WO2015017673A1 - Procédés de production de compositions de vaccin contre la grippe - Google Patents

Procédés de production de compositions de vaccin contre la grippe Download PDF

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Publication number
WO2015017673A1
WO2015017673A1 PCT/US2014/049192 US2014049192W WO2015017673A1 WO 2015017673 A1 WO2015017673 A1 WO 2015017673A1 US 2014049192 W US2014049192 W US 2014049192W WO 2015017673 A1 WO2015017673 A1 WO 2015017673A1
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sucrose
filter
viral harvest
filtration
centrifugation
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PCT/US2014/049192
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English (en)
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Weidong Cui
Ashish BEZAWADA
Guangyu Zhu
Phuong TRAN
Loleta CHUNG
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Medimmune, Llc
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Publication of WO2015017673A1 publication Critical patent/WO2015017673A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16251—Methods of production or purification of viral material

Definitions

  • influenza virus vaccines capable of producing a protective immune response specific for such different influenza viruses have been produced for over 50 years and include, e.g., whole virus vaccines, split virus vaccines, surface antigen vaccines and live attenuated virus vaccines.
  • whole virus vaccines split virus vaccines
  • surface antigen vaccines live attenuated virus vaccines.
  • live attenuated virus vaccines have the advantage of being also able to stimulate local mucosal immunity in the respiratory tract.
  • a vaccine comprising a live attenuated virus that is capable of being quickly and economically produced and that is capable of easy storage/transport is thus quite desirable.
  • a vaccine capable of being stored/transported at refrigerator temperatures e.g., approximately 2-8 °C
  • influenza vaccines are propagated in embryonated hen eggs. Although influenza virus grows well in hen eggs, the production of vaccine is dependent on the availability of such eggs. Because the supply of eggs must be organized, and strains for vaccine production selected months in advance of the next flu season, the flexibility of this approach can be limited, and often results in delays and shortages in production and distribution. Additionally, influenza vaccines produced in eggs are typically subjected to several purification processes which can have a negative impact on yield and/or can significantly increase production time. Therefore, methods to improve influenza vaccine production efficiency (e.g., purification efficiency) are desirable to achieve, for example, higher yields and faster production. Summary
  • an influenza virus composition comprising a) clarifying a viral harvest comprising influenza viruses by filtration, thereby producing a clarified viral harvest; b) concentrating the clarified viral harvest, thereby producing a concentrated viral harvest; c) subjecting the concentrated viral harvest to centrifugation, thereby producing a further clarified viral harvest; and d) sterilizing by sterile filtration the further clarified viral harvest, thereby producing a sterilized viral harvest.
  • an influenza virus composition comprising: a) clarifying a viral harvest comprising influenza viruses by filtration, thereby producing a clarified viral harvest; b) subjecting the clarified viral harvest to centrifugation, which centrifugation comprises continuous zonal centrifugation performed over a sucrose density gradient, where the sucrose density gradient is generated by combining a volume of a 60% (w/w) sucrose composition and a volume of a 10% (w/w) sucrose composition, where the volume of the 60% (w/w) sucrose composition is equal to or greater than the volume of the 10% (w/w) sucrose composition; thereby producing a further clarified viral harvest; and c) sterilizing by sterile filtration the further clarified viral harvest, thereby producing a sterilized viral harvest.
  • FIG. 1 shows a flow diagram of an existing live attenuated influenza virus monovalent bulk (LAIV-MB) manufacturing process (left) and a modified LAIV-MB manufacturing process (right).
  • LAIV-MB live attenuated influenza virus monovalent bulk
  • FIG. 2 shows a tangential flow filtration (TFF) set up.
  • FIG. 3 presents equations useful for calculating transmembrane pressure (TMP).
  • FIG. 4 shows a summary of process parameters.
  • FIG. 5 shows a summary of results from TFF concentration.
  • FIG. 6 shows a characterization of TFF concentrated clarified harvest fluid (CHF).
  • FIG. 7 shows permeate flow per unit area vs. time using a GE Healthcare HF cartridge.
  • FIG. 8 shows permeate flow per unit area vs. time using a Spectrum Labs single-use HF cartridge.
  • FIG. 9 shows flux vs. TMP curves for A/South Dakota/6/07.
  • FIG. 10 shows flux vs. TMP curves for B/Malaysia/2506/04.
  • FIG. 1 1 shows flux vs. TMP curves for A/Uruguay/716/07.
  • FIG. 12 shows flux vs. TMP curves for B/Florida/4/2006.
  • FIG. 13 shows a summary of optimal TMP values at different shear rates and corresponding flux rates.
  • FIG. 14 shows average flux and TMP vs. shear rate.
  • FIG. 15 shows a summary of certain process development studies.
  • FIG. 16 shows recovery and material balances after TFF concentration for a supplemental characterization study (phase 1 ).
  • FIG. 17 shows recovery and material balances after TFF concentration for a supplemental characterization study (phase 2).
  • FIG. 18 shows impurity profiles for a supplemental characterization study (phase 1 ).
  • FIG. 19 shows impurity profiles for a supplemental characterization study (phase 2).
  • FIG. 20 shows TFF experimental conditions and average permeate flux.
  • FIG. 21 shows a contour plot of shear rate, TMP and average permeate flux.
  • FIGS. 22A and 22B show potency assay data for GE HF TFF processes.
  • FIG. 23 shows a summary of process parameters for certain pilot-scale studies.
  • FIG. 24 shows impact of loading flow rate on virus recovery in an ultracentrifugation process.
  • FIG. 25 shows certain operational parameters for concentration and ultracentrifugation.
  • FIG. 26 shows doses per batch comparison for A/Victoria strain (TFF vs. non-TFF). TFF batches include the two shown on the right.
  • FIG. 27 shows doses per batch comparison for B/Wisconsin strain (TFF vs. non-TFF).
  • FIG. 28 shows doses per batch comparison for A/California strain (TFF vs. non-TFF).
  • FIG. 29 shows impurity removal data for TFF and non-TFF batches for A/Victoria strain.
  • FIG. 30 shows impurity removal data for TFF and non-TFF batches for A/California strain.
  • FIG. 31 shows impurity removal data for TFF and non-TFF batches for B/Wisconsin strain.
  • FIG. 32 shows filterability and potency change of cold adapted influenza virus (CAIV) after pre- clarification through 47 mm pre-filters.
  • CAIV cold adapted influenza virus
  • FIG. 33 shows a filterability comparison between clarification processes with and without pre- clarification filtration.
  • FIG. 34 shows a potency change comparison between clarification processes with and without pre-clarification filtration.
  • FIG. 35 shows a summary of average potency of five CAIV strains before and after pre- clarification filtration and clarification filtration.
  • FIG. 36 shows potency and virus recovery data for 8 micrometer clarification batches compared to previous commercial batches.
  • FIG. 37 shows filters and operation conditions used for a cross-flow microfiltration (CF-MF) study.
  • CF-MF cross-flow microfiltration
  • FIG. 38 shows permeate flux of CF-MF using a GE 0.45 micrometer hollow fiber cartridge without permeate control.
  • FIG. 39 shows permeate flux of CF-MF using a GE 0.45 micrometer hollow fiber cartridge with permeate control at 45 LMH.
  • FIG. 40 shows permeate flux of CF-MF using a Pall 0.65 micrometer hollow fiber cartridge without permeate control.
  • FIG. 41 shows potency of pooled harvest fluid (PHF) and clarified harvest fluid (CHF) from a hollow fiber CM-MF process.
  • FIG. 42 shows permeate flux of flat sheet cassette TFF (Sartorius 0.45 micrometer flat sheet with permeate control at 3 psi.
  • FIG. 43 shows permeate flux of flat sheet cassette TFF (Millipore 0.65 micrometer flat sheet with permeate control at various pressures).
  • FIG. 44 shows potency of pooled harvest fluid (PHF) and clarified harvest fluid (CHF) from a CF-MF process using flat sheet cassettes.
  • FIG. 45 shows permeate flux of a Pall KLEENPAK 0.65 micrometer capsule with no permeate control in a CF-MF process using ca A convinced/716/07.
  • FIG. 46 shows permeate flux of a Pall KLEENPAK 0.65 micrometer capsule with permeate control at 150 LMH in a CF-MF process using ca A/Uruguay/716/07.
  • FIG. 47 shows potency of pooled harvest fluid (PHF) and clarified harvest fluid (CHF) from a Pall KLEENPAK capsule TFF process.
  • FIG. 48 shows virus potency and filtration throughput of the Millipore MILLISTAK+ DOHC and C0HC filtration process.
  • FIG. 49 shows a summary of linear flux and corresponding flow rates used in certain
  • FIG. 50 shows a summary of filtration throughput at a flux of 250 LMH when filtration end pressure reached 30 psi.
  • FIG. 51 shows a summary of potency change after MILLISTAK+ DOHC depth filtration at a flux of 250 LMH and at a filtration end pressure of 30 psi.
  • FIG. 52 shows an effect of flux on filtration throughput and potency recovery of MILLISTAK+ DOHC depth filtration.
  • FIG. 53 shows throughput of MILLISTAK+ DOHC filtrate on a 0.8/0.45 micrometer
  • FIG. 54 shows potency of monovalent bulk (MVB) stored in a 125 mL bottle and a 1 L bag at 2- 8°C over a period of 14 days.
  • FIG. 55 shows filtration throughput after filtering through a MILLISTAK+ DOHC depth filter at 250 LMH.
  • FIG. 56 shows potency data for filtration through a MILLISTAK+ DOHC depth filter at 250 LMH.
  • FIG. 57 shows sucrose and PBS volume of three gradient buffer compositions.
  • FIG. 58 shows volume of 60% and 10% sucrose and total time for rotor speed maintained at 35,000 rpm.
  • FIG. 59 shows a sucrose gradient generated from gradient buffer 1 (GB 1 ) with 0, 1 , 3, 5 and 12 hour run times at 35,000 rpm on a Hitachi CP40Y ultracentrifuge.
  • FIG. 60 shows sucrose gradients generated from GB 1 , GB 2 and GB 3 with 1 hour run times at 35,000 rpm on a Hitachi CP40Y ultracentrifuge.
  • FIG. 61 shows sucrose gradients generated from GB 1 , GB 2 and GB 3 with 3 hour run times at 35,000 rpm on a Hitachi CP40Y ultracentrifuge.
  • FIG. 62 shows total volume of 60% sucrose recovered with different gradient buffers used and total ultracentrifuge run time.
  • FIG. 63 shows sucrose gradient profiles for certain batches using GB 1 and GB 3.
  • FIG. 64 shows sucrose concentration of centrifuge fractions using GB 3 (1.2 L 60% sucrose, 1 .6 L 10% sucrose and 0.4 L PBS).
  • FIG. 65 shows ultracentrifugation process times.
  • FIG. 66 shows sucrose gradient concentration of GB 1 (1.5 L 60% sucrose, 1 .3 L 10% sucrose and 0.4 L PBS) and total centrifuge run time.
  • FIG. 67 shows sucrose gradient concentration of GB 2 (1.35 L 60% sucrose, 1.45 L 10% sucrose and 0.4 L PBS) and total centrifuge run time.
  • FIG. 68 shows sucrose gradient concentration of GB 3 (1.2 L 60% sucrose, 1 .6 L 10% sucrose and 0.4 L PBS) and total centrifuge run time.
  • FIG. 69 shows a flow diagram of an existing influenza virus production method (left) and an influenza virus production with certain optional modifications outlined in dashed boxes (right).
  • FIG. 70 shows CHF concentration and loading flow rate for various strains.
  • FIG. 71 shows loading flow rate, volume and total process time of concentrated CHF, and the percentage of virus lost in the flow-through.
  • FIG. 72 shows the impact of loading flow rate on virus lost in the flow-through.
  • FIG. 73 shows volume and fluorescent focus assay (FFA) titer of concentrated CHF and centrifuge flow-through.
  • influenza virus production includes one or more purification processes.
  • influenza viruses in a viral harvest may be purified using methods such as filtration and/or centrifugation.
  • Such purification methods reduce or substantially eliminate contaminants (e.g., cellular debris, bioburden, host cell proteins and/or host cell nucleic acid) present in a viral harvest.
  • contaminants e.g., cellular debris, bioburden, host cell proteins and/or host cell nucleic acid
  • Such methods can be costly and time consuming, and/or can have a negative impact viral yield, potency, and/or stability.
  • modified purification methods which can improve purification efficiency and increase viral yield without negatively impacting viral potency or stability.
  • influenza viruses suitable as vaccines, including live attenuated influenza vaccines, such as those suitable for administration in an intranasal vaccine formulation.
  • Influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope lined by a matrix protein.
  • Influenza A and influenza B viruses each contain eight segments of single stranded negative sense RNA.
  • the influenza A genome encodes eleven polypeptides. Segments 1 -3 encode three polypeptides, making up a RNA-dependent RNA polymerase. Segment 1 encodes the polymerase complex protein PB2.
  • the remaining polymerase proteins PB1 and PA are encoded by segment 2 and segment 3, respectively.
  • segment 1 of some influenza strains encodes a small protein, PB1-F2, produced from an alternative reading frame within the PB1 coding region.
  • Segment 4 encodes the hemagglutinin (HA) surface glycoprotein involved in cell attachment and entry during infection.
  • Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, the major structural component associated with viral RNA.
  • Segment 6 encodes a neuraminidase (NA) envelope glycoprotein.
  • Segment 7 encodes two matrix proteins, designated M1 and M2, which are translated from differentially spliced mRNAs.
  • Segment 8 encodes NS1 and NS2, two nonstructural proteins, which are translated from alternatively spliced mRNA variants.
  • the eight genome segments of influenza B encode 1 1 proteins.
  • the three largest genes code for components of the RNA polymerase, PB1 , PB2 and PA.
  • Segment 4 encodes the HA protein.
  • Segment 5 encodes NP.
  • Segment 6 encodes the NA protein and the NB protein. Both proteins, NB and NA, are translated from overlapping reading frames of a biscistronic mRNA.
  • Segment 7 of influenza B also encodes two proteins: M1 and M2. The smallest segment encodes two products, NS1 which is translated from the full length RNA, and NS2 which is translated from a spliced mRNA variant.
  • influenza virus vaccines are produced in embryonated hen eggs (e.g., specific pathogen free (SPF) embryonated hen eggs) using strains of virus selected based on empirical predictions of relevant strains.
  • SPF pathogen free
  • reassortant viruses are produced that incorporate selected hemagglutinin and neuraminidase antigens in the context of an approved attenuated, temperature sensitive, and/or cold-adapted master strain.
  • influenza viruses are recovered and, optionally, inactivated, e.g., using formaldehyde and/or beta-propiolactone; or are used in live attenuated vaccines.
  • influenza vaccine in this manner has several significant concerns. For example, contaminants remaining from the hen eggs can be highly antigenic and/or pyrogenic, and can frequently result in significant side effects upon administration. Thus, certain methods include purification methods that reduce such contaminants and/or replacement of some or all of egg components with animal free media. Virus strains designated for vaccine production typically are selected and distributed months in advance of the next flu season to allow time for production and inactivation of influenza vaccine. Thus, improvements in production efficiency and/or stability at certain temperatures (e.g., refrigerator temperature of about 2-8 °C), are desirable.
  • temperatures e.g., refrigerator temperature of about 2-8 °C
  • Recombinant and reassortant vaccines also may be produced in cell culture (e.g., using a vector system described, for example, in U.S. patent no. 8,012,736) using any appropriate type of host cell.
  • Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, avian or mammalian cells, including human cells.
