Vaccine Production

State of the Art and Future Needs in Upstream Processing
  • Yvonne Genzel
  • Udo Reichl
Part of the Methods in Biotechnology book series (MIBT, volume 24)

Abstract

The production of viral vaccines in animal cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine definition requires a specific production and purification process. Media have to be selected as well as the production vessel, production conditions, and type of process. Here, we describe different issues that have to be considered during virus-production processes by discussing the influenza virus production in a microcarrier system in detail as an example. The use of serum-containing as well as serum-free media, but also the use of stirred tank bioreactors or wave bioreactors, is considered.

Key Words

Vaccine virus influenza large-scale production microcarrier Madin-Darby canine kidney cell adherent virus harvest virus quantification wave bioreactor stirred tank bioreactor 

1 Introduction

1.1 State of the Art

Most commercial viral vaccines for human or veterinary use comprise either attenuated (live) viruses, inactivated (killed) viruses, or purified viral components (subunit vaccines) (1,2). In addition, new technologies are being developed such as viral vectors, vaccines consisting only of defined viral proteins (produced as recombinant surface proteins in yeast), virus-like particles (VLPs), or vaccines containing viral DNA bound to gold particles (DNA vaccines). For large-scale production viruses are typically produced in animals, fertilized eggs, cell lines, tissues, or by culture of genetically modified cells. Depending on the type of vaccine and the virus type, different production processes have to be used.

In animal cell culture viruses are produced either in primary, diploid, or continuous (transformed) cell lines (3). Often adherent epithelial-like cells are used, which are first grown to high cell numbers and subsequently infected, resulting in a biphasic batch process. However, some viruses only replicate in actively growing and dividing cells; thus virus infection is in parallel to the inoculation of cells (seeFig. 1).
Fig. 1.

Overview of inactivated cell culture derived viral vaccine production processes (continuous and diploid cell lines). For primary cell lines scale-up steps are omitted as cells are harvested from tissues directly into the production scale. STR, stirred tank bioreactor.

Safety is of utmost importance for any vaccine, whereas product specifications and production technologies partly differ between veterinary and human vaccines. For cell-culture-derived viruses, well-characterized cell substrates have to be used. Starting materials of animal origin should be avoided if possible, and media including any additives should preferably not contain any ingredients known to cause toxic, allergic, or other undesirable reactions in humans and animals. The titer per dose is defined, as well as the amount of remaining DNA, host cell protein, and endotoxin per dose after purification to reduce cross reactions. The product must be stable, conveniently applicable, and booster vaccinations possibly not be needed. Especially new vaccine types need to be cheap, and development time as well as the time needed for product-to-market must be short.

1.2 Future Needs

Because of new threats like fast-developing pandemics, newly emerging viruses, or bioterrorism, as well as new application fields for viral vaccines in gene or cancer therapy and low-cost vaccines for the third world, certain questions should be reconsidered.

  • Is it economically feasible to optimize existing processes?

  • Can this be done without new approval?

  • Can existing cells lines be genetically modified to high-producer cells?

  • Is it possible to design cells to express desired traits such as suspension growth in protein-free media with optimal virus replication?

  • Is it possible to develop completely defined media?

  • Will batch-to-batch consistency and reproducibility no longer be an issue?

  • Are faster reaction times for adaptation of the process to new virus types possible?

  • Will high-density cultures and perfusion systems allow higher productivity?

1.3 Influenza as an Example

At present not many details of large-scale vaccine-production processes can be found in the literature. Therefore, in this chapter the upstream processing of one viral vaccine production process, namely influenza, is described in detail.

Inactivated human influenza vaccines are still mostly produced in the allantoic cavity of embryonated hen’s eggs. Veterinary influenza vaccines, however, have been produced in large-scale mammalian cell culture systems for many years (4,5). Pros and cons for both production systems can be cited (6, 7, 8).

