a 33 ⁇ l°C, 5 ⁇ l% CO 2 incubator and incubated for a minimum of 10 minutes.
• a virus growth media is dispensed into each well of the cell plate before inoculation with the virus.
• the virus is serially diluted across a 96-well plate containing a Vera cell monolayer and then the inoculated cells are incubated for 6 days.
• the plates are subsequently washed, fixed with paraformaldehyde, and incubated with Numax® (RSV-F specific MAb).
• Cell yield and cell density can be measured by any method well-known in the art, for example, but not limited using a hemacytometer or the Cedex cell counting and viability testing system (Innovatis Inc., Malvern, PA).
• cell densities in microcarrier cultures can be determined by counting nuclei released by 0.1% crystal violet in 0.1 M citric acid solution (Hu and Wang, 1987, Biotechnol Bioeng 30: 548-557).
• kits of the invention comprises serum- free medium and lipid concentrate.
• the serum-free medium in the kit is OptiPROTM SFM or VP-SFM or SFM4MegaVirTM and the lipid concentrate is CDLC.
• the kit may comprise, in one or more containers, serum-free medium, lipid concentrate, and vials of cells that can be infected with an enveloped virus.
• the kit contains one or more vials of Vera cells.
• the kit comprises serum- free medium, lipid concentrate, one or more vials of cells that can be infected with an enveloped virus, and one or more vials of an enveloped virus.
• the kit contains one or more vials of MEDI-534 or MEDI-559.
• the kits of the invention include a manual for conducting a method of the present invention.
• the manual describes the propagation of a virus as follows: cells in which the virus is known to grow well are grown under their optimal growth conditions, e.g., with serum and at 37 0 C; cells are placed into the CDLC enriched medium, e.g., medium without serum or substantially free of serum, and the cells are infected with the virus; the infected cells are cultured at a lower temperature than the pre-infection culture, e.g., at 33 0 C or 3O 0 C.
• a method for propagating a virus in Vera cells comprising:
• a culturing the Vera cells in a bioreactor at a first temperature, comprising seeding a cell culture medium containing chemically-defined lipid concentrate (CDLC) and microcarriers with the Vera cells; b. infecting the Vera cells cultured in step (a) at a second temperature at a multiplicity of infection of about 0.001 to about 0.10, wherein said second temperature is lower than said first temperature; and c. recovering the virus from the cell culture of step (c), wherein said recovered virus yields a viral titer of at least 7.0 logio TCID 50 /ml.
• CDLC chemically-defined lipid concentrate
• bioreactor is a single use bioreactor (SUB) system.
• the chemically defined lipid concentrate comprises one or more of Pluronic F-68, Ethyl Alcohol, Cholesterol, Tween 80, DL- alpha-Tocopherol Acetate, Stearic Acid, Myristic Acid, Oleic Acid, Linoleic Acid, Palmitic Acid, Palmitoleic Acid, Arachidonic Acid, and Linolenic Acid.
• the chemically defined lipid concentrate comprises Pluronic F-68, Ethyl Alcohol, Cholesterol, Tween 80, DL-alpha- Tocopherol Acetate, Stearic Acid, Myristic Acid, Oleic Acid, Linoleic Acid, Palmitic Acid, Palmitoleic Acid, Arachidonic Acid, and Linolenic Acid.
• the chemically defined lipid concentrate comprises 100,000 mg/L of Pluronic F-68, 100,00 mg/L of Ethyl Alcohol, 220 mg/L of Cholesterol, 2,200 mg/L of Tween 80, 70 mg/L of DL-alpha- Tocopherol Acetate, 10 mg/L of Stearic Acid, 10 mg/L of Myristic Acid, 10 mg/L of Oleic Acid, 10 mg/L of Linoleic Acid, 10 mg/L of Palmitic Acid, 10 mg/L of Palmitoleic Acid, 2 mg/L of Arachidonic Acid, and 10 mg/L of Linolenic Acid.
• step (a) utilizes agitation of the culture at an agitation rate between about 50 to about 150 rpm.
• step (a) utilize a dissolved oxygen (DO) amount of between about 35% to about 100%.