  • Host cells may include, for example, Vero (African green monkey kidney) cells, BHK (baby hamster kidney) cells, CHO cells, Hep-2 cells, HeLa cells, LLC-MK2 cells, primary chick kidney (PCK) cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) cells, human diploid lung fibroblast cell lines (e.g., MRC-5 and WI-38), human retinoblastoma cell lines, fetal rhesus lung cell lines (e.g., FRhl_2), human kidney cell lines (e.g., PER.C6 and 293 (293T)), and COS cells (e.g., COS1 , COS7 cells).
  • Vero African green monkey kidney
  • BHK baby hamster kidney
  • CHO cells Hep-2 cells
  • HeLa cells LLC-MK2 cells
  • PCK primary chick kidney
  • MDCK Madin-Darby
  • reassortant influenza A and/or influenza B viruses can be produced in cells using an eight plasmid system from cloned cDNA (see e.g., U.S. patent no. 8,012,736). Such reassortants are optionally further amplified in hen eggs.
  • cell cultures are maintained in a system, such as a cell culture incubator, under controlled humidity and C0 2 , at constant temperature using a temperature regulator, such as a thermostat to insure that the temperature does not exceed 35 °C.
  • a temperature regulator such as a thermostat to insure that the temperature does not exceed 35 °C.
  • Such cell culture methods can be modified using methods described herein in whole or part.
  • influenza viruses correspond to one or more influenza B viruses. In some embodiments, the influenza viruses correspond to one or more influenza A viruses.
  • the methods include producing recombinant and/or reassortant influenza viruses capable of eliciting an immune response upon administration, e.g., intranasal administration, to a subject. In some embodiments, the viruses are inactivated prior to administration. In some embodiments, live-attenuated viruses are administered. In certain embodiments, viruses include an attenuated influenza virus, a cold adapted influenza virus, a temperature sensitive influenza virus, or a virus with any combination of these desirable properties. In some embodiments, an influenza virus incorporates an influenza B/Ann
  • Arbor/1/66 strain virus e.g., a cold adapted, temperature sensitive, attenuated strain of B/Ann Arbor/1/66.
  • an influenza virus incorporates an influenza A/Ann Arbor/6/60 strain virus, e.g., a cold adapted, temperature sensitive, attenuated strain of A/Ann Arbor/6/60.
  • viruses are artificially engineered influenza viruses incorporating one or more substituted amino acids which influence certain biological properties of a donor strain, e.g., ca A/Ann Arbor/6/60 or ca B/Ann Arbor/1/66.
  • Such substituted amino acids may correspond to unique amino acids of ca A/Ann Arbor/6/60 or ca B/Ann Arbor/1/66, e.g., in an A strain virus: PB1 391 (K391 E), PB1 581 (E581 G), PB1 661 (A661 T), PB2 265 (N265S) and NP 34 (D34G); and, in a B strain virus: PB2 630 (S630R); PA 431 (V431 M); PA 497 (Y497H); NP 55 (T55A); NP 114 (V1 14A); NP 410 (P410H); NP 509 (A509T); M1 159 (H159Q) and M1 183 (M 183V).
  • a strain virus PB1 391 (K391 E), PB1 581 (E581 G), PB1 661 (A661 T), PB2 265 (N265S) and NP 34 (D34G); and, in a B strain
  • a or B viruses may already have the recited residues at the indicated positions. In such instances, the substitutions can be made such that the resulting virus will have all of the above substitutions.
  • Reassortant viruses may be produced by introducing vectors including the six internal genes of a first viral strain selected for its favorable properties regarding vaccine production, in combination with the genome segments encoding the surface antigens (HA and NA) of a selected, e.g., pathogenic strain. Such reassortants are sometimes referred to as 6:2 reassortants. In some instances, seven complementary gene segments (i.e., 6 internal genes and 1 surface antigen) of a first strain are introduced in combination with either an HA or NA encoding segment. Such reassortants are sometimes referred to as 7:1 reassortants. In certain instances, an HA segment can be selected from a pathogenically relevant influenza A strain (e.g., H1 , H3) or influenza B strain.
  • a pathogenically relevant influenza A strain e.g., H1 , H3
  • the HA segment can be selected from an emerging pathogenic influenza strain such as an H2 influenza strain (e.g., H2N2), an H5 influenza strain (e.g., H5N1 ) or an H7 influenza strain (e.g., H7N7).
  • the NA segment can be selected from a pathogenically relevant or emerging pathogenic influenza A strain or influenza B strain, and may be selected from any NA subtype (e.g., N1 , N2, N3 , N7).
  • the internal gene segments are derived from the influenza B/Ann Arbor/1/66, A/Ann Arbor/6/60 or other suitable master strain.
  • the master strain is selected from the group consisting of A/Ann Arbor/6/60, B/Ann Arbor/1/66, PR8,
  • the master strain is derived from a strain selected from the group consisting of A/Ann Arbor/6/60, B/Ann Arbor/1/66, PR8, B/Leningrad/14/17/55, LEN-B14/5/1 , B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55 and B/England/2608/76.
  • the master strain may be derived from any of the above strains by the introduction of one or more amino acid substitutions that confer a desirable phenotype such as attenuation, temperature sensitivity and/or cold-adaptation, as describe above and as described, for example in U.S. patent no. 8,354, 1 14.
  • temperature sensitive indicates that the virus exhibits a 100 fold or greater reduction in titer at a higher temperature, e.g., 39°C relative to a lower temperature, e.g., 33°C for influenza A strains, and that the virus exhibits a 100 fold or greater reduction in titer at a higher temperature, e.g., 37°C relative to a lower temperature, e.g., 33°C for influenza B strains.
  • the term “cold adapted” indicates that the virus exhibits a higher growth rate at a lower temperature, e.g., 25°C within 100 fold of its growth at a higher temperature, e.g., 33°C.
  • the term “attenuated” indicates that the virus replicates in the upper airways of ferrets but is not detectable in lung tissues, and does not cause influenza-like illness in the animal. Growth indicates viral quantity as indicated by titer, plaque size or morphology, particle density or other measures known in the art.
  • Influenza vaccine production typically includes multiple manufacturing steps including, for example, co-infection, reassortment, selection and cloning of reassortants, purification and expansion of reassortants, harvesting, purification of a viral harvest, stabilization, and potency/sterility assays.
  • Various aspects of vaccine production are described, for example, in U.S. patent no. 7,262,045; U.S. patent no. 8,247,207; U.S. patent no. 8,012,736; U.S. patent no. 7,465,456; U.S. patent no. 8,354, 1 14; U.S. patent no. 7,601 ,356; U.S. patent no. 8,357,376; U.S. patent no.
  • Viruses e.g., reassortant influenza viruses grown in eggs or host cells
  • Viruses may be harvested (i.e., removed from the eggs or host cells) and subjected to one or more purification processes which may include, for example, clarification, concentration, centrifugation and/or sterilization.
  • Certain aspects of viral purification can be modified to increase production efficiency (e.g., higher yield, faster production, less waste, and the like). Such modified aspects of viral purification are described herein.
  • purification of a viral harvest comprises a) subjecting a concentrated viral harvest to centrifugation, thereby producing a clarified viral harvest; and, optionally, b) sterilizing by sterile filtration the clarified viral harvest, thereby producing a sterilized viral harvest.
  • a viral harvest herein generally comprises influenza viruses.
  • a viral harvest is initially clarified before or during a concentration step.
  • purification of a viral harvest comprises a) concentrating a viral harvest, thereby producing a concentrated viral harvest; b) subjecting the concentrated viral harvest to centrifugation, thereby producing a clarified viral harvest; and, optionally, c) sterilizing by sterile filtration the clarified viral harvest, thereby producing a sterilized viral harvest.
  • a viral harvest is initially clarified before or during concentration.
  • purification of a viral harvest comprises a) concentrating a viral harvest, where the viral harvest optionally is a clarified viral harvest, thereby producing a concentrated viral harvest; and b) subjecting the concentrated viral harvest to centrifugation, thereby producing a clarified viral harvest.
  • purification of a viral harvest comprises a) clarifying a viral harvest comprising influenza viruses, thereby producing a clarified viral harvest; b) concentrating the clarified viral harvest, thereby producing a concentrated viral harvest; c) subjecting the concentrated viral harvest to centrifugation, thereby producing a further clarified viral harvest; and, optionally, d) sterilizing by sterile filtration the further clarified viral harvest, thereby producing a sterilized viral harvest.
  • purification of a viral harvest comprises a) clarifying a viral harvest comprising influenza viruses, thereby producing a clarified viral harvest; b) subjecting the clarified viral harvest to centrifugation, which centrifugation comprises continuous zonal centrifugation performed over a sucrose density gradient, where the sucrose density gradient is generated by combining a volume of a 60% (w/w) sucrose composition and a volume of a 10% (w/w) sucrose composition, where the volume of the 60% (w/w) sucrose composition is equal to or greater than the volume of the 10% (w/w) sucrose composition; thereby producing a further clarified viral harvest; and, optionally, c) sterilizing by sterile filtration the further clarified viral harvest, thereby producing a sterilized viral harvest.
  • a viral purification process comprises an initial clarification of a viral harvest.
  • Methods useful for the initial clarification of a viral harvest include, but are not limited to, centrifugation, dialysis, and membrane filtration, which includes, but is not limited to, methods such as single pass, dead-end, direct flow filtration (DFF) in which liquid flows directly through the filter medium, depth filtration, and crossflow or tangential flow filtration (TFF) in which liquid flows tangential to (along) the surface of the membrane.
  • DFF direct flow filtration
  • TFF crossflow or tangential flow filtration
  • Membranes for use in filtration applications are available from commercial sources. Certain methods for the initial clarification of a viral harvest, and modifications thereto, are described herein in Example 2.
  • a viral harvest is clarified by filtration.
  • Filtration typically involves use of membranes which generally are defined by the size of the material they remove from a solution. For example, from the smallest to largest pore size, filtration membranes include reverse osmosis membranes, nanofiltration membranes, ultrafiltration membranes, and microfiltration membranes. Filtration using such membranes separates molecules according to their molecular weight by using membranes with specific pore sizes. For example, filtration with reverse osmosis membranes that have pore sizes less than 0.001 micrometers generally is intended for separation of molecules that have a molecular weight less than 200 Daltons.
  • Filtration with nanofiltration membranes that have pore sizes from 0.001 - 0.008 micrometers, inclusive generally is intended for separation of molecules that have a molecular weight from 200 Daltons to 15 kilodaltons (kD, kDa) inclusive.
  • Filtration with ultrafiltration membranes that have pore sizes from 0.005 - 0.1 micrometers, inclusive generally is intended for separation of molecules that have a molecular weight from 5 kDa - 300 kDa, inclusive.
  • Filtration with microfiltration membranes that have pore sizes from 0.05 - 3.0 micrometers, inclusive is intended for separation of molecules that have a molecular weight from 100 kDa - 3000 kDa and larger.
  • membrane-filtration can separate molecules of interest (e.g., viruses) from other cellular components based on size exclusion by utilizing membranes that have a particular Molecular Weight Cut-Off (MWCO) that is determined by the pore size of the membrane.
  • MWCO Molecular Weight Cut-Off
  • the MWCO also called Nominal Molecular Weight Limit (NMWL) or Nominal Molecular Weight Cut- Off (NMWCO)
  • NMWL Nominal Molecular Weight Limit
  • NMWCO Nominal Molecular Weight Cut- Off
  • the MWCO is defined as the molecular weight of the molecule that is 90% retained by the membrane.
  • the MWCO may not be an exact metric, but is nevertheless a useful metric and is commonly employed by filter manufacturers.
  • Membranes may be used as flat sheets or in a spirally wound configuration, for example. Hollow fibers may also be used depending on the type of filtration method. Any number of potential membrane materials may be used including, but not limited to, regenerated cellulose, polyether sulfone (which may or may not be modified to alter its inherent hydrophobicity), polyvinylidene fluoride (PVDF), and ceramic and metal oxide aggregates, as well as polycarbonate, polypropylene, polyethylene and PTFE (TEFLON®). In some embodiments, combinations of filtration methods and membrane types may be used. The capacity of certain filters, columns, etc., comprising separation membranes can be adjusted depending on the volume and/or concentration of material being processed.
  • clarification of a viral harvest comprises use of one or more filter species.
  • a filter species may be distinct from another filter species based on pore size, membrane material, filter manufacturer, membrane area, layers of membrane, filter capacity and the like or a combination thereof.
  • clarification of a viral harvest comprises use of at least two filter species.
  • clarification of a viral harvest may comprise use of at least three filter species, at least four filter species, at least five filter species, at least six filter species or more.
  • clarification of a viral harvest comprises use of at least three filter species.
  • one or more filter species is a pre-filter.
  • a pre-filter generally is used in a filtration process prior to the use of one or more other filter species (e.g., downstream filters), and can remove certain cell debris components from a viral harvest (e.g., host cell debris).
  • a pre-filter may have a pore size that is larger than one or more downstream filters.
  • a pre-filter has a pore size ranging from about 3 microns to about 20 microns.
  • a pre-filter may have a pore size of about 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 microns.
  • a pre-filter has a pore size of about 8 microns.
  • a pre-filter has a pore size of about 10 microns.
  • Filtration throughput refers to the passage of a solution (e.g., viral harvest fluid) through a filter (e.g., one or more clarification filters such as the one or more clarification filters downstream of a pre-filter) for a certain duration, flow rate and/or volume of solution passaged before filtration slows or ceases due to, for example, filter clogging.
  • a filter e.g., one or more clarification filters such as the one or more clarification filters downstream of a pre-filter
  • filtration throughput may be increased when a pre-filter is used relative to filtration throughput when a pre-filter is not used.
  • filtration throughput is increased by at least about 1 .5-fold.
  • filtration throughput may be increased by at least about 2-fold, 2.5- fold, 3-fold, 3.5-fold, 4-fold or more.
  • filtration throughput is increased by at least about 3-fold.
  • one or more other filter species is used after a pre-filter to remove other cell debris components, bacteria, other bioburden, and the like from a viral harvest.
  • the one or more other filter species have pore sizes that are smaller than a pre- filter.
  • the one or more other filter species are selected from filters having pore sizes ranging from about 0.2 microns to about 3.0 microns.
  • the one or more other filter species may have pore sizes of about 0.3, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1.1 , 1.2, 1 .3, 1 .4, 1 .5, 1.6, 1.7, 1.8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 microns.
  • one or more other filter species have a pore size of 1 .2 microns.
  • one or more other filter species have a pore size of 0.8 microns.
  • one or more other filter species have a pore size of 0.45 microns.
  • one or more other filter species comprise one or more membrane layers. In some embodiments, one or more other filter species comprise two membrane layers (e.g., paired filters). For example, one or more other filter species may comprise two membrane layers, each having a different pore size (e.g., 0.8 microns and 0.45 microns). In some embodiments, the one or more other filter species comprise one or more filters having a pore size of 1 .2 microns and one or more filters having two membrane layers, each having a pore size of 0.8 microns and 0.45 microns.
  • clarification of a viral harvest comprises use of one or more membrane filters.
  • Membrane filters (sometimes referred to as screen filters) generally have pores of a certain size that allow certain particles to pass through.
  • clarification of a viral harvest comprises use of one or more depth filters.
  • Depth filters generally include filters that comprise a porous filtration medium (e.g., fibers, or fibrous materials) to retain particles throughout the medium, rather that just on the surface of the medium. Such filters often can retain a large mass of particles before becoming clogged.
  • a depth filter is a stacked depth filter.
  • clarification of a viral harvest comprises use of a combination of one or more membrane filters and one or more depth filters.