Not many continuous cell lines are good candidates as a host system. These cell lines have to fulfil the production requirements and be well characterized. Most cells used for virus production need immobilization on a growth surface, resulting in static systems (cell cubes, cell factories), roller bottle systems, or microcarrier cultures. Fetal calf serum (FCS) is still added to many cell lines for better growth and high virus yields, thus bringing animal-derived protein into the production system. FCS not only makes these processes expensive, but also involves the risk of prion contamination. Three cell lines, Madin-Darby canine kidney cells (MDCK) (6,9, 10, 11), African green monkey kidney cells (Vero) (12), and PER.C6 cells (human cell line derived from primary culture of human fetal retinoblast immortalized upon transfection with an E1 minigene of adenovirus type 5) (13), have been cited in the literature for the production of influenza A and B viruses, which meet regulatory requirements. These three cell lines have all been adapted to grow in serum-free media, such as Ex-Cell MDCK for MDCK cells (JRH Biosciences technical bulletin) or EpiSerf (Gibco) (6), Ex-Cell VPRO and Ex-Cell 525 (13) for PER.C6 cells, and Dulbecco’s modified Eagle’s medium for Vero cells (12).

2 Materials

2.1 Cell Culture

  1. 1.

    Adherent MDCK cells from ECACC (Nr. 841211903) cultivated in serum-containing conditions (seesteps 2 and 3). Adapted to growth in serum-free medium (SFM) (seestep 4).

     
  2. 2.

    Serum-containing cell growth medium (CGM): GMEM (Gibco no. 22100-093, powder dissolved in tissue-culture Milli-Q water culture water) supplemented with glucose (final concentration 5.5 g/L) (Sigma no. G-8270), 10% FCS (Gibco no. 10270-106) and 2 g/L peptone (autoclaved 20% solution, International Diagnostics Group no. MC33) and 4.0 mg/mL NaHCO3 (Merck, p.a.); pH adjusted to 6.8 with HCl; sterile filtered (0.22 µm); storage at 4°C (seeNotes 1 and 2).

     
  3. 3.

    Virus maintenance medium (VMM): CGM without serum containing low levels of porcine trypsin (12.5 mg/L; Gibco no. 27250-018) to facilitate infection of cells; sterile filtered (0.22 µm); storage at 4°C (seeNotes 1 and 2).

     
  4. 4.

    SFM: Ex-Cell MDCK (JRH Bioscience, no. 14580-1000M) supplemented with glutamine (2 mM final concentration; Sigma no. G3126), stock solution (200 mM, sterile filtered [0.22 µm], stable at 4°C for 3 months); for virus infection, addition of low levels of porcine trypsin (12.5 mg/L; Gibco no. 27250-018) (seeNotes 1 and 2).

     
  5. 5.

    Trypsin solution for cell detachment: 0.5 g trypsin (1∶250, powder; (Gibco no. 27250-018, 300 USP) and 0.2 g ethylene diamine tetraacetic acid (EDTA) (Sigma no. 101K0012) in 100 mL phosphate-buffered saline (PBS), filtered (0.22 µm) and stored at −20°C (stable for 1 yr) or at 4°C (stable for 3 mo) (seeNote 2).

     
  6. 6.

    PBS solution: NaCl (8.00 g/L), KCl (0.20 g/L), KH2PO4 (0.20 g/L), Na2HPO4 (1.15 g/L) in tissue-culture Milli-Q water, autoclaved before storage at room temperature (seeNote 2).

     

2.2 Cultivation Methods/Cell Growth

  1. 1.

    Static cultivation flasks and roller bottles (RB) 850 cm2 (Greiner).

     
  2. 2.

    Microcarriers: Cytodex 1 (GE Healthcare).

     
  3. 3.