• DO dissolved oxygen
• microcarrier concentration is between about 1 to about 4 g/L.
• step (a) is at a pH of between about 6.6 to about 7.6.
• step (b) 17. The method of embodiment 1, wherein after step (a) but prior to step (b) about 50% to about 90% of the cell culture medium is exchanged.
• paramyxovirus is a recombinant parainfluenza virus or a recombinant respiratory syncytial virus or a recombinant metapneumo virus .
• bovine parainfluenza virus further comprises one or more human parainfluenza virus nucleotide sequences.
• step (a) 32.
• said Vera cells in step (a) are cultured to a cell density of at least about 8 x 10 5 cells/ml.
• 33. The method of embodiment 1, wherein the volume of said cell culture of step (a) is at least 1.5 L.
• a Vera cell culture supernatant comprising virus in a cell culture medium substantially free of serum, wherein said supernatant yields a viral titer of at least 7.0 logio TCIDso/ml.
• bovine parainfluenza virus further comprises one or more human parainfluenza virus nucleotide sequences.
• step (a) wherein said culture in step (a) is split 1 : 1 or 1 :5 with fresh culture medium containing new microcarriers.
• a vial of Vero cells (ATCC CCL-81, passage 121) was thawed in DMEM + 5% (v/v) FBS and then passaged four times in the FBS-supplemented medium prior to direct adaptation to serum free growth in OptiPROTM SFM that is free from animal derived components.
• the serum- free Vero cells were banked after 10-15 passages in OptiPROTM SFM.
• the Vero cells used in the experiments provided below originated from the OptiPROTM SFM banks.
• the anchorage-dependent Vero cells were routinely seeded at 5 x 10 4 cells/mL in corresponding culture volumes (35 mL for T-75 flasks, 100 mL - for T-225 flasks and 350 mL - for 850 cm 2 roller bottles (RBs) and passaged 3-5 days post-seeding (dps). For cultures passaged 4-5 dps, a complete medium exchange was performed on each culture three dps. In preparation for subculturing, the spent media was aspirated, and the cells were rinsed twice with DPBS.
• the cultures were incubated at 37 0 C with a 0.05% solution of trypsin-EDTA (3 mL for T-75 flasks, 6 mL for T-225 flasks and 10 mL for RBs) After the cells had detached, an equal volume of lima bean trypsin inhibitor (Worthington Biochemical Corporation, Lakewood, NJ) was added to quench trypsin activity.
• trypsin-EDTA 3 mL for T-75 flasks, 6 mL for T-225 flasks and 10 mL for RBs
• an equal volume of lima bean trypsin inhibitor (Worthington Biochemical Corporation, Lakewood, NJ) was added to quench trypsin activity.
• T-flask cultures were maintained in 37°C/5%CO2/95%Rh incubators and RB cultures were placed on a roller bottle apparatus operated at 0.3 rpm in a 37 0 C incubator.
• the cells were pre-adapted to the media tested in each experiment for at least one passage before initiating the experiment, and the cultures were discarded when the cell passage number exceeded 165.
• Glutamine-free culture media were always supplemented with 4mM L-glutamine before each use.
• the cell culture reagents and supplies were sourced from GIBCO/Invitrogen (Carlsbad, CA) and tissue culture wares were purchased from Corning (Corning, NY), unless specified otherwise.
• MEDI-534 virus has been previously detailed (Tang et al., 2003). To generate the virus seed stock used for infection in all the experiments, MEDI-534 obtained by plasmid rescue (Tang et al., 2003, supra) was added at a multiplicity of infection (MOI) of 0.001 to T-225 flask cultures of Vero cells growing for three days in OptiPROTM SFM. The culture media were collected four dpi and stabilized with 10% (v/v) sucrose phosphate prior to aliquoting into multiple 1 mL cryo vials. The virus seed stocks were stored at -8O 0 C and thawed only immediately before use.