  • a depth filter is used prior to a membrane filter. In some embodiments, a depth filter is used after a membrane filter. In some embodiments, a depth filter is used after a first membrane filter and before a second membrane filter. In some embodiments, one or more depth filters is used as a pre-filter. In some embodiments, one or more depth filters is used in combination with one or more paired membrane filters (e.g., 0.8/0.45 micron filter).
  • filtration throughput is increased when a depth filter is used relative to filtration throughput when a depth filter is not used. In some embodiments, filtration throughput is increased by at least about 1.5-fold. For example, filtration throughput may be increased by at least about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold or more. In some embodiments, filtration throughput is increased by at least about 3-fold.
  • a viral purification process comprises concentration of a viral harvest.
  • a viral purification process may comprise concentration of a clarified viral harvest, such as a viral harvest clarified by a filtration process described above.
  • Methods useful for concentrating a viral harvest include, but are not limited to, dialysis, tangential flow filtration (TFF), ultrafiltration (UF) and diafiltration (DF; continuous or
  • TFF may incorporate both UF, which can be used to concentrate, and DF, which can be used to exchange buffers.
  • Tangential flow filtration sometimes referred to as crossflow filtration, is a process whereby a feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) is recirculated back to the feed reservoir.
  • TFF Tangential flow filtration
  • the use of TFF, in certain instances, may result in additional purification by the fractionation process that washes smaller molecules (e.g., contaminants) through a membrane and leaves larger molecules of interest (e.g., virus) in the retentate.
  • a viral purification process may incorporate the use of any suitable TFF system known in the art and any TFF components (e.g., cartridges) by various manufacturers.
  • TFF components e.g., cartridges
  • Non- limiting examples of certain TFF systems and components useful for concentrating a viral harvest are described herein in Example 1.
  • a TFF process comprises use of a hollow fiber cartridge (i.e., a filter membrane composed of a collection of hollow fibers (e.g., polysulphone)).
  • a hollow fiber cartridge i.e., a filter membrane composed of a collection of hollow fibers (e.g., polysulphone)
  • the hollow fiber cartridge has a pore size ranging from about 500 kD to about 750 kD. In some embodiments, the hollow fiber cartridge has a pore size of about 500 kD. In some embodiments, the hollow fiber cartridge has a pore size of about 750 kD. In some
  • the hollow fiber cartridge has a membrane with a nominal internal diameter (ID) of about 0.5 mm and pore size of about 500 kD.
  • ID nominal internal diameter
  • product flows tangentially across the surface of the hollow fiber filter membranes at a defined flow rate.
  • the inlet and outlet pressures are controlled to provide a constant differential pressure. This differential pressure enables concentration whereby waste and impurities (which generally are smaller than 500 kD or smaller than 750 kD) pass through the pores and enter the waste stream (permeate) while virus particles (which are typically bigger than 500 kD or 750 kD) are retained in the product solution.
  • waste material which is generally less than 500 kD or 750 kD
  • Virus particles which are larger than 500 kD or 750 kD
  • an ultracentrifuge for example, once the concentration process is complete.
  • a TFF process typically includes several operational parameters, some of which may be modified to achieve an optimal concentration process. Certain operational parameters are described below and non-limiting examples of modifications thereof are described herein in Example 1.
  • Shear rate (s "1 ) is the ratio of velocity and distance. Shear rate can be controlled, and an increased shear rate typically ensures the efficiency of the filter is maintained over the lifetime of a concentration process. An optimized shear should prevent filter blockage and thus ensures effective concentration times are maintained, although shear may be dependent on the limits of certain equipment and changes in the nature of the product.
  • the shear rate for a hollow fiber (HF) cartridge for example, can be calculated based on the flow rate through the fiber lumen as follows:
  • a TFF process is performed using a shear rate ranging from about 8,000 s "1 to about 22,000 s ' In some embodiments, a TFF process is performed using a shear rate ranging from about 10,000 s "1 to about 16,000 s ' For example, a TFF process may be performed at a shear rate of about 1 1 ,000 s "1 ; 12,000 s "1 ; 13,000 s “1 ; 14,000 s "1 ; or 15,000 s '
  • Transmembrane pressure is the average applied pressure from the feed to the filtrate side of the membrane.
  • An optimal TMP generally ensures the rate of concentration is maximized and controlled within an acceptable timeframe and within certain physical limits of the equipment, and prevents damage to the filter or the virus that is being concentrated.
  • TMP may be measured as pounds per square inch (psi) or pounds per square inch gage (psig) and can be calculated as follows:
  • TMP [(P in + P ret )/2] - P perm ,
  • a TFF process is performed using a transmembrane pressure (TMP) ranging from about 10 psig to about 20 psig.
  • TMP transmembrane pressure
  • a TFF process may be performed at a TMP of about 1 1 psig, 12 psig, 13 psig, 14 psig, 15 psig, 16 psig, 17 psig, 18 psig, or 19 psig.
  • Flux (filtrate flux rate) is the volume of the permeate flowing through the defined filter membrane area during a given time and is expressed as LMH (liters per square meter per hour).
  • a TFF process is performed at a filtrate flux rate of at least about 25 LMH.
  • a TFF process may be performed at a filtrate flux rate of about 30 LMH, 40 LMH, 50 LMH, 60 LMH, 70 LMH, 80 LMH, 90 LMH, 100 LMH, 150 LMH, 200 LMH or more.
  • Load factor is defined as the ratio of feed volume to filter surface area and is expressed as L/m 2 (liters per square meter).
  • a TFF process is performed using a load factor ranging from about 50 L/m 2 to 100 L/m 2 of clarified viral harvest per square meter.
  • a TFF process may be performed at a load factor of about 55 L/m 2 , 60 L/m 2 , 70 L/m 2 , 80 L/m 2 , or 90 L/m 2 .
  • TFF systems may be run so as to maintain a constant filtrate flux rate (i.e., flux) or to maintain a constant transmembrane pressure (TMP).
  • flux and/or TMP may be regulated, for example, to prevent membrane fouling.
  • a viral harvest (e.g., clarified viral harvest) is concentrated (e.g., by a TFF process) at least about 2-fold (e.g., 200 L clarified viral harvest concentrated to 100 L clarified viral harvest).
  • a clarified viral harvest may be concentrated about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold or more.
  • the volume of clarified viral harvest that can be purified is greater relative to the volume of clarified viral harvest that can be purified in a method that does not comprise concentrating a clarified viral harvest.
  • certain starting volumes of a viral harvest e.g., clarified viral harvest
  • concentration e.g., by a TFF process
  • 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L or more clarified viral harvest may be concentrated in a viral purification method provided herein.
  • a viral purification process comprises concentrating a viral harvest prior to centrifugation.
  • viral yield is increased relative to viral yield of a method that does not comprise concentrating a clarified viral harvest prior to centrifugation.
  • a viral yield may be increased al least about 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
  • the amount of clarified viral harvest i.e., the number of virus particles in a viral harvest
  • the amount of clarified viral harvest subjected to centrifugation is greater (e.g., less viral harvest is wasted or discarded) relative to the amount of clarified viral harvest subjected to centrifugation in a method that does not comprise concentrating the clarified viral harvest prior to centrifugation.
  • the amount of clarified viral harvest subjected to centrifugation may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% greater relative to the amount of clarified viral harvest subjected to centrifugation in a method that does not comprise
  • all or substantially all (e.g., about 90% or greater) of the clarified viral harvest is subjected to centrifugation.
  • a viral purification process comprises centrifugation of a viral harvest. In some embodiments, a clarified viral harvest is subjected to centrifugation. In some
  • a concentrated viral harvest is subjected to centrifugation.
  • Centrifugation may include continuous zonal centrifugation, which may also be referred to as ultracentrifugation, continuous flow zonal centrifugation, continuous flow zonal ultracentrifugation, and the like. Any centrifuge device suitable for the methods described herein may be used. Non-limiting examples of certain centrifugation devices, processes and modifications thereto are described herein in Example 3.
  • Centrifugation may be performed at any temperature, rotor speed and/or duration suitable for virus purification.
  • centrifugation may be performed at room temperature or below.
  • centrifugation may be performed at about 2 °C to about 25 °C.
  • centrifugation may be performed at about 2 °C to about 14 °C.
  • centrifugation may be performed at about 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 1 1 °C, 12 °C, or 13 °C.
  • centrifugation performed at a speed of about 25,000 RPM to about 50,000 RPM.
  • centrifugation performed at a speed of about 30,000 RPM to about 40,000 RPM.
  • centrifugation may be performed at a speed of about 31 ,000 RPM, 32,000 RPM, 33,000 RPM, 34,000 RPM, 35,000 RPM, 36,000 RPM, 37,000 RPM, 38,000 RPM, or 39,000 RPM.
  • centrifugation has a run time of at least about 6 hours.
  • centrifugation may have a run time of about 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12, hours, 13 hours, 14 hours, 15 hours or longer.
  • centrifugation has a run time of at least about 9 hours.
  • centrifugation has a run time of at least about 12 hours.
  • a method comprises loading a viral harvest (e.g., concentrated viral harvest, clarified viral harvest) into a centrifuge device at a particular loading flow rate.
  • a viral harvest e.g., concentrated viral harvest, clarified viral harvest
  • adjusting the loading flow rate e.g., decreasing
  • the loading flow rate is lower relative to a loading flow rate for centrifugation in a method that does not comprise concentrating the clarified viral harvest prior to centrifugation.
  • the loading flow rate may be less than about 200 mL/min, 190 mL/min, 180 mL/min, 170 mL/min, 160 mL/min, 150 mL/min, 140 mL/min, 130 mL/min, 120 mL/min, 1 10 mL/min or 100 mL/min.
  • the loading flow rate ranges from about 120 mL/min to about 160 mL/min.
  • the loading flow rate ranges from about 140 mL/min to about 180 mL/min. In some
  • the loading flow rate is about 180 mL/min, 170 mL/min, 160 mL/min, 150 mL/min, 140 mL/min, 130 mL/min, or 120 mL/min.
  • centrifugation comprises continuous zonal centrifugation. In some embodiments, centrifugation is performed over a sucrose density gradient. In some
  • the sucrose density gradient is a 0% to 100% sucrose gradient. In some embodiments, the sucrose density gradient is a 0% to 90% sucrose gradient. In some embodiments, the sucrose density gradient is a 0% to 80% sucrose gradient. In some embodiments, the sucrose density gradient is a 0% to 70% sucrose gradient. In some embodiments, the sucrose density gradient is a 0% to 60% sucrose gradient. In some embodiments, the sucrose density gradient is a 10% to 100% sucrose gradient. In some embodiments, the sucrose density gradient is a 10% to 90% sucrose gradient. In some embodiments, the sucrose density gradient is a 10% to 80% sucrose gradient. In some embodiments, the sucrose density gradient is a 10% to 70% sucrose gradient.
  • the sucrose density gradient is a 10% to 60% sucrose gradient. In some embodiments, a sucrose density gradient is generated using two different sucrose concentrations. In some embodiments the sucrose is in a buffer. In a specific embodiment, the sucrose is in a phosphate buffer (e.g., a phosphate-glutamate buffer, or PBS) In some embodiments, a sucrose density gradient is generated using two different sucrose
  • a sucrose density gradient is generated using 1 ) a sucrose concentration of 60% and 2) a sucrose concentration of 10%.
  • a sucrose density gradient is generated using a volume of a 10% sucrose (w/w) composition that is greater than the volume of a 60% sucrose (w/w) composition.
  • a sucrose density gradient is generated using equal or substantially equal volumes of a 60% sucrose (w/w) composition and a 10% sucrose (w/w) composition.
  • a sucrose density gradient is generated using a volume of a 60% sucrose (w/w) composition that is greater than the volume of a 10% sucrose (w/w) composition.
  • a sucrose density gradient may be generated where the volume of a 60% sucrose (w/w) composition is at least about 1 .1 , 1 .2, 1.3, 1.4, 1.5, 1 .6, 1.7, 1 .8, 1 .9, 2.0 or more times greater than the volume of a 10% sucrose (w/w) composition.
  • a sucrose density gradient is generated where the volume of a 60% sucrose (w/w) composition is at least about 1 .1 times greater than the volume of a 10% sucrose (w/w) composition.
  • a sucrose density gradient is generated using volumes of a 60% sucrose (w/w) composition, a 10% sucrose (w/w) composition and buffer (e.g., PBS) at a ratio of 1 .3-1.6 to 1 .2- 1.5 to 0.4, respectively.
  • a sucrose density gradient is generated using volumes of a 60% sucrose (w/w) composition, a 10% sucrose (w/w) composition and PBS at a ratio of 1.5 to 1 .3 to 0.4, respectively.
  • a viral harvest (e.g., a clarified viral harvest, a further clarified viral harvest) is collected from the sucrose density gradient at certain gradient coordinates.
  • a viral harvest (e.g., a clarified viral harvest, a further clarified viral harvest) is collected from the sucrose density gradient at gradient coordinates between about 30% to about 55% sucrose.
  • a viral harvest (e.g., a clarified viral harvest, a further clarified viral harvest) is collected from the sucrose density gradient at gradient coordinates between about 34-36% to about 48-50% sucrose.
  • a viral harvest (e.g., a clarified viral harvest, a further clarified viral harvest) is collected from the sucrose density gradient at gradient coordinates between about 35% to about 49% sucrose.
  • centrifuge peak fractions are pooled and/or diluted as described, for example in U.S. Patent No. 8,247,207. Peak fractions may be identified, for example, by a hemagglutinin assay.
  • sucrose concentration is determined for a peak fraction or peak fraction pool according to, for example, a refractive index (Rl) reading. Peak fractions or peak fraction pools also may be sampled for potency. Fractions or pools with certain sucrose concentrations and/or potencies may be diluted by addition of a buffer, in certain embodiments.
  • Buffers can be sterile and/or cold (e.g., 2-8°C) and may include, for example, a phosphate buffer.
  • a phosphate buffer e.g., a phosphate-glutamate buffer (PBG buffer; e.g., at pH 7.2) may be used to dilute peak fractions or peak fraction pools.
  • PBG buffer components may be added to a peak fraction or peak fraction pool to achieve final
  • PBG buffer components may be added to a peak fraction or peak fraction pool to achieve a final concentration of about 0.2 M sucrose, about 0.1 M phosphate, and about 0.005 M glutamate.
  • Dilution of a peak fraction or peak fraction pool may be about a 1 :2 dilution, a 1 :3 dilution, a 1 :4 dilution, a 1 :5 dilution, 1 :6 dilution, a 1 :7 dilution, a 1 :8 dilution, a 1 :9 dilution, or a 1 :10 dilution, for example.
  • a diluted centrifuge peak fraction or a diluted centrifuge peak fraction pool can be sampled for potency and/or bioburden, as described, for example in U.S. Patent No. 8,247,207.
  • bioburden as described, for example in U.S. Patent No. 8,247,207.
  • dilution may be performed simultaneously with or prior to addition of a stabilizer, as described below. In certain embodiments, dilution is performed before sterilization, as described below.
  • a viral harvest (e.g., further clarified viral harvest) is sterilized after centrifugation. Sterilization can be performed as a terminal filtration step and/or using one or more other sterilization methods. Methods useful for the sterilization of vaccine components (e.g., viruses) include, but are not limited to, irradiation, filtration, chemical treatment, and other suitable procedures.
  • a further clarified viral harvest is sterilized by filtration.
  • Filtration methods useful for sterilization include, but are not limited to, single pass, dead-end, direct flow filtration (DFF) and tangential flow filtration (TFF), some of which are described above, using, for example, one or more sterilization grade filters (e.g., pore size of about 0.2 microns).
  • a method provided herein further comprises combining a sterilized viral harvest with a stabilizer.
  • the sterilized viral harvest is combined with a stabilizer to obtain a final concentration of 6-8% sucrose weight/volume (w/v), 1 -2% arginine w/v, 0.05-0.1 % monosodium glutamic acid w/v and 0.5-2% gelatin hydrolysate.
  • the final concentration is 6.84% sucrose weight/volume (w/v), 1.21 % arginine w/v, 0.094% monosodium glutamic acid w/v, and 1 % gelatin hydrolysate.
  • the sterilized viral harvest is combined with a stabilizer to obtain a final concentration of 6-8% sucrose weight/volume (w/v), 1-2% arginine w/v, and 0.5-2% gelatin hydrolysate. In some embodiments, the final concentration is 6.84% sucrose weight/volume (w/v), 1.21 % arginine w/v, and 1 % gelatin hydrolysate. In some embodiments, a viral harvest is combined with a stabilizer (or certain components of a stabilizer) prior to, during or after sterilization.
  • a method provided herein further comprises blending a sterilized viral harvest with at least one other sterilized viral harvest, thereby producing a blended viral harvest.
  • a sterilized viral harvest is blended with two other sterilized viral harvests, thereby producing a trivalent blended viral harvest.
  • a trivalent blended viral harvest may comprise two influenza A viruses and one influenza B virus, or may comprise one influenza A virus and two influenza B viruses.
  • a sterilized viral harvest is blended with three other sterilized viral harvests, thereby producing a quadrivalent blended viral harvest.
  • a quadrivalent blended viral harvest may comprise two influenza A strains and two influenza B strains; three influenza A strains and one influenza B strain; or one influenza A strain and three influenza B strains.
  • liquid vaccines e.g., live attenuated influenza virus vaccines
  • formulations thereof that are substantially stable at temperatures ranging from 4°C and 8°C.
  • liquid vaccine formulations produced by the methods herein are substantially stable at temperatures ranging from 2-8°C or at 4°C for a period of at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 36 months, or at least 48 months, in that there is an acceptable loss of potency (e.g., influenza virus potency loss) at the end of such time, for example, a potency loss of between 0.5-1.0 logs or a potency loss of less than 10%, or less than 20%, or less than 30%, or less than 40%, or less than 50%, or less than 60%, or less than 70%, or less than 80%, or less than 90%
  • a liquid vaccine formulation produced by the methods herein has a potency loss of less than 1 .0 logs when stored for a period of 3 months at 4°C to 8°C. In some embodiments, a liquid vaccine formulation produced by the methods herein has a potency loss of less than 1.0 logs when stored for a period of 6 months at 4°C to 8°C. In some embodiments, a liquid vaccine formulation produced by the methods herein has a potency loss of less than 1 .0 logs when stored for a period of 12 months at 4°C to 8°C.
  • a liquid vaccine formulation produced by the methods herein has a potency loss of less than 1 .0 logs when stored for a period of 3 to 12 months at 4°C to 8°C. In some embodiments, a liquid vaccine formulation produced by the methods herein has a potency loss of less than 1 .0 logs when stored for a period of 6 to 12 months at 4°C to 8°C. In some embodiments, a liquid vaccine formulation produced by the methods herein has a potency loss of less than 1 .0 logs when stored for a period of 3 to 6 months at 4°C to 8°C. Viral potency (and potency loss) can be measured, for example, by TCID 50 or
  • FFA Fluorescent Focus Assay
  • liquid vaccine formulations comprise live influenza viruses.
  • formulations may comprise one or more of the following: an attenuated influenza virus, a cold-adapted influenza virus, a temperature-sensitive influenza virus, an attenuated cold- adapted temperature sensitive influenza virus, an influenza A virus, and an influenza B virus.
  • liquid vaccine formulations comprise one or more stabilizers which may include, for example, one or more of the following: arginine (e.g., 0.5-1 %, 1 -2%; 1 %; 1 .2%; 1.5%, 0.75-2%); poloxamer; sucrose (e.g., 2-8%; 2%; 6-8%; 3%; 4%; 5%; 6%; 7%, or 8%); hydrolyzed gelatin (e.g., 1 %; 0.5-2%; 1.5%; 0.5%; 0.75%); and glutamate (e.g., 0.05-0.1 %, 0.02-0.15%, 0.03%, 0.04%, 0.06%, 0.02-0.3%, or 0.094%).
  • arginine e.g., 0.5-1 %, 1 -2%; 1 %; 1 .2%; 1.5%, 0.75-2%)
  • poloxamer e.g., 2-8%; 2%; 6-8%; 3%; 4%; 5%; 6%;
  • Certain formulations also may comprise one or more buffers such as, for example, one or more of the following: phosphate buffer (mono or dibasic or both) (e.g., 10-200mM, pH 7-7.5; 100 mM, pH 7.2; 100 mM, pH 7- 7.3); potassium phosphate (e.g., at least 50 mM, or at least 100mM, or at least 200mM, or at least 250mM); and histidine buffers (e.g., 25 - 50 mM histidine, pH 7-7.5; 50- 100mM histidine, pH 7-7.5; at least 50 mM histidine, or at least 100mM histidine, or at least 200mM histidine, or at least 250mM histidine).
  • vaccine formulations comprise one or more of the following in the final formulations: sucrose: 6-8% weight/volume (w/v); arginine monohydrochloride 1 -2% w/v;
  • glutamic acid monosodium monohydrate 0.05-0.1 % w/v; gelatin hydrolysate, porcine Type A (or other sources) 0.5-2% w/v; potassium phosphate dibasic 1-2%; and potassium phosphate monobasic 0.25-1 % w/v.
  • vaccine formulations comprise one or more of the following: sucrose: 6.84% weight/volume (w/v); arginine monohydrochloride 1 .21 % w/v; glutamic acid, monosodium monohydrate 0.094 w/v; gelatin hydrolysate, porcine Type A (or other sources) 1 % w/v; potassium phosphate dibasic 1 .13%; and potassium phosphate monobasic 0.48% w/v.
  • vaccine formulations comprise all of the following: sucrose: 6.84% weight/volume (w/v); arginine monohydrochloride 1.21 % w/v; glutamic acid, monosodium monohydrate 0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources) 1 % w/v; potassium phosphate dibasic 1 .13%; and potassium phosphate monobasic 0.48% w/v.
  • vaccine formulations comprise all of the following (within 10% variation of one or more component): sucrose: 6.84% weight/volume (w/v); arginine monohydrochloride 1.21 % w/v; glutamic acid, monosodium monohydrate 0.094% w/v; gelatin hydrolysate, porcine Type A (or other sources) 1 % w/v; potassium phosphate dibasic 1.13%; and potassium phosphate monobasic 0.48% w/v.
  • vaccine formulations comprise all of the following (within 10% variation of one or more component): sucrose: 6.84% weight/volume (w/v); arginine monohydrochloride 1.21 % w/v; gelatin hydrolysate, porcine Type A (or other sources) 1 % w/v.
  • formulations are in a buffer (e.g., a potassium phosphate buffer (pH 7.0-7.2)).
  • vaccine formulations may comprise trace amounts of EDTA.
  • vaccine formulations may comprise no EDTA.
  • FluMist ® is a live, attenuated vaccine that protects children and adults from influenza illness.
  • FluMist ® is a live, attenuated vaccine that protects children and adults from influenza illness.
  • the methods and compositions herein may be adapted to, or used with, production of FluMist ® vaccine.
  • the methods and compositions herein are adaptable to production of similar or different viral vaccines and their compositions.
  • FluMist ® vaccine strains typically contain, for example, hemagglutinin (HA) and neuraminidase (NA) gene segments derived from the wild-type strains to which the vaccine is addressed along with six gene segments, PB1 , PB2, PA, NP, M and NS, from a common master donor virus (MDV), also referred to herein as a donor strain or backbone strain.
  • Influenza A strains of FluMist ® can include, for example, MDV-A as the master donor virus.
  • MDV-A was created by serial passage of a wild-type A/Ann Arbor/6/60 (A/AA/6/60) strain in primary chicken kidney tissue culture at successively lower temperatures (see e.g., Maassab (1967) Nature 213:612-4). MDV-A replicates efficiently at 25°C (ca, cold adapted), but its growth is restricted at 38°C and 39°C (ts, temperature sensitive). Additionally, this virus does not replicate in the lungs of infected ferrets (att, attenuation). The ts phenotype is believed to contribute to the attenuation of the vaccine in humans by restricting its replication in all but the coolest regions of the respiratory tract. The stability of this property has been demonstrated in animal models and clinical studies.
  • the ts property of MDV-A does not revert following passage through infected hamsters or in shed isolates from children (see e.g., Murphy & Coelingh (2002) Viral Immunol. 15:295-323).
  • Reassortants carrying the six internal genes of MDV-A and the two HA and NA gene segments of a wild-type virus consistently maintain ca, ts and att phenotypes (see e.g., Maassab et al. (1982) J. Infect. Dis. 146:780-900).
  • Certain systems and methods described previously are useful for the rapid production in cell culture of recombinant and reassortant influenza A and B viruses, including viruses suitable for use as vaccines, including live attenuated vaccines, such as vaccines suitable for intranasal administration (e.g., FluMist ® ).
  • Certain methods provided herein, are optionally used in conjunction with or in combination with such cell culture methods involving, e.g., reassortant influenza viruses for vaccine production to produce viruses for vaccines in a more stable, consistent and efficient manner. Examples
  • Example 1 Concentration of clarified harvest fluid by tangential flow filtration (TFF)
  • This example describes certain improvements to the purification process for influenza viruses. Improved purification methods are described for a live attenuated influenza virus monovalent bulk (LAIV-MB) manufacturing process, however such methods may be applied to any influenza virus manufacturing process. Improvements include introduction of a tangential flow filtration (TFF) step, as illustrated in FIG. 1 and described in detail below.
  • FFF tangential flow filtration
  • Study #1 Concentration of clarified harvest fluid of cold-adapted influenza virus using tangential flow filtration (TFF) with hollow fiber cartridges
  • TFF tangential flow filtration
  • CHF clarified harvest fluid
  • CAIV-MB cold-adapted influenza vaccine - monovalent bulk
  • a TFF concentration step was introduced prior to an ultracentrifugation step, in an approximately 10-fold scaled down model of an existing CAIV-MB manufacturing process.
  • product-containing fluid is passed tangentially to the filter membrane at a fixed shear rate.
  • the difference in pressure between the inlet and outlet can be controlled to provide a constant driving force for the filtration to occur.
  • the product is collected either in permeate or in retentate.
  • each pass of the CHF through the HF cartridge resulted in a portion of the impurities that were smaller than 500 kD to pass through the filter pores as permeate, while the virus particles (i.e., bigger than 500 kD) were retained in the retentate.
  • each pass through the filter module resulted in a concentration of virus particles present in the CHF.
  • the process was continued until a desired concentration of CHF volume was obtained.
  • the process parameters of shear and transmembrane pressure (TMP) were not optimized for this study. However, to minimize the possibility of fouling the cartridge, a shear rate of 10,000 ⁇ 1 ,000 s "1 was chosen.
  • the TMP was chosen as 20 ⁇ 2 psig based on prior TFF studies.
  • CAIV strains were used (e.g., A/Uruguay/716/07, A/South Dakota/6/07, B/Florida/4/2006 and B/Malaysia/2506/04).
  • MPX22803-M Polycarbonate Connector, 3/8" HB non- valved Insert (Qosina, Edgewood, NY, Cat. No. MPX22603-M); Polysulfone MPX Cap Body with Lock, 1 ⁇ 2 " ID (Qosina, Edgewood, NY, Cat. No. MPXK32003); Polysulfone MPX Cap Body with Lock, 3/8" ID (Qosina, Edgewood, NY, Cat. No. M PC2206T03M); Platinum Cured Silicone Tubing, MasterflexTM l/P 73 Tubing, 3/8" ID x 1 ⁇ 2 " OD, (Cole Parmer Instrument Co., Vernon Hills, IL, Cat. No.
  • SARTOCLEAN CA Sterile filter Capsule (Sartorius, Edgewood, NY, Cat No. 5621304E0-OO); Hollow Fiber Module, 500-kD, 0.5 mm Fiber ID (GE Health Care, Piscataway, NJ, Cat. No. UFP- 500-C-5A, S/N: 91982101 153); and Hollow Fiber Modules, 500-kD, 0.5 mm Fiber ID, (Spectrum Labs, Collinso Dominguez, CA, Cat. No.: M6-500S-100-01 S, M7-500S-100-01 N & M8-500S- 300-01 N).
  • Each CAIV strain was propagated and harvested essentially as described in U.S. patent no. 8,247,207 to provide the clarified harvest fluid (CHF) material for TFF experimental runs that were performed.
  • the TFF set up included a peristaltic pump, a Flex Stand, an HF cartridge with appropriately sized permeate and retentate lines connected to appropriate containers and analog pressure gauges to measure pressure at the inlet and outlet of the HF cartridge (FIG. 2). The feed and retentate pressure were monitored and
  • Equation 1 the permeate pressure was neither monitored nor controlled. Because the permeate pressure was equal to atmospheric pressure, the permeate pressure was zero and could be neglected from Equation 1 to give Equation 2 for calculating TMP. Equations 1 and 2 are presented in FIG. 3. Thus, the term TMP as used for this study refers to the average of the feed and retentate pressures.
  • HF cartridges from GE Healthcare and Spectrum Labs were prepared and evaluated according to manufacturers' instructions. For example, for the evaluation of HF cartridges from GE Healthcare, the following procedure was followed for the preparation and use of the HF cartridges.
  • Flush HF cartridge 1 Flush the HF cartridge with clean water using a shear rate of 10,000 sec "1 or greater with the permeate line closed and retentate line fully open in order to establish the cross flow before the permeate line is fully opened. Adjust the TMP to 20 ⁇ 2 psig.
  • HA Hemagglutination Assay
  • Samples were withdrawn from the retentate bags using a 10 ml. syringe connected to the Luer- lock port on each bag. Before collecting the samples, the bags were inverted at least 10 times to ensure the contents were well mixed. Samples (15-30 ml.) were withdrawn from the bags using a 10 ml. syringe. 9 ml. of each sample collected was stabilized with 1 ml. of 10X SP before aliquotting 1 ml. into a 2 ml. CRYOVIAL. The samples were stored at -80 °C and submitted for potency testing (potency was measured by Fluorescent Focus Assay (FFA) analysis, essentially as described in U.S. patent no. 7,262,045).
  • FFA Fluorescent Focus Assay
  • Concentration of CHF 3-5 fold by TFF resulted in little to no change in the density and viscosity of the concentrated CHF from that of the pre-concentrated CHF.
  • the table presented in FIG. 5 summarizes the results of the TFF concentration step for the six runs in this study.
  • the table presented in FIG. 6 characterizes the TFF concentration step.
  • the concentration in volume is the ratio of the volume of CHF to that of TFF retentate.
  • the concentration in potency is the ratio of potency in FFU/mL of the TFF retentate to that of potency in FFU/mL of the CHF.
  • FIG. 7 and FIG. 8 show permeate flow rates versus time for the GE Healthcare and Spectrum Labs cartridges, respectively.
  • Shear rates below 10,000 sec "1 may have contributed to fouling of the filter membrane as evidenced by lower permeate flow rates and consequently longer processing time for B/Florida/4/2006 -PD14Aug08 (see FIG. 4 and FIG. 8). Accordingly, shear rates of 10,000 sec “1 or greater (e.g., 10,000 sec "1 to 18,000 sec “1 or greater) may be necessary to prevent fouling and obtain high permeate flow rates.
  • TMP provides the driving force for filtration to take place across the filter membrane.
  • filtrate flux typically increases with increased TMP.