• MOI multiplicity of infection
• T-75 flasks were seeded with 1.75 x 10 6 Vero cells in 35 mL OptiPROTM SFM (unless specified otherwise) and maintained in a 37°C/5%CO 2 /95%Rh incubator. At the time of infection, the spent medium was removed from each flask and the cells were rinsed with 2 x 10 mL DPBS. One flask in each medium condition was trypsinized and the cells were counted. To infect the cultures, DMEM (unless stated otherwise) containing MEDI-534 at the appropriate MOI was added to the remaining flasks. Post-infection, the T-flasks were maintained in humidified incubators with 5% CO 2 overlay.
• each 850cm 2 roller bottle (RB) was seeded with 1.5 x 10 7 Vera cells in the growth medium of choice and maintained at 37 0 C with constant rotation at 0.3 rpm.
• the basal growth medium was either OptiPROTM SFM or virus production serum- free medium (VP-SFM), also a commercially available ADCF SFM from GIBCO/Invitrogen (Carlsbad, CA).
• the basal growth medium was supplemented with fetal bovine serum (FBS) sourced from JRH Biosciences, Inc. (Lenexa, KS) or with a chemically defined lipid concentrate (CDLC) purchased from GIBCO/Invitrogen.
• FBS fetal bovine serum
• CDLC chemically defined lipid concentrate
• the growth medium was either OptiPROTM supplemented with 0.5% (v/v) FBS or VP-SFM supplemented with 1% (v/v) CDLC.
• CytodexTM 1 microcarriers were rehydrated and sterilized according to the manufacturer's recommendations (Amersham Biosciences AB, Uppsala, Sweden). The microcarrier beads were then rinsed once with the appropriate growth medium before use.
• each 250 mL glass spinner flask (Bellco Biotechnology, Inc., Vineland, NJ) was filled with 200 mL of the chosen growth medium containing 2 g/L CytodexTM 1 and incubated at 37°C/5%CO 2 /95%Rh with 60 rpm agitation.
• 2 x 10 7 Vera cells were added per vessel. All cultures were incubated pre-infection at 37°C/5%C0 2 /95%Rh with agitation maintained at 60 rpm.
• To generate growth curves well-mixed samples were taken from the spinner flasks daily for nuclei counting.
• Bioreactor experiments were conducted in 3 L stirred tank bioreactors (Applikon, Foster City, CA) with dissolved oxygen (DO) maintained at 50% of air saturation. Each bioreactor was equipped with an ADI 1030 Bio Controller (Applikon) and an ADI 1035 Bio Console (Applikon). CytodexTM 1 microcarriers were prepared for use following the manufacturer's instructions. Three hours pre-seeding, three bioreactors were each filled with 2 L VP-SFM supplemented with 1% (v/v) CDLC and 2 g/L CytodexTM 1. The bioreactor contents were warmed to 37 0 C with heating blankets and agitated at 60 rpm with single marine impellers.
• the pH setpoints in the three bioreactors were 7.0, 7.2, and 7.4, respectively.
• the culture pH was controlled at the designated levels by the CO 2 percentage in the inlet gas and by the addition of 1 N NaOH solution after the CO 2 percentage in the inlet gas was reduced to 0%.
• all the bioreactors in each experiment were inoculated with cells pooled from multiple RBs. Bioreactor contents were sampled daily for nuclei counting to generate growth curves.
• sucrose phosphate was added to stabilize the virus samples.
• microcarrier beads in the samples were allowed to settle and the culture supernatants collected were stabilized with 10% (v/v) sucrose phosphate. All sucrose phosphate stabilized virus samples were immediately stored at -8O 0 C until analyses.
• T-flask experiments were conducted to investigate the combined effects of time of infection and post-infection cultivation temperature on MEDI-534 production (Fig. 2a & 2b).
• dps days post-seeding
• the number of Vera cells at infection increased from 0.6 x 10 7 cells/flask to 1.7 x 10 7 cells/flask.
• the peak virus titers achieved in cultures infected at three dps (Fig. 2a) were slightly higher that that measured in cultures infected at five dps (Fig. 2b). Therefore, increasing in the Vera cell number at infection did not enhance MEDI-534 production.
• a T-flask experiment assessed the impact of pre-infection culture media on virus production (Table I).
• Vera cultures were supplemented with FBS at the time of seeding.
• Addition of FBS to OptiPROTM SFM almost doubled the cell yields measured at three dps, and increased the maximum virus titers at least fivefold.