  • the filtrate flux typically levels off after a certain TMP value.
  • a typical flux vs. TMP curve is shown in FIG. 9. The first part of the curve where the flux increases with the TMP is the pressure dependent regime. The "plateau" phase of the curve where the flux is practically unaffected by increase in TMP is the pressure independent regime.
  • the TFF process is operated at pressures corresponding to the initial onset of the pressure-independent region i.e., the TMP corresponding to the "knee" of the Flux vs. TMP curve.
  • the filtrate flux rate also can be influenced by shear rate, which is a measure of the tangential or cross-flow.
  • Materials used for study #2A included the following: 1 X SP (sucrose-phosphate) buffer, (100 mM potassium phosphate and 200 mM sucrose) (HYCLONE, Logan, UT, cat no.
  • Materials used for study #2B included the following: 1 X PBS (phosphate buffered saline) buffer, (8.4 mM sodium phosphate, dibasic, 1.6 mM potassium phosphate, monobasic, 150 mM sodium chloride) (HYCLONE, Logan, UT, cat. no. SH3A1798.01 ); 5 L LabtainerTM BIOPROCESS container (HYCLONE, Logan, UT, cat no. SH30712.01 ); Polysulfone connector, 1/4" HB non- valved insert (Qosina, Edgewood, NY, cat. no. MPC22004T39M); Polysulfone connector, 3/8" HB non-valved insert (Qosina, Edgewood, NY, cat. no.
  • MPC22006T39M Polysulfone sealing cap with lock, 1/4" ID (Qosina, Edgewood, NY, cat. no. MPCK32039); Polysulfone in-line hose barb, 3/8" ID (Qosina, Edgewood, NY, cat. no. MPC17006T39); Polysulfone in-line hose barb, 1/4" ID (Qosina, Edgewood, NY, cat. no. MPC17004T39); Platinum cured silicone tubing, Masterflex L/S 73 tubing, (Cole Parmer Instrument Co., Vernon Hills, IL, cat. no. 96410-73); ca B/Brisbane/60/2008 (lot no.
  • PD-10Mar10 ca A/Uruguay/716/07 (lot no. PD-17Mar10); ca A/California/07/09 (lot no. PD-24Mar10); ca A/Uruguay/716/07 (lot no. PD-30Mar10); ca A/California/07/09 (lot no. PD-06Apr10); SARTOPORE 2, 0.45/0.2 ⁇ m, sterile filter capsule (Sartorius, Edgewood, NY, cat no. 5441307H8G-OO); MiniKros ® Plus, hollow fiber module, 500 kD, 0.5 mm fiber ID, 320 fiber count, and 1050 cm 2 (Spectrum Labs, Collinso Dominguez, CA, cat.
  • Equipment used for both study #2A and study #2B included the following: Biosafety cabinet (Baker Co., Stanford, MN, model: STERILGARD III Advance); Pipet aid (VWR International, Brisbane, CA, cat. no.: 14006-026); FlexStandTM system (GE Healthcare, Piscataway, NJ, cat. no.: FS01 S); Pressure gauge, 0-60 psig (Anderson Instrument Co., Fultonville, NY, cat. no.: 3004300); Weighing balance, 0-35 kg (Sartorius, Edgewood, NY, model: EB35EDE-1 );
  • Peristaltic pump (Spectrum Labs, Collinso Dominguez, CA, model: KROSFLO, MINIKROS Pilot system).
  • the TFF set up included a peristaltic pump, a FLEXSTAND, a hollow fiber (HF) cartridge with permeate and retentate lines connected to appropriate containers and analog pressure gauges to measure the pressure at the inlet and outlet of the HF cartridge.
  • Each CAIV strain was propagated in specific pathogen-free (SPF) eggs. At the end of secondary incubation the inoculated eggs were de-capped and harvested. The harvested egg allantoic fluid was pooled to make the Pooled Harvest Fluid (PHF). The PHF was filtered using a SARTOPORE 2, 0.2/0.45- ⁇ filter to obtain the CHF. This process was used for the different CAIV strains (ca A/South Dakota/6/07, ca B/Malaysia/2506/04, ca B/Florida/4/2006 ca
  • the HF cartridge was flushed with 500 ml. or greater (2 mL/cm 2 or greater of surface area) of clean water.
  • the clean water flush was performed at a shear rate of 14,000 s '
  • the feed flow rate was increased to correspond to a shear rate of 16,000 s '
  • the feed line was then disconnected from the clean water vessel and connected to the CHF bag.
  • the optimization of the TMP was performed at flow rates corresponding to four different shear rates: 16,000 s "1 , 14,000 s "1 , 12,000 s "1 and 10,000 s '
  • the TMP was increased in 5 psig increments from 10 psig to 25 psig.
  • the permeate (filtrate) flow was allowed to stabilize for at least 5 minutes before recording the permeate flux data.
  • the permeate flux rate was expressed as LMH (liters per square meter per hour).
  • the HF cartridge was flushed with 4 L or greater (2 mL/cm 2 or greater of surface area) of clean water.
  • the clean water flush was performed at a shear rate of 10,000 s '
  • the cartridge was then tested for integrity and flushed with at least 2 void volumes of 1X PBS buffer solution to equilibrate the cartridge with that buffer.
  • the entire TFF set up was moved to a 5 ⁇ 3 °C environment or left at room temperature.
  • the permeate valve completely closed, the CHF was then re-circulated through the TFF set up and the required TMP was adjusted.
  • the permeate valve was gradually opened while simultaneously restricting the retentate flow to maintain the desired TMP.
  • the concentration process was continued until a 4- to 5-fold reduction in volume of the CHF was obtained.
  • the concentration process was stopped and the system was flushed with 2 void volumes of 1X PBS buffer.
  • the CHF/retentate bag was then disconnected from the system and the TFF concentration process was considered complete.
  • the average values of the TMP and the flux across the four strains are plotted against the shear rate to depict the overall trend.
  • the best flux rates were observed when the concentration process was operated at a shear rate range of 14,000 s "1 to 16,000 s "1 and a TMP range of 15-16 psig (the region indicated by the shaded area in FIG. 14, i.e., the range of TMP values (y-axis) for the TMP vs. shear rate curve that span the shaded region (along the x-axis), corresponding to the leveled region of the flux vs. shear rate curve).
  • the preliminary parameter study was followed by a pilot-scale study.
  • the TFF concentration process was operated at a shear rate of 14,000 s "1 and a TMP of 15 psig.
  • the first run was performed at room temperature (i.e., 18 to 20°C) and the average flux observed was 95 LMH.
  • the other four runs were performed in a 2 to 8°C refrigerator.
  • the lowest, highest and average fluxes for the four runs were 33, 85 and 59 LMH, respectively, for the 2 to 8°C refrigerator runs.
  • FIGS. 16-19 Additional details regarding the supplementary characterization study such as recovery, material balances and impurity profiles are presented in FIGS. 16-19.
  • tangential flow filtration (TFF) procedure uses disposable hollow fiber (HF) cartridges from Spectrum Labs for concentration of clarified harvest fluid (CHF) of live attenuated influenza virus (LAIV).
  • CHF clarified harvest fluid
  • LAIV live attenuated influenza virus
  • alternate disposable HF cartridges were evaluated.
  • disposable HF cartridges manufactured by GE Healthcare were evaluated for their suitability for the concentration of CHF.
  • the clarified harvest fluid (CHF) was concentrated using a TFF process as described below:
  • a GE HF cartridge (0.5 mm ID, 500 kDa
  • ultrafiltration membrane was affixed to a GE Flex StandTM with the inlet and outlet lines connected to the CHF/retentate bag and the permeate line connected to the permeate collection vessel.
  • a peristaltic pump was used to pump CHF from the CHF/retentate bag or 1 X PBS from a bag or bottle through the HF cartridge.
  • the HF cartridge was equilibrated using 1 X PBS (0.5 mL/cm 2 or greater of surface area).
  • CHF was circulated at the set shear rate and TMP. Once the flow was fully established, the permeate valve was gradually opened to a fully open position. The TMP was re-established to the set value by adjusting the retentate valve after the permeate valve was fully open.
  • Shear rates from 8000 to 18000 s "1 and TMP from 9 to 18 psi were evaluated at load factors 200 to 31 1 L/m 2 and concentration factors of 4.7 to 7.2.
  • the average permeate flux was evaluated by setting the TFF process parameters to shear rates at 8000 s "1 , 10000 s “1 , 14000 s “1 , 16000 s “1 , or 18000 s "1 and the TMPs at 9 psi, 10 psi, 12 psi, 15 psi, or 18 psi.
  • FIG. 20 presents a table summarizing experimental conditions of the TFF processes and the corresponding average permeate fluxes.
  • FIG. 21 shows a contour plot of shear rates, TMPs, and average permeate fluxes.
  • FIGS. 22A and 22B present a table summarizing potency assay data of the GE HF TFF process.
  • the CHF of four different LAIV strains (A/Uruguay/716/07, A/California/07/09, B/Florida/04/06, and B/Brisbane/60/08) was concentrated 4.7 to 7.2- fold with shear rates ranging from 8,000 to 18,000 s "1 and TMPs ranging from 9 to 18 psi.
  • An average permeate flux of 50 LMH or greater was observed for 22 of the TFF processes.
  • An average permeate flux of 41 and 48 LMH was observed during the concentration of
  • B/Brisbane/60/08 at a shear rate of 8,000 s "1 and TMP of 10 psi (run 1 ), and during the concentration of B/Florida/04/06 at a shear rate of 10,000 s "1 and TMP of 9 psi (run 2), respectively.
  • FIG. 21 shows that when the TFF process is operated at a shear rate of 10,000 s "1 or greater, an average permeate flux of 50 LMH or greater was achieved with a TMP ranging from 9 to 18 psi. This indicates that 200 L of CHF can be concentrated 4 to 7- fold within 2.6 hr using a 1.15 m 2 HF cartridge.
  • FIG. 21 also shows an optimal operation region with shear rate ranging from 1 1 ,000 to 18,000 s "1 and TMP ranging from 10 to 14 psi, in which an average permeate flux of 58 LMH or greater, was obtained.
  • the highest average permeate flux of 64 LMH or greater was achieved.
  • Certain parameters, e.g., optimal shear rate can vary depending on the TFF materials used. For example, column packing density and/or membrane pore density can vary amongst TFF cartridge manufacturers, and parameters such as shear rate can be adjusted accordingly.
  • potency assay results showed that the potency of the permeate from all the 24 TFF processes was less than 3.3 log-io FFU/mL (below the assay detection limit), which indicated that no virus leaked through the GE HF membrane.
  • the acceptance criteria for selection of tubing are a minimum operation time of 6 hours without damage or imminent signs of damage, animal derived component free (ADCF) manufacture and low degree of spallation. From this study Pure Weld ® tubing was chosen and displayed no imminent signs of damage on the outer or inner wall after 6.5 hours of operation and had a low level of tubing degradation (spallation). Furthermore, to minimize vibrations, the process was best operated at flow rates that corresponded to shear rates of 1 1 ,000 ⁇ 1 ,000 s "1 and TMP of 13 ⁇ 1 psi. This indicates that while the TFF column can be run over a wide range of conditions, the actual run conditions may be limited by other equipment limits. For example, use of alternative tubing and/or pump system could expand and/or shift the operating range.
  • Pilot-scale studies A pilot-scale study was performed to evaluate the scalability of the TFF process described above with GE HF cartridges using the SciPure ® 200 system. A total of four runs with four LAIV strains (ca B/Brisbane/60/2008 (Victoria lineage), ca A/Victoria/361/201 1 (H3N2), ca
  • FIG. 23 presents a table summarizing the results. No virus was detected in the permeate samples (which confirms that the membrane was integral and adequately sized), and a 10-fold concentration was performed in less than 2 hours of processing time for all four runs.
  • the stationary rotor was completely filled with approximately 3.2 L of PBS through the bottom port of the ultracentrifuge. A portion of the PBS was then displaced with 1 .6 L of 10% sucrose followed by 1 .2 L of 60% sucrose. After the 60% sucrose was pumped into the rotor, the top and bottom tubing were clamped off, and the rotor was immediately accelerated to 7000 rpm. When the rotor reached 7,000 rpm, PBS was pumped into the rotor at 100 -120 mL/min through the bottom port of the ultracentrifuge. Once the centrifuge tubing pressure was stable, the flow rate was increased and the rotor was accelerated to 35,000 rpm.
  • the CHF that was concentrated approximately 3 or 5-fold in volume by TFF was loaded into the ultracentrifuge with the flow rates ranging from 75 to 275 mL/min as shown in FIG. 70.
  • the flow-through was collected when concentrated CHF was pumped into the
  • the virus banding started with PBS flow at 100 -120 mL/min for one hour.
  • the bottom inlet line of the centrifuge was clamped, and the rotor was decelerated to 7000 rpm under normal brake. Once the speed reached 7000 rpm, the rotor was stopped in free coasting mode. Once the rotor completely stopped, the sucrose gradient was offloaded at 100 mL/min from the bottom port of the ultracentrifuge. The sucrose gradient was collected into three pools according to certain cut-off densities.
  • the sucrose gradient with a density greater than 1.2276 g/cm 3 (greater than 49.2% sucrose solids) was directed into a high sucrose density pool (P1 ) biotainer.
  • the virus peak within a density range from 1 .1525 to 1 .2276 g/cm 3 (equivalent to 34.8% - 49.2% sucrose solids) was collected into a virus peak pool (P2) biotainer.
  • the sucrose gradient with density less than 1.1525 g/cm 3 (less than 34.8% sucrose solids) was collected in a low sucrose density pool (P3) biotainer.
  • the purified and concentrated virus in the P2 biotainer was further processed to produce a diluted centrifuge pool (DCP) and monovalent bulk (MB).
  • DCP diluted centrifuge pool
  • MB monovalent bulk
  • the amount of virus that was captured in the sucrose gradient ranged from 97.5% to 76.1 % when the loading flow rate of the TFF concentrated CHF varied from 75 to 275 mL/min (the amount of virus lost in the flow-through ranged from 2.5% to 23.9%, respectively). Less virus was lost and, thus, higher virus recovery was observed at lower loading flow rates. A virus recovery of 90.0% or greater was achieved when a loading flow rate ranging from 120 to 160 mL/min was applied. For example, the total time to process 13.4 L of concentrated CHF at 140 mL/min was 1.6 hour, and 96.8% of virus capture in the sucrose gradient was achieved.
  • the percentage of virus loss was calculated base on the amount of virus loaded into the ultracentrifuge and the amount present in the flow-through (FIG. 73).
  • the virus lost in the flow- through ranged from 2.5% to 23.9% as the loading flow rate of the concentrated CHF varied from 75 to 275 mL/min, respectively.
  • Increasing the loading flow rate led to a higher loss of virus in the flow-through as indicated by a linear fit of the data with 95% confidence (FIG. 72). More than 16% of virus was lost in the flow-through when the flow rate was higher than 200 mL/min. Therefore, a lower loading flow rate was chosen to achieve higher virus capture in the sucrose gradient during centrifugation of the
  • the centrifuge pool was diluted and filtered through a 0.2 ⁇ filter to obtain the monovalent bulk (MB).
  • the impurity content of the MB solution met in-process control (IPC) specifications according to current IPC limits for CHF that has been concentrated to 5.3-fold by TFF concentration, without affecting the IPC specifications for the impurity content of the MB.
  • IPC in-process control
  • a maximum 5 fold concentration limit is applied for a TFF process (e.g., 200 L CHF maximum volume concentrated to minimum TFF retentate volume of 40L).
  • TFF concentration was selected based on results from previous studies and are shown in FIG. 25. TFF engineering and validation runs (full-scale batches)
  • TFF virus strain
  • non-TFF batches The number of doses created for each virus strain (TFF and non-TFF batches) is shown in FIGS. 26-28.
  • the figures show the mean and range profiles for the number of doses generated for each non-TFF batch compared with individual TFF batches for each strain. Data for impurity performance from PHF to monovalent bulk for each TFF batch is compared with commercial non-TFF batches of the same strain in FIGS. 29-31 .
  • Example 2 Modified clarification methods
  • This example describes certain improvements to the purification process for influenza viruses. Improved purification methods are described for a live attenuated influenza virus monovalent bulk (LAIV-MB) manufacturing process, however such methods may be applied to any influenza virus manufacturing process. Improvements include clarification using modified filtration methods described in detail below. Pre-filter for improved clarification of an influenza virus
  • the manufacture of a refrigerator-stable, liquid formulation of an influenza virus drug substance often includes a sequence of downstream processing steps: clarification filtration,
  • the drug substance is also called the monovalent bulk (MB), which contains a single strain of the cold adapted, live-attenuated, influenza virus (CAIV).
  • MB monovalent bulk
  • CAIV live-attenuated influenza virus
  • Certain clarification filtration processes include filtering pooled virus harvest fluid (PHF) through a 1 .2 ⁇ filter (e.g., Milligard ® ) followed by a 0.8 ⁇ /0.45 m filter (e.g., SARTOPORE 2).
  • a single filtration rig can include two-10 inch 1.2 ⁇ filters (1 .6 m 2 total effective filter area) in parallel and one-20 inch 0.8 ⁇ /0.45 ⁇ filter (1 .2 m 2 total effective filter area) in succession.
  • filter clogging near a production volume of 80 to 90 liters (L) can occur for certain CAIV strains.
  • Polyethylene terephthalate glycol modified (PETG) bottle (Nalgene, Rochester, NY, Cat. No.: 2019-1000); ca A/South Dakota/6/07, Batch number: 141900666A; ca A/Uruguay/716/07, Batch number: 141900675A; ca A/Mississippi/4/08, Batch number: 2000018430; ca B/Florida/4/2006, Batch number: 141900641A; and ca B/Bangladesh/3333/07, Batch number: 2000018547.
  • PETG Polyethylene terephthalate glycol modified
  • BIOPROCESSING Systems Middleton, WC, Cat. No.: 080-699PSX
  • 3/8" Barb pressure sensor flow cell (SCILOG BIOPROCESSING Systems, Middleton, WC, Cat. No.: 080-694PSX);
  • HACH 21 OOP turbidity Meter HACH Company, Loveland, CO, Part number: 46500-00
  • -80 °C freezer Revco Technologies, Asheville, NC, Model No.: UL T2586-9-D35
  • Milligard ® 1.2 ⁇ OptiscaleTM disposable capsule filter (Millipore Corporation, Billerica, MA, Cat. No.: SW19A47HH3); SARTOPORE 2 0.8/0.45 m SARTOSCALE disposable capsule filter (Sartorius Stedim Biotech, Goettingen, Germany, Cat. No.: 5445306GS-FF); Milligard ® Opticap ® XL2, (Millipore Corporation, Billerica, MA, Cat. No.: KW19A02HH1 ); SARTOPORE 2 MIDICAP, (Sartorius Stedim Biotech, Goettingen, Germany, Cat.
  • VAF virus-infected allantoic fluid
  • Each filter was wetted and equilibrated before use in the clarification filtration.
  • Each filter capsule assembly was completely bled before filtration.
  • the pre-clarification filtration rig assembly included a tubing assembly, a pressure monitoring SCIPRESS pressure sensor, and a pre-filter. Filters were first wetted with purified water and then equilibrated with 1X PBS individually. Buffer was drained out of the filter before each filtration. Air bubbles were removed through the filter vent port by briefly tapping the rig assembly. CAIV clarification filtration at a linear flux rate of up to 6 L/min/m 2 (LPM/m 2 ) typically results in minimal potency loss of 0.1 log 10 FFU/mL or less. Thus, 5 LPM/m 2 was used in this study.
  • PHF was pumped by a Watson Marlow Bredel pump through the pre-clarification filtration rig assembly at a linear flux rate of 5 LPM/m 2 and the filtrate was collected in a 1-L PETG bottle. Pressure was monitored upstream of the pre-filter during the filtration process. A constant flow method was used to assess the filterability of the pre-clarification fluid in this study. The filtrations proceeded until the differential pressure plateaued or reached 30 psi.
  • the clarification filtration rig included a tubing assembly, a pressure monitoring SCI PRESS pressure sensor, and a 1 .2 ⁇ filter (Milligard ® ) followed by a 0.8/0.45 ⁇ filter (SARTOPORE 2). Filtrate obtained from the clarification filtration was pumped by a Watson Marlow Bredel pump through the pre-clarification filtration rig assembly at a linear flux rate of 5 LPM/m 2
  • FAA Fluorescent Focus Assay
  • FIG. 32 presents a table summarizing potency change and filterability of PHF after pre- clarificationfiltration through the four pre-filters chosen for this study. Potency data are presented in FIG. 35.
  • the range in potency change for each pre-filter after pre- clarification filtration was 0 to -0.2 logTM FFU/mL for 10 ⁇ (POLYGARD CN), +0.1 to -0.2 logTM FFU/mL for 8 ⁇ (SARTOPURE PP2), -0.1 to -0.2 logTM FFU/mL for 20 ⁇ (SARTOPURE PP2), and 0 to -0.1 logTM FFU/mL for stainless steel 42 ⁇ mesh filter.
  • the positive increase in potency was due to assay variation.
  • the overall potency changes for all four pre-filters among the five CAIV strains were similar and were all -0.2 log-io FFU/mL or less after pre-clarification filtrations.
  • the filterability of CAIV in each pre-filter varied.
  • the filterability of the five CAIV strains on the 10 ⁇ pre-filter ranged between 185 to 314 L/m 2 .
  • EFA effective filter area
  • Two 10-inch capsules (0.6 m 2 EFA per capsule) or one 20-inch capsule (1 .2 m 2 EFA per capsule) provided sufficient filter area to cover the minimum required EFA.
  • a bigger capsule with 1.6 m 2 EFA per capsule (30-inches) also accommodated the increasing batch size.
  • fewer 8 ⁇ pre-filters were required based on a 160-L batch size.
  • a similar number filters as the 8 ⁇ pre-filter were needed to process this batch size.
  • Other filter configurations could be determined based on the results as demonstrated above (e.g., determine minimum filterability for a given filter and calculate a minimum EFA for a given batch size).
  • the stainless steel 42 ⁇ mesh has the largest pore size compared to other pre-filters. No pressure drop across this pre-filter was observed throughout filtration. Thus, the absolute filterability on this pre-filter could not be determined.
  • FIG. 33 presents a table summarizing filterability of the five CAIV strains after clarification filtrations through a 47 mm 1.2 ⁇ filter (Milligard ® ) followed by a 47 mm 0.8/0.45 ⁇ filter (SARTOPORE 2) with and without a preceding pre-clarification step.
  • Clarification filtration performance was investigated for certain TFF engineering and validation batches (described in Example 1 ).
  • Intermediate potency samples from pooled harvest fluid (PHF) and clarified harvest fluid (CHF) samples were collected from each of the batches for which the 8 ⁇ filter (SARTOPURE PP2 8 ⁇ ) was used in the process validation.
  • the results are presented in FIG. 36, which also includes a virus recovery assessment across the clarification filtration process by using the volume of material generated at the PHF and CHF process stages. Virus recovery data from these batches was compared with commercial material from each strain for which intermediate potency testing was completed. This allowed a direct comparison with the virus recovery during clarification both with and without the 8 ⁇ filter in place.
  • the virus recovery for the A/Victoria process validation batch was towards the upper end of the recovery range observed during commercial batch manufacture whereas the for the A/California strain the recovery was on the lower end of commercial batch virus recovery.
  • pooled harvest fluid PHF
  • PHF pooled harvest fluid
  • 1.2- ⁇ filters e.g., Milligard ® (Millipore)
  • filtration area of each filter is 0.8 m 2
  • 0.8/0.45- ⁇ filter e.g., Sartopore ® 2 (Sartorius); 1.2 m 2 filtration area.
  • the 1.2- ⁇ filters clogged at a filtration volume as low as 80 L (equivalent to a throughput of 50 L/m 2 ) when processing certain LAIV strains.
  • a pre-filter with a pore size of 8 m to 20 m e.g., POLYGARD CN 10- ⁇ (Millipore) filter; SARTOPURE PP2 8- ⁇ or 20- ⁇ filter (Sartorius)
  • POLYGARD CN 10- ⁇ (Millipore) filter e.g., SARTOPURE PP2 8- ⁇ or 20- ⁇ filter (Sartorius)
  • PUMPSIL tubing with 1.6 mm I.D. and 2.4 mm wall thickness cat. No. 913.A016.024 (Watson Marlow, Wilmington, MA)I PUMPSIL tubing with 4.8 mm I.D. and 2.4 mm wall thickness, cat. No. 913.A048.024 (Watson Marlow, Wilmington, MA); MASTERFLEX platinum-cured silicone tubing L/S size 16, cat. No. 96410-16 (Cole Parmer, Vernon Hills, IL); MASTERFLEX platinum-cured silicone tubing L/S size 36, cat. No. 96410-36 (Cole Parmer, Vernon Hills, IL); 2 mL
  • B/Brisbane/60/2008 batch number: 14190099A.
  • BIOPROCESSING Systems, Middleton, WC BIOPROCESSING Systems, Middleton, WC
  • SCIPRESS monitor cat. No. 080-690 (SCILOG BIOPROCESSING Systems, Middleton, WC); 3/8" Barb pressure sensor flow cell, cat. No. 080- 694PSX (SCILOG BIOPROCESSING Systems, Middleton, WC); HACH 21 OOP turbidity Meter, part number 46500-00 (HACH Company, Loveland, CO); and -80 °C Freezer, model No.
  • Millipore Milligard ® 1.2- ⁇ OptiScaleTM disposable capsule filter cat. No.: SW19A47HH3 (Millipore Corporation, Billerica, MA); Sartorius SARTOPORE 2 0.8/0.45- ⁇ SARTOSCALE disposable capsule filter, cat. No. 5445306G-FF; (Sartorius Stedim Biotech, Goettingen, Germany); GE XAMPLER laboratory scale microfiltration cartridges, model No.
  • CFP-4-E-3X2MA polysulfone, pore size 0.45 ⁇ , fiber ID 1 mm, membrane area 0.023 m 2 , flow path length 60 cm (GE Healthcare Bio-Sciences, Piscataway, NJ); Pall MICROZA hollow fiber microfiltration module, part No. UJP-0047R, polyvinylidene difluoride, pore size 0.65 ⁇ , fiber ID 1 .1 mm, membrane area 0.02 m 2 , flow path length 31.4 cm (Pall Corporation, Covina, CA); Sartorius SARTOCON Slice 200, cat. No.
  • PSM80C12P2 polyethersulfone, pore size 0.8 ⁇ , filtration area 0.02 m 2 (Pall Corporation, Covina, CA); Pall KLEENPAK capsule (pleated cross flow), polyethersulfone, pore size 0.65 ⁇ , filtration area 0.06 m 2 (Pall
  • Millipore MILLISTAK+DOHC disposable capsule filter cat. No. SG3J017A03, cellulose fibers with inorganic filter aid, pore size 9.00-0.55 ⁇ , filtration area 23 cm 2 (Millipore Corporation, Billerica, MA); Millipore MILLISTAK+COHC disposable capsule filter, cat. No. MC0HC23HH3, cellulose fibers with inorganic filter aid, pore size 2.5-0.2 ⁇ , filtration area 23 cm 2 (Millipore Corporation, Billerica, MA).
  • VAF virus-infected allantoic fluid
  • Each filter was wetted as recommended by each filter manufacturer and equilibrated using 1X PBS before use in clarification filtration.
  • Each filter capsule assembly was completely bled before filtration.
  • DFF Direct flow clarification filtration
  • the clarification filtration rig included a tubing assembly, a pressure monitoring SCI PRESS pressure sensor, a 1 .2- ⁇ filter (Milligard ® ) followed by a 0.8/0.45 ⁇ filter (SARTOPORE 2) serving as a control, or a depth filter (MILLISTAK+DOHC or MILLISTAK+COHC).
  • PPM/m 2 L/min/m 2
  • MILLISTAK+ 1 1 .5 mL/min through the 23-cm 2 depth filters
  • FIG. 37 presents a table summarizing three different filter formats and operating conditions used in this study for evaluating the performance of cross-flow microfiltration in the clarification of PHF.
  • Filter choices were based, in part, on the availability of suitable small-scale configurations for initial evaluation.
  • Selection of filter pore size was based, in part, on the largest available pore size for the chosen filter.
  • Operating conditions used in this study were selected based, in part, on the manufacturer's recommendation.
  • a pressure monitoring SCIPRESS pressure sensor was placed upstream of the filter inlet, downstream of the retentate outlet, and downstream of the permeate outlet.
  • the PHF was pumped using a Watson Marlow Bredel pump or the MINIKROS pilot system peristaltic pump through the inlet of the microfiltration filter at the chosen operating condition, and clarified filtrate was collected at the permeate outlet into 1-L PETG bottles as clarified harvest fluid.
  • the filtrations were performed until the permeate flux plateaued or until the PHF was exhausted.
  • the transmembrane pressure (TMP) and permeate flux were monitored and measured during each filtration process.
  • permeate control was also examined in this study.
  • a Watson Marlow pump was placed downstream of the permeate outlet to control the permeate flow at a constant flow rate of 150 mL/min (equivalent to 9 LMH). Clarified filtrate was collected at the permeate outlet into 1 -L PETG bottles as clarified harvest fluid. The filtrations were performed until an air bubble was observed at the permeate outlet.
  • Samples taken from the filtration process were stabilized with 10X SP (to a final concentration of 1X SP) and then frozen in 1-mL aliquots and stored in a -80 °C freezer. Viral potency was analyzed using a fluorescent focus assay (FFA), with six and twelve replicates read per sample.
  • FFA fluorescent focus assay
  • FIG. 39 shows that a steady permeate flux of about 47 LMH was achieved on the 0.45- ⁇ hollow fiber cartridge (GE) when the permeate flux was controlled at 45 LMH by a peristaltic pump. Similarly, a steady permeate flux was expected for the 0.65- ⁇ hollow fiber cartridge (Pall) with controlled permeate flow.
  • FIG. 44 presents a table showing potency data for a CF-MF process using flat sheet cassettes.
  • DFF control filtration
  • 1.2- ⁇ filter Milligard ®
  • SARTOPORE 2 0.8/0.45- ⁇ filter
  • FIG. 46 shows that permeate flux decline was alleviated by applying permeate control at 150 LMH, and a more steady permeate flux was achieved by controlling permeate at a lower flux (the first flux decline shown in FIG. 46 was due to kinked tubing).
  • FIG. 48 presents a table showing throughput and potency data of DFF depth filtration processes using a 9.0-0.55 ⁇ depth filter (Millipore MILLISTAK+DOHC (pore size range: 9.00-0.55- ⁇ ) and COHC (pore size range: 2.5-0.2- ⁇ )) for ca A/Uruguay/716/07 and ca B/Brisbane/60/2008, respectively.