• FBS supplementation at the 0.5%, 2% and 5% levels produced comparable cell yields (1.0-1.1) x 10 7 cells/flask) and virus titers ((7.6-7.8) logio TCID 50 /mL), suggesting that increasing the FBS concentration will not further improve cell growth or virus production.
• OptiPROTM SFM is a chemically undefined SFM that has exhibited discernible lot-to-lot variability in supporting Vera cell growth and MEDI-534 production. Since the first and second RB experiments used different lots of OptiPROTM SFM, the higher peak virus titers achieved with OptiPROTM SFM in the second RB experiment ((7.3 ⁇ 0.2) logio TCID5o/mL) versus the first experiment ( Figure 3; (6.8 ⁇ 0.2) logio TCIDso/mL) may have resulted from the lot-to-lot variability in OptiPROTM SFM, the inter-assay variability in the TCID50 assay, or both. Table II. Comparison of cell yield and MEDI-534 production in five distinct pre- infection media
• MEDI-534 production is apparently influenced by the pre- infection culture media; maximum virus titer was detected at four dpi and five dpi for the OptiPROTM ⁇ 0.5% (v/v) FBS and VP-SFM 1% (v/v) CDLC cultures, respectively (Fig. 5b).
• the maximum virus titers (represented as mean ⁇ standard deviation) measured in VP-SFM + 1% (v/v) CDLC, VP-SFM and WME corresponded to (7.5 ⁇ 0.2) log 10 TCID 50 /mL, (7.6 ⁇ 0.1) log 10 TCID 50 /mL and (8.1 ⁇ 0.2) logio TCID 50 /mL, respectively.
• serum-supplemented media were not tested. Consequently, ensuing infections with MEDI- 534 employed WME exclusively as the virus production medium.
• microcarrier process parameters including seeding density, serum- free growth medium, agitation rate, microcarrier type and bead concentration — showed that the current operating conditions (inoculate 1 x 10 5 cells/mL in VP-SFM + 1% CDLC containing 2 g/L CytodexTM 1 with 60 rpm agitation) gave the best results.
• Bioreactor experiments were performed to assess the scalability of the serum- free MEDI-534 production process and evaluate the dependence of cell growth and virus production on culture pH.
• Three parallel bioreactor cultures maintained pH setpoints at 7.0, 7.2 and 7.4, respectively.
• Pre-infection cell growth (Fig. 9a) and virus production profiles (Fig. 9b) in the bioreactor cultures were similar to that observed in the VP-SFM ⁇ 1% CDLC spinner flask cultures (Fig. 8).
• a repeat of this bioreactor experiment generated the same trends with maximum virus titers of 8 logio TCIDso/mL.
• Vero cell line (ATCC CCL-81) was adapted to serum free growth condition and banked.
• Cells derived from the working cell bank (WCB 29AprO3 PN532AC (SF) 03BA01 PJS) were used in all experiments.
• the anchorage-dependent Vero cells were routinely seeded at 5 x 10 cells/mL — in corresponding culture volumes of 35 mL for T-75 flasks, 100 mL for T-225 flasks, and 300 mL for 850cm 2 roller bottles (RBs) — and passaged every 3-4 days.
• spent media were aspirated, cells were rinsed with DPBS and detached from the flasks by treating with appropriate amount of TrypLE solution (Invitrogen, Carlsbad, CA) at 37 0 C.
• Equal volume of lima bean trypsin inhibitor (Worthington Biochemical Corporation, Lakewood, NJ) was added to neutralize TrypLE activity.
• Vera cells All uninfected Vera cells were cultured in VP-SFM (Invitrogen, Carlsbad, CA) supplemented with 4mM L-glutamine and 1% of chemically defined lipid concentrate (CDLC, Invitrogen, Carlsbad, CA), T-flask cultures were maintained in 37°C/5%CO2/95%Rh incubators and RB cultures were placed on a roller bottle apparatus operated at 0.3 rpm in a 37 0 C incubator.
• VP-SFM Invitrogen, Carlsbad, CA
• CDLC chemically defined lipid concentrate
• MEDI-560 is a derivative of cp45, a live, attenuated vaccine candidate for hPIV3 virus.