  • FIG. 48 presents a table showing throughput and potency data of DFF depth filtration processes using a 9.0-0.55 ⁇ depth filter (Millipore
  • MILLISTAK+DOHC MILLISTAK+DOHC
  • MILLISTAK+COHC 2.5-0.2 ⁇ depth filter
  • a substantial increase in throughput was obtained from the depth filtration processes: a 3.9-fold increase using a 9.0-0.55 ⁇ depth filter and a 1.7-fold increase using a 2.5-0.2 ⁇ depth filter compared to a control DFF (control filtrations).
  • tests with ca A/Uruguay/716/2007 showed that throughput of the 9.0-0.55 ⁇ depth filter increased to 428 L/m 2 compared to an existing filtration process (1 1 1 L/m 2 ) while no further potency loss was observed (e.g., -0.1 log-io FFU/mL after the 9.0-0.55 ⁇ depth filtration vs. -0.2 log-io FFU/mL after the current filtration).
  • the 9.0-0.55 ⁇ depth filter achieved a high filtration throughput and low virus potency loss, it was chosen for further testing as a potential substitute to the current 1.2- ⁇ filter (Milligard ® ) for clarifying LAIV PHF.
  • PHF pooled virus harvest fluid
  • LAIV live- attenuated influenza virus
  • a 9.0-0.55 ⁇ depth filter achieved higher filtration throughput and similar virus potency recovery, while maintaining ease of operation as well as scale-up compared to the existing clarification process described above.
  • This study further evaluated the performance of the 9.0-0.55 ⁇ depth filter by measuring filtration throughput and potency recovery at an increased filtration scale (i.e., from 23 cm 2 to 1 100 cm 2 ).
  • PEF Pooled harvest fluids (PHF) of six live-attenuated influenza virus (LAIV) strains, A/California/07/2009, A/Uruguay/716/07, A/Perth/16/2009, B/Brisbane/60/2008,
  • a mini capsule filter of 23 cm 2 and lab scale pod filters of 270, 540, and 1 100 cm 2 filtration areas were evaluated for filtration throughput and potency recovery at flux ranges of 100- 300 LMH.
  • a 1.2- ⁇ membrane filter (Milligard ® ) was used in this study as a control for comparison (filtration area: 17.7 cm 2 ).
  • the filtrate of the depth filtration was filtered through a 0.8/0.45- ⁇ filter (SARTOPORE 2) and the corresponding throughput and potency recovery also were determined.
  • the stability of monovalent bulk (MVB) produced in subsequent steps following the depth filtration was assessed at 2-8 °C in 125-mL bottles and 1 -L bags for a period of 14 days.
  • BioProcess ContainerTM cat. No. SH30712.02 (HyClone ® , Logan, UT); 50-L BioProcess ContainerTM, cat. No. SH30712.04 (HyClone ® , Logan, UT); 60% Sucrose in PBS, cat. No.
  • SH3A1800.01 HyClone ® , Logan, UT
  • 10% Sucrose in PBS cat. No. SH3A1799.01
  • HyClone ® , Logan, UT Centrifuge Diluent Phosphate Buffer (CDPB), cat. No. SH3A 1801.01 (HyClone ® , Logan, UT); 10-mL Pipettes, cat. No.: 53283-708 (VWR International, Brisbane, CA); Lancet (LIFESCAN, Model: One Touch, FinePointTM); A/California/07/2009, batch number:
  • BIOPROCESSING Systems Middleton, WC
  • Refrigerator Incubator model No. 2005 (VWR International, Brisbane, CA); -80 °C Freezer, model No. UL T2586-9-D35 (Revco Technologies, Asheville, NC); Pipette aid, cat. No.: 14006-026 (VWR International, Brisbane, CA); Hitachi CC40 continues flow ultracentrifuge (Hitachi, Japan); Automatic Centrifuge Offloading System (Medlmmune, Inc., Santa Clara, CA); WAVE MIXER, model MIXER 20/50P (Wave Biotech, Bridgewater, NJ, Model); Egg incubator, model NMC2500 (Natureform Inc., Jacksonville, FL); Egg candler, cat. No.: N4130 (FIBREOPTIC LLLUMINATOR, FIBREOPTIC LIGHTGUIDES, Australia); and Egg puncher, model No.: E90 (Glas Col, Terre Haute, IN).
  • Millipore Milligard ® 1.2- ⁇ OptiScaleTM disposable capsule filter cat. No.: SW19A47HH3 (Millipore Corporation, Billerica, MA); Millipore Millistak+ ® D0HC disposable capsule filter, cat. No. SG3J017A03, cellulose fibers
  • MD0HC054H1 cellulose fibers with inorganic filter aid, pore size 9.00-0.55 ⁇ , filtration area 540 cm 2 (Millipore Corporation, Billerica, MA); Millipore Millistak+ ® D0HC disposable capsule filter, cat. No. MD0HC01 FS1 , cellulose fibers with inorganic filter aid, pore size 9.00-0.55 ⁇ , filtration area 0.1 1 m 2 (Millipore Corporation, Billerica, MA); Sartorius SARTOPORE 2 0.8/0.45- ⁇ 300 capsule filter, cat. No.
  • 5441306G5-OO (Sartorius Stedim Biotech, Goettingen, Germany); Sartorius SARTOPORE 2 0.8/0.45- ⁇ MIDICAP filter, cat. No. 5441306G8-OO (Sartorius
  • a 1 .2- ⁇ filter (Milligard ® ) and a 0.8/0.45- ⁇ filter (SARTOPORE 2) were prepared according to previous methods. 9.0-0.55 ⁇ depth filters (Millistak+ ® DOHC) were wetted as referenced in the filter manufacturer's specification and equilibrated using 1 X PBS before being used in the clarification filtration. Each filter assembly was completely bled before filtration.
  • the clarification filtration rig included a tubing assembly, a pressure monitoring SCI PRESS pressure sensor and a 1.2- ⁇ filter (Milligard ® ).
  • the 1.2- ⁇ filter membrane was used as a control in this study to determine the throughput of an existing clarification filtration process.
  • the clarification filtration rig included a tubing assembly, a pressure monitoring SCI PRESS pressure sensor and a 9.0-0.55 ⁇ depth filter.
  • the depth filters included three ports, one inlet, one vent, and one outlet; while the capsule filter has one inlet and one outlet.
  • a pilot pod filter holder was used for the operation of pod filters larger than 1 100 cm 2 .
  • the vent port was opened while the outlet port was closed.
  • PHF was pumped through the clarification filtration rig assembly at a designated flux (outlined in FIG. 49) using a Watson Marlow Bredel pump. Different fluxes were evaluated within a flux range of 100-300 LMH.
  • Virus filtrate after filtration using a 9.0-0.55 ⁇ depth filter was pumped using a Watson Marlow Bredel pump through a clarification filtration rig that included a tubing assembly, a pressure monitoring SCI PRESS pressure sensor and a 0.8/0.45- ⁇ filter (SARTOPORE 2).
  • the clarified harvest fluid (CHF) was collected in a HyClone ® bag. Filtrations were performed until the differential pressure plateaued or reached 30 psi.
  • CHF was loaded into a Hitachi CC40 ultracentrifuge at 100 mL/min while 240 mL/min was used to load A/Perth/16/2009.
  • About 100 ml. and 800 ml. of MVB were transferred into a 125-mL PC bottle and a 1-L HyClone ® bag, respectively. Both the bottle and the bag were stored in a refrigerator at 2-8 °C for stability study over a 14 day period. Potency analysis
  • the filtration throughput of LAIV using 9.0-0.55 ⁇ depth filters is summarized in the table presented in FIG. 50. Throughput values were expressed as averages of the experimental results (shown in FIG. 55) measured when filtration differential pressure reached 30 psi, except those for A/Uruguay/716/07. The filtration throughput tracked closely across the various tested filter areas and formats (from the 23 cm 2 mini capsule to the 270, 540, and 1 100 cm 2 pod filters). Compared to control filtration using a 1.2- ⁇ filter (Milligard ® ), the throughput of a 9.0-0.55 ⁇ depth filter was higher with a 1.8-fold increase for B/Malaysia/2506/04 and more than a 3-fold increase for all other LAIV strains. On average, the throughput improvement was 3.4-fold.
  • the potency change after depth filtration was similar across different filter areas and formats.
  • the potency drop after depth filtration was 0.2 log- ⁇ FFU/mL or less, except for the filtration of A convinced/716/07 using the 540 cm 2 filter and B/Malaysia/2506/04 using the 270 cm 2 filter, which showed a potency drop of 0.3 log 10 FFU/mL.
  • the potency drop after depth filtration was similar to that after filtration using a 1.2- ⁇ filter (Milligard ® ).
  • FIG. 52 shows that throughput of depth filtration using a 9.0-0.55 ⁇ depth filter (Millistak+ ® D0HC) was consistently greater than 200 L/m 2 at a flux range of 100-300 LMH across different filter sizes; while the potency change was -0.2 log-io FFU/mL or less after each filtration.
  • a flux in the range of 100-300 LMH can be used for depth filtration.
  • FIG. 53 shows that the throughput of filtering D0HC-CF through a 0.8/0.45- ⁇ filter
  • MVB stability following the use of a 9.0-0.55 pm depth filter in the clarification process
  • the clarified virus fluids of A/Perth/16/2009, A/California/07/2009, and B/Brisbane/06/2008 were loaded into a CC40 ultracentrifuge after filtration through a 9.0-0.55 ⁇ depth filter followed by a 0.8/0.45- ⁇ filter (SARTOPORE 2).
  • SARTOPORE 2 The stability of monovalent bulk (MVB) produced by this process was assessed over a 14-day period at 2-8 °C in two types of containers: 125-mL PC bottle (used in an existing MVB process) and 1-L HyClone ® bag (an alternative container to the 125-mL PC bottle).
  • FIG. 54 summarizes the potency of MVB during a 14-day testing period in the 125-mL PC bottle and in the 1 -L HyClone ® bag. The data showed a similar potency drop for the MVB stored in the 125-mL PC bottle and in the 1-L bag for each LAIV strain.
  • Filtration throughput was greater than 300 L/m 2 and a 0.1 log-io FFU/mL or less in potency loss was observed after subsequent filtration of the depth filtrate through the 0.8/0.45- ⁇ filter.
  • the monovalent bulk (MVB) produced following the use of depth filtration in the clarification process showed the same stability when stored in a 125-mL PC bottle and in a 1 -L bag at 2-8 °C for a period of 14 Days. Therefore, a 9.0-0.55 ⁇ depth filter can be substituted for the 1 .2- ⁇ membrane filter in the clarification of PHF.
  • Example 3 Sucrose gradient optimization This example describes certain improvements to the purification process for influenza viruses. Improved purification methods are described for a live attenuated influenza virus monovalent bulk (LAIV-MB) manufacturing process, however such methods may be applied to any influenza virus manufacturing process. Improvements include optimization of a sucrose gradient described in detail below.
  • the ultracentrifugation process sometimes used in the manufacture of cold adapted influenza virus (CAIV) monovalent bulk typically uses a sucrose gradient to concentrate and isolate the virus from clarified harvest fluid (CHF).
  • CHF clarified harvest fluid
  • the sucrose gradient decays over time during the centrifugation process due to the diffusion of sucrose from high concentration to low
  • PBS Phosphate Buffered Saline
  • MEDI part No. 4101086 HYCLONE, Logan, UT, Cat. No. 20012- 043
  • 60% Sucrose in PBS MEDI Part No. 4101084 (HYCLONE, Logan, UT, Cat. No.
  • Equipment used in this example included: Hitachi large scale continuous flow ultracentrifuge (Hitachi, Japan, Model CP40Y); Leica AR600 automatic refractometer (Leica Microsystems Inc., Buffalo, NY, Model AR600); Biosafety cabinet (Baker Co., Stanford, MN, Model STERILGARD III Advance); Peristaltic pump (Watson Marlow Inc., Wilmington, MA, Models 505Di/RL);
  • MASTERFLEX EASYLOAD II pump Cold-Parmer, Vernon Hills, IL, Model No. 77521-40
  • GB1-3 Three compositions of gradient buffers (GB1-3) were used to generate sucrose gradient profiles at different centrifuge run times.
  • GB 1 was produced using 60% sucrose, 10% sucrose and PBS at a ratio of 1.5:1 .3:0.4
  • GB 2 was produced using 60% sucrose, 10% sucrose and PBS at a ratio of 1.35:1.45:0.4
  • GB 3 was produced using 60% sucrose, 10% sucrose and PBS at a ratio of 1.2:1 .6:0.4.
  • PBS was pumped into the bottom port of the ultracentrifuge at 200 - 300 mL/min while rotor speed was maintained at 35,000 rpm for 0, 1 , 3, 5 or 12 hours as described in the table presented in FIG. 58.
  • PBS was re-circulated until a total time rotor spinning at 35,000 rpm was reached. After spinning the rotor at 35,000 rpm for each of the times listed below, the rotor was decelerated to 7,000 rpm under normal braking then coasted from 7,000 rpm to a complete stop.
  • FIG. 1 1 -4 and 6-9, FIG.
  • a development tubing rig with short tubing length was used to load the gradient buffer and offload the sucrose gradient.
  • the empty tubing was connected to the bottom port of the ultracentrifuge before the start of offloading.
  • a centrifuge tubing assembly that mimics an existing tubing rig was used.
  • sucrose gradient was pumped out from the bottom port of the ultracentrifuge at 100 mL/min.
  • the sucrose gradient was collected at 100 ml. per fraction in 125 ml. bottles and tested for refractive index (index - temperature compensated (TC)) and solids - TC to determine the sucrose concentration. Diffusion of sucrose in the gradient based on total centrifuge run time
  • FIG. 59 shows sucrose gradients generated from GB 1 (60% sucrose, 10% sucrose and PBS at a ratio of 1 .5:1 .3:0.4 (e.g., 1 .5 L of 60% sucrose, 1.3 L of 10% sucrose and 0.4 L of PBS)) with 0, 1 , 3, 5, and 12 hour total run times at 35,000 rpm.
  • the inlet line was filled with PBS, which mixed with the sucrose upon offloading. This resulted in a low sucrose concentration for the first fraction collected.
  • the data showed the 60% sucrose continued to diffuse over time during the ultracentrifuge operation from 0 hour to 12 hour run times.
  • the peak sucrose concentration recovered was 56% after 12 hours with the rotor spinning at 35,000 rpm (FIG. 66). Additionally, the concentration of the sucrose gradient front recovered depended on the concentration of initial 60% sucrose which can be varied from 58% to 63% solids - TC. Thus, the concentration of sucrose gradient front recovered can be higher or lower based on the starting concentration of 60% sucrose. In this study, the 60% sucrose ranged from 62% to 63% solids - TC; and the concentration of sucrose gradient front recovered was around 62%.
  • FIGS. 60 and 61 show sucrose gradients generated from GB 1 , GB 2 and GB 3 when rotor speed was maintained at 35,000 rpm for 1 and 3 hours.
  • the volume of 60% sucrose concentration recovered (60% solids-TC or higher) is plotted against the total run time at 35,000 rpm for the three gradient buffer compositions (GB 1 , GB 2, GB 3).
  • the diffusion rate of the sucrose gradient front was estimated by the volume of the fractions that had 60% sucrose concentration.
  • the correlation coefficient of the regression line was greater than 0.95 indicating the gradient front movement within the rotor fit well to the linear model.
  • sucrose gradient profiles of a 30,000 egg batch (UK-300597) and a 15,000 egg batch (UK-300639) from an existing manufacture (FIG. 65) using GB 3 (60% sucrose, 10% sucrose and PBS at a ratio of 1.2:1 .6:0.4 (e.g., 1 .2 L of 60% sucrose, 1 .6 L of 10% sucrose, 0.4 L of PBS)) are compared to the 12 hour-run using GB 1 (60% sucrose, 10% sucrose and PBS at a ratio of 1.5:1 .3:0.4 (e.g., 1 .5 L of 60% sucrose, 1 .3 L of 10% sucrose and 0.4 L of PBS)).
  • the sucrose gradient profile from an existing manufacture (batch 300597) had a front offloading sucrose concentration at approximately 49% compared with 56% recovered from the
  • Example 4 Examples of embodiments A1.
  • a method for making an influenza virus composition comprising subjecting a concentrated viral harvest comprising influenza viruses to centrifugation, thereby producing a clarified viral harvest.
  • a method for making an influenza virus composition comprising:
  • A5 The method of any one of embodiments A1 to A4, further comprising after centrifugation sterilizing by sterile filtration the viral harvest, thereby producing a sterilized viral harvest.
  • A6 A method for making an influenza virus composition comprising subjecting a clarified and concentrated viral harvest comprising influenza viruses to centrifugation, thereby producing a further clarified viral harvest.
  • a method for making an influenza virus composition comprising:
  • the method of embodiment A9, wherein the TFF process comprises use of a hollow fiber cartridge.
  • A1 1 The method of embodiment A10, wherein the hollow fiber cartridge has a pore size ranging from about 500 kD to about 750 kD.
  • A12 The method of embodiment A1 1 , wherein the hollow fiber cartridge has a pore size of about 500 kD.
  • A13 The method of embodiment A1 1 , wherein the hollow fiber cartridge has a pore size of about 750 kD.
  • A14 The method of any one of embodiments A9 to A13, wherein the TFF process is performed using a shear rate ranging from about 10,000 s "1 to about 16,000 s '
  • TFF transmembrane pressure
  • A16 The method of any one of embodiments A9 to A15, wherein the TFF process is performed using a load factor ranging from about 50 L to about 100 L of clarified viral harvest per square meter.
  • A17 The method of any one of embodiments A9 to A16, wherein the TFF process is performed at a filtrate flux rate of at least about 25 LMH.
  • A23 The method of embodiment A22, wherein the viral harvest is concentrated at least about 10-fold.
  • A24 The method of embodiment A23, wherein the viral harvest is concentrated at least about 20-fold.
  • A25 The method of embodiment A24, wherein the viral harvest is concentrated at least about 50-fold.
  • A27 The method of any one of embodiments A1 to A26, wherein viral yield is increased relative to viral yield of a method that does not comprise concentrating the viral harvest prior to centrifugation.
  • A28 The method of embodiment A27, wherein the viral yield is increased at least about 2%.
  • A29 The method of embodiment A28, wherein the viral yield is increased at least about 5%.
  • A30 The method of embodiment A29, wherein the viral yield is increased at least about 10%.
  • A32 The method of embodiment A31 , wherein the viral yield is increased at least about 20%.
  • A33 The method of embodiment A32, wherein the viral yield is increased at least about 50%.
  • A35 The method of any one of embodiments A1 to A34, wherein at least about 100 L of viral harvest is concentrated.
  • A36 The method of embodiment A35, wherein at least about 150 L of viral harvest is concentrated.
  • A37 The method of embodiment A36, wherein at least about 200 L of viral harvest is concentrated.
  • A38 The method of embodiment A37, wherein at least about 400 L of viral harvest is concentrated.
  • A43 The method of any one of embodiments A1 to A42, wherein all or substantially all of the viral harvest is subjected to centrifugation.
  • A44 The method of any one of embodiments A1 to A43, wherein the centrifugation is performed at about 2 °C to about 25 °C.
  • A45 The method of embodiment A44, wherein the centrifugation is performed at about 2 °C to about 14 °C.
  • A46 The method of any one of embodiments A1 to A45, wherein the centrifugation is performed at a speed of about 30,000 RPM to about 40,000 RPM.
  • A47 The method of any one of embodiments A1 to A46, comprising prior to or during centrifugation loading the concentrated viral harvest into a centrifuge device at a particular loading flow rate.
  • A52 The method of embodiment A51 , wherein the loading flow rate is less than about 130 mL/min.
  • A53 The method of embodiment A52, wherein the loading flow rate is less than about 120 mL/min.
  • A55 The method of embodiment A47 or A48, wherein the loading flow rate ranges from about 120 mL/min to about 160 mL/min.
  • A56 The method of embodiment A47 or A48, wherein the loading flow rate ranges from about 140 mL/min to about 180 mL/min.
  • A58 The method of embodiment A57, wherein the continuous zonal centrifugation is performed over a sucrose density gradient.
  • A59. The method of embodiment A58, wherein the sucrose density gradient is a 0% to 100% sucrose gradient.
  • sucrose density gradient is a 0% to 60% sucrose gradient.
  • sucrose density gradient is a 10% to 60% sucrose gradient.
  • sucrose density gradient is generated using equal or substantially equal volumes of a 60% sucrose (w/w) composition and a 10% sucrose (w/w) composition.
  • sucrose density gradient is generated using a volume of a 60% sucrose (w/w) composition that is greater than the volume of a 10% sucrose (w/w) composition.
  • A65 The method of embodiment A63, wherein the sucrose density gradient is generated using volumes of a 60% sucrose (w/w) composition, a 10% sucrose (w/w) composition and PBS at a ratio of 1.3-1.6 to 1 .2-1.5 to 0.4, respectively.
  • A66 The method of embodiment A63 or A65, wherein the sucrose density gradient is generated using volumes of a 60% sucrose (w/w) composition, a 10% sucrose (w/w) composition and PBS at a ratio of 1.5 to 1 .3 to 0.4, respectively.
  • A67 The method of any one of embodiments A62 to A66, wherein the centrifugation has a run time of at least about 9 hours.
  • A69 The method of any one of embodiments A58 to A68, wherein after centrifugation the viral harvest is collected from the sucrose density gradient at gradient coordinates between about 35% to about 49% sucrose.
  • A70 The method of any one of embodiments A1 to A69, wherein after centrifugation the viral harvest is diluted with a buffer.
  • A71 The method of embodiment A5 or any one of embodiments A8 to A69, wherein after centrifugation and before sterilizing, the viral harvest is diluted with a buffer.
  • A72 The method of embodiment A70 or A71 , wherein the buffer is a phosphate buffer.
  • A73 The method of any one of embodiments A7 to A72, wherein the clarifying in (a) comprises use of at least two filter species.
  • A76 The method of embodiment A74, wherein the initial clarification comprises use of at least three filter species.
  • A77 The method of any one of embodiments A73 to A76, wherein the filter species comprise at least one pre-filter.
  • A81 The method of any one of embodiments A77 to A80, wherein filtration throughput is increased when a pre-filter is used relative to filtration throughput when a pre-filter is not used.
  • A82 The method of embodiment A81 , wherein filtration throughput is increased by at least about 1 .5-fold.
  • A84 The method of any one of embodiments A80 to A83, wherein the one or more other filter species have pore sizes ranging from about 0.2 microns to about 3.0 microns.
  • A86 The method of embodiment A84 or A85, wherein the one or more other filter species are selected from filters having pore sizes of about 1 .2 microns, 0.8 microns and 0.45 microns.
  • A87 The method of any one of embodiments A73 to A86, wherein at least one filter is a depth filter.
  • A88 The method of embodiment A87, wherein the depth filter is a stacked depth filter.
  • A89 The method of embodiment A87 or A88, wherein filtration throughput is increased when a depth filter is used relative to filtration throughput when a depth filter is not used.
  • A90 The method of any one of embodiments A1 to A89, further comprising after centrifugation combining the viral harvest with a stabilizer.
  • A93 The method of embodiment A91 , wherein the final concentration is 6.84% sucrose weight/volume (w/v), 1 .21 % arginine w/v, 0.094% monosodium glutamic acid w/v, and 1 % gelatin hydrolysate.
  • A94 The method of embodiment A92, wherein the final concentration is 6.84% sucrose weight/volume (w/v), 1 .21 % arginine w/v, and 1 % gelatin hydrolysate.
  • influenza virus composition is a refrigerator-stable influenza virus composition
  • influenza virus composition exhibits a potency loss of less than 1 .0 log over a 6 to 12 month period when stored at 4°C to 8°C.
  • influenza viruses comprise live influenza viruses.
  • influenza viruses comprise reassortant influenza viruses.
  • reassortant influenza viruses comprise hemagglutinin and/or neuraminidase antigens in the context of an attenuated and/or
  • A101 The method of embodiment A99, wherein the master strain is derived from a master strain selected from the group consisting of A/Ann Arbor/6/60, B/Ann Arbor/1/66, PR8,
  • centrifugation blending the viral harvest with at least one other viral harvest, thereby producing a blended viral harvest.
  • A103 The method of embodiment A5 or A8, further comprising blending the sterilized viral harvest with at least one other sterilized viral harvest, thereby producing a blended viral harvest.
  • A104 The method of embodiment A102 or A103, wherein the viral harvest is blended with two other viral harvests, thereby producing a trivalent blended viral harvest.
  • A105 The method of embodiment A102 or A103, wherein the viral harvest is blended with three other viral harvests, thereby producing a quadrivalent blended viral harvest.
  • A106 The method of embodiment A105, wherein the quadrivalent blended viral harvest comprises two influenza A strains and two influenza B strains.
  • A109 The method of any one of embodiments A1 to A108, which comprises formulating the viral harvest, whereby an influenza virus composition suitable for intranasal administration is produced.
  • A1 10 The method of any one of embodiments A1 to A108, which comprises formulating the viral harvest, whereby an influenza virus composition suitable for administration to a human is produced.
  • a method for making an influenza virus composition comprising:
  • centrifugation comprises continuous zonal centrifugation performed over a sucrose density gradient, wherein the sucrose density gradient is generated by combining a volume of a 60% (w/w) sucrose composition and a volume of a 10% (w/w) sucrose composition, wherein the volume of the 60% (w/w) sucrose composition is equal to or greater than the volume of the 10% (w/w) sucrose composition; thereby producing a further clarified viral harvest; and, optionally,
  • sucrose density gradient is a 0% to 60% sucrose gradient.
  • sucrose density gradient is a 10% to 60% sucrose gradient.
  • sucrose density gradient is generated using volumes of a 60% sucrose (w/w) composition, a 10% sucrose (w/w) composition and PBS at a ratio of 1.5 to 1 .3 to 0.4, respectively.
  • TFF transmembrane pressure
  • B25 The method of embodiment B24, wherein the clarified viral harvest is concentrated at least about 5-fold.
  • B26 The method of embodiment B25, wherein the clarified viral harvest is concentrated at least about 6-fold.
  • B28 The method of embodiment B27, wherein the clarified viral harvest is concentrated at least about 10-fold.
  • B29 The method of embodiment B28, wherein the clarified viral harvest is concentrated at least about 20-fold.
  • B33 The method of embodiment B32, wherein the viral yield is increased at least about 2%.
  • B34 The method of embodiment B33, wherein the viral yield is increased at least about 5%.
  • B35 The method of embodiment B34, wherein the viral yield is increased at least about 10%.
  • B36 The method of embodiment B35, wherein the viral yield is increased at least about 15%.
  • B37 The method of embodiment B36, wherein the viral yield is increased at least about 20%.
  • B38 The method of embodiment B37, wherein the viral yield is increased at least about 50%.
  • B39 The method of embodiment B38, wherein the viral yield is increased at least about 70%.
  • B47 The method of any one of embodiments B1 to B46, wherein the centrifugation in (b) is performed at a speed of about 30,000 RPM to about 40,000 RPM.
  • B48 The method of any one of embodiments B13 to B47, comprising in (b) loading the concentrated viral harvest into a centrifuge device at a particular loading flow rate.
  • B58 The method of any one of embodiments B1 to B57, wherein the clarifying in (a) comprises use of at least two filter species.
  • B59 The method of embodiment B58, wherein the clarifying in (a) comprises use of at least three filter species.
  • B61 The method of embodiment B60, wherein the pre-filter has a pore size ranging from about 3 microns to about 20 microns.
  • B63 The method of embodiment B60, B61 or B62, wherein the pre-filter functions as a pre-filter for one or more other filter species.
  • B64 The method of any one of embodiments B60 to B63, wherein filtration throughput is increased when a pre-filter is used relative to filtration throughput when a pre-filter is not used.
  • B68 The method of embodiment B67, wherein the one or more other filter species are selected from filters having pore sizes of about 0.8-3.0 microns and 0.2-1.0 microns.
  • B69 The method of embodiment B67 or B68, wherein the one or more other filter species are selected from filters having pore sizes of about 1 .2 microns, 0.8 microns and 0.45 microns.
  • B70 The method of any one of embodiments B58 to B69, wherein at least one filter is a depth filter.
  • B71 The method of embodiment B70, wherein the depth filter is a stacked depth filter.
  • B73 The method of any one of embodiments B1 to B72, further comprising after (b) or (c) combining the viral harvest with a stabilizer.
  • B74 The method of embodiment B73, wherein the sterilized viral harvest is combined with a stabilizer to obtain a final concentration of 6-8% sucrose weight/volume (w/v), 1 -2% arginine w/v, 0.05-0.1 % monosodium glutamic acid w/v and 0.5-2% gelatin hydrolysate.
  • B77 The method of embodiment B75, wherein the final concentration is 6.84% sucrose weight/volume (w/v), 1 .21 % arginine w/v, and 1 % gelatin hydrolysate.
  • B78 The method of any one of embodiments B1 to B77, wherein the influenza virus composition is a refrigerator-stable influenza virus composition
  • influenza viruses comprise live influenza viruses.
  • influenza viruses comprise reassortant influenza viruses.
  • reassortant influenza viruses comprise hemagglutinin and/or neuraminidase antigens in the context of an attenuated and/or

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Abstract

L'invention concerne des procédés de production de virus de la grippe. Généralement, la production de virus de la grippe comprend un ou plusieurs processus de purification. L'invention concerne également des procédés de purification qui permettent d'améliorer l'efficacité de purification et d'augmenter le rendement viral sans impacter négativement la stabilité ou la puissance virale.
PCT/US2014/049192 2013-08-01 2014-07-31 Procédés de production de compositions de vaccin contre la grippe WO2015017673A1 (fr)

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CN105106949A (zh) * 2015-10-19 2015-12-02 北京健翔和牧生物科技有限公司 一种鸡新城疫、禽流感二联灭活苗的制备方法
CN105288611A (zh) * 2015-10-19 2016-02-03 北京健翔和牧生物科技有限公司 一种鸡新城疫、传染性支气管炎二联活疫苗的制备方法
US10703072B2 (en) 2015-10-13 2020-07-07 Saint-Gobain Glass France Heatable laminated vehicle window with improved heat distribution
CN112704733A (zh) * 2020-12-29 2021-04-27 深圳康泰生物制品股份有限公司 四价流感病毒鸡胚尿囊液纯化工艺、四价流感病毒裂解疫苗及其制备方法

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US20120115206A1 (en) * 2004-12-23 2012-05-10 Medlmmune Way Non-tumorigenic mdck cell line for propagating viruses

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10703072B2 (en) 2015-10-13 2020-07-07 Saint-Gobain Glass France Heatable laminated vehicle window with improved heat distribution
CN105106949A (zh) * 2015-10-19 2015-12-02 北京健翔和牧生物科技有限公司 一种鸡新城疫、禽流感二联灭活苗的制备方法
CN105288611A (zh) * 2015-10-19 2016-02-03 北京健翔和牧生物科技有限公司 一种鸡新城疫、传染性支气管炎二联活疫苗的制备方法
CN112704733A (zh) * 2020-12-29 2021-04-27 深圳康泰生物制品股份有限公司 四价流感病毒鸡胚尿囊液纯化工艺、四价流感病毒裂解疫苗及其制备方法

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