• MEDI-560 virus seed stocks were stored at -8O 0 C and thawed only immediately before use. T-flask Experiment
• Infection parameters for MEDI-560 production were first screened in T25 flask. T-25 flasks were seeded with 6 x 10 5 Vera cells in 12 mL of VP-SFM supplemented with 4mM L-glutamine and 1% CDLC and maintained in a 37°C/5%CO 2 /95%Rh incubator. Infection was done on day 3 post seeding. Cell counts in two T25 flasks were measured by detaching cells with TrypLE solution and counting cells on Vi-CeIl Cell Viability Analyzer (Beckman Coulter, Miami, FL. Model Vi-CeIl XR).
• the average cell count from the two flasks was used to calculate the amount of virus seed to be used for infection based on the multiplicity of infection (MOI) of 0.01 TCIDso/cell.
• Duplicate T25 flasks were infected with MEDI-560 in three infection media (SFM4MegaVir (Hyclone, Logan, UT), William's medium E (Lonza), and Ex-Cell Vera (SAFC Biosciences JRH), all supplemented with 4mM L-glutamine), two temperatures (32°C and 30 0 C) and harvested at three time points (5, 6, and 7 days post infection or dpi) as shown in Table IV. Cultures were maintained in incubator with 5%CO 2 /95%Rh.
• Vero cells harvested from RB cultures were seeded at 2e5 cells/mL density in Vero cell growth medium (VP-SFM supplemented with 4mM L-GIn and 1% CDLC) with 4 g/L of Cytodex 1 microcarriers or seeded at Ie5 cells/mL with 2 g/L of Cytodex 1 microcarriers (the modified process) in 1.5 to 2L working culture volume in the 3L bioreactors. pH was controlled at 7.1 ⁇ 0.05 by the addition of NaOH solution and sparging Of CO 2 . Temperature was maintained at 37 0 C. Dissolved oxygen floated from 100% during the early culture time and was maintained at 50% of air saturation as the cell grew, by sparging pure oxygen. Agitation speed was set at 125 rpm.
• Infection was done on day 5 post seeding. After collecting a sample, all control loops were disabled and microcarrier beads were allowed to settle for > 30 minutes. Then, a partial medium exchange was performed. Spent growth medium was pumped out and the same volume of fresh infection medium was added through one of the medium addition ports. The extent of medium exchange was 66%. During the infection phase, pH was controlled at 7.1 ⁇ 0.05. Temperature was maintained at 3O 0 C. Dissolved oxygen was maintained at 50% of air saturation and agitation was maintained at 100 rpm. The cells were infected at an MOI (Multiplicity of Infection) of 0.01 TCID 50 /cell. Collection of Infected Culture Samples for Virus Quantification
• Tables V and VI summarize the main differences in the cell growth and infection conditions amongst the three different bioreactor production runs.
• Figure 16 shows the cell growth profiles of the three bioreactor cultures during the pre-infection phase with cell density measured in cells per milliliter (cells/mL).
• Figure 17 shows the cell growth profile with cell density measured in cells per square centimeter (cells/cm 2 ).
• agitation was maintained at 100 rpm during the first day of culturing to allow cells attaching to the microcarrier beads. Agitation was increased to and maintained at 125 rpm from days 2 to 3. However, cell growth in SUB lags behind the 1.5L Applikon bioreactor culture, 3L120407-R10. In an attempt to improve cell growth, agitation was reduced to and maintained at 100 rpm from days 4 to 5 post seeding. Cells in the SUB culture grew similarly to the 1.5L bioreactor culture, 3L120407-R10, during the first day. However, cell growth in SUB was considerably slower than the 1.5L control bioreactor after day 1. It has been shown that agitation rate significantly affect MDCK cell growth in SUB.
• 3L260307-R9 had an initial glutamine concentration of 5.6 mM, higher than the calculated concentration of 4 mM ( Figure 19 A-B). Consumption of glutamine is slower in the SUB than the other two perhaps because it had the lowest cell density. Ammonium ion production profile for 3L120407-R10 and SUB120407 are very similar. 3L260307-R9 produced more ammonium ion, which is likely due to the fact that it had the highest cell density. MEDI-560 Production in Bioreactors
• Virus production in the three bioreactor runs was measured using a TCID50 assay and are summarized in Table VII.
• Vero cells need to be detached from the microcarrier beads upon which they are attached and then attach to freshly added microcarrier beads in order to grow.
• trypsin/EDTA has been used to detach the Vero cells from the microcarrier beads to allow expansion (Sugawara K., et al., Biologicals 2002, 30, 303-314).
• this approach involves removal of culture medium and the utilization of a large amount of trypsin/EDTA. It is cumbersome, costly and increases the risk of contamination.
• a direct bead to bead transfer method was developed to expand Vera cell cultures in bioreactors without using trypsin or using a trypsin-like enzyme to detach the Vera cells from the microcarrier beads. Vera cells were allowed to directly migrate from the microcarrier beads they are attached to freshly added beads in order to expand and grow. To test if cells from a expanded bioreactor culture using the direct bead-to-bead transfer method have comparable virus productivity as the cultures seeded with cells from roller bottles, the following comparative study was performed.
• Vera cell line (ATCC CCL-81) was adapted to serum free growth condition and banked.
• Cells derived from the working cell bank (WCB 29AprO3 PN532AC (SF) 03BA01 PJS) were used in all experiments.
• Vera cells All uninfected Vera cells were cultured in VP-SFM (Invitrogen, Carlsbad, CA) supplemented with 4mM L- glutamine and 1% of chemically defined lipid concentrate (CDLC, Invitrogen, Carlsbad, CA), T-flask cultures were maintained in 37°C/5%CO2/95%Rh incubators and RB cultures were placed on a roller bottle apparatus operated at 0.3 rpm in a 37 0 C incubator.
• VP-SFM Invitrogen, Carlsbad, CA
• CDLC chemically defined lipid concentrate
• MEDI-560 and MEDI-559 were used in the experiment.
• the virus seed stocks were stored at -8O 0 C and thawed only immediately before use.
• Vera cells harvested from RB cultures were seeded at 2e5 cells/mL density in Vera cell growth medium (VP-SFM supplemented with 4mM L-GIn and 1% CDLC) with 4 g/L of Cytodex 1 microcarriers or seeded at Ie5 cells/mL with 2 g/L of Cytodex 1 microcarriers in 1.5 to 2L working culture volume in the 3L bioreactors. pH was controlled at 7.1 ⁇ 0.05 by the addition of NaOH solution and sparging Of CO 2 . Temperature was maintained at 37 0 C. Dissolved oxygen floated from 100% during the early culture time and was maintained at 50% of air saturation as the cell grew, by sparging pure oxygen. Agitation speed was set at 125 rpm.
• Vera cells were cultured in an Applikon bioreactor in 1.5L working volume as described above for 3 days when cell density reached > Ie6 cells/mL.
• For expansion at 1 : 1 split ratio 75OmL to IL of the 3 day culture was transferred to a new bioreactor containing an equal volume of fresh growth medium with 4 g/L of fresh Cytodex 1 beads.
• For expansion at 1 :5 split ratio 30OmL of the Vera cell culture was transferred to a fresh bioreactor containing 1.2L of fresh growth medium with 4 g/L of fresh Cytodex 1 beads.
• Vero cells cultured in 3L bioreactors using the platform process parameters were expanded to 15L bioreactors in two separate experiments described below in Table X.
• Cells were transferred from the 3L bioreactor and expanded to 15L bioreactor at the split ratio of 1 :5. Cells reached > Ie6 nuclei/mL by day 4 or 5 as seen in cultured expanded in 3L bioreactors ( Figure 22).
• Expanded Vero cell cultures in 15L Applikon bioreactors were infected with MEDI-559 or MEDI-560 at the MOI of 0.01. Peak titers obtained in the 15L expanded cultures were comparable to that from the 3L bioreactor cultures (Table X)
• Table X Experiments Design and Results of Expansion Vero Cells from 3L to 15L Applikon Bioreactors
• Vero cells expanded using the bead to bead transfer method described above produced comparable RSV vaccines as the Vero cell culture seeded with cells derived from roller bottles.

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