    Stirred tank bioreactor (STR): a 5-L bioreactor (Biostat C, B. Braun Biotech) with temperature, pO2, and pH sensors as well as a dip tube; a PCS7 system (Siemens). Stirrer with two inclined paddle impellers (paddle: 8.5 cm length; 2 cm width; distance from bottom: 9 cm; distance between impellers: 4 cm), four baffles (30 cm in length).

     
  4. 4.

    Wave bioreactor (System 20P, Wave Biotech AG) with 19-in. instrument rack (Wave Biotech AG) and cellbags (CB2L, Wave Biotech AG, LDPE material).

     

2.3 Virus Infection

  1. 1.

    Equine influenza strain A/Equi 2 (H3N8) Newmarket 1/93 or human influenza A/PR/8/34 (H1N1) (NIBSC). Virus seed was stored at aliquots of 10 mL (2.1–2.4 log HA units/100 µL; equine influenza: 2.0–4.0 × 107 viruses/mL; human influenza: 3.2 × 107 viruses/mL from TCID50) at −70°C. Influenza A viruses are classified as S2 pathogens and have to be handled under S2 biohazard laboratory conditions. All active virus should be handled under the laminar flow box. Virus can be inactivated under acidic conditions, by heat, or by corresponding disinfectants and inactivation agents.

     

2.4 Virus Harvest, Clarification, and Inactivation

  1. 1.

    Filters for harvesting: 5- and 1-µm Polyfil II depth filters (P05/P01, Porvair plc); 0.65-µm Flotrex AP depth filters (FAP10/FAP96, GE Infrastructure); 0.45-µm Memtrex AP membrane filter (MMP94, GE Infrastructure).

     
  2. 2.

    Binary ethyleneimine (BEI) inactivation: 2-bromoethylamine hydrobromide (Sigma, no. B-9258); hydroxyethyl piperazine ethane sulfonate (HEPES) buffer (pH 7.5; 0.5 M) (e.g., Sigma, no. H4034).

     
  3. 3.

    β-Propiolactone inactivation: β-propiolactone (Serva Electrophoresis, no. 33672.01); HEPES buffer (pH 7.5; 0.5 M) (e.g., Sigma, no. H4034).

     
  4. 4.

    Sterility test: casein peptone soy peptone medium (CASO): 30 g/L CASO (Fluka no. VM92175923) in tissue-culture Milli-Q water (25 mL per flask), autoclaved.

     
  5. 5.

    Innocuity assay: two 75-cm2 T-flasks confluently grown with MDCK cells (CGM medium).

     

2.5 Virus Quantitation

  1. 1.

    Hemagglutination assay (HA): round-bottomed 96-well microtiter plates (with lid for active samples), a purified chicken erythrocyte solution (Chicken blood is diluted 1∶2 with alsevers solution) (20.5 g/L glucose [Roth], 8.0 g/L sodium citrate [Merck], 0.55 g/L citric acid [Merck], 4.2 g/L NaCl [Merck] in tissue-culture Milli-Q water), then washed three times with PBS and set to 1.9–2.1 × 107 red blood cells/mL with PBS), PBS, plate reader photometer (e.g., Rainbow Spectra, Tecan Instruments) (700 nm) (seeNote 3).

     
  2. 2.

    Virus titration (TCID50): VMM with addition of gentamycin (final concentration 0.1 g/L), PBS, 96-well plate (confluently grown with MDCK cells), ice-cold acetone-solution (80%), primary antibody (40 µL per well of a 1∶5 dilution (filtered PBS) of pretreated primary antibody: equine influenza A anti-goat produced in goat (nanoTools). (As the primary antibody was obtained against equine influenza A plus MDCK cell debris a pretreatment by incubation of a 1∶100 dilution [PBS] of the antibody solution with a confluent MDCK cell monolayer for 30 min at 37°C is needed.) Secondary antibody (40 µL per well of a 1∶500 dilution with filtered PBS; Molecular Probes, # A-11015), fluorescecnce microscopy (seeNote 3).

     

2.6 On-Line and Off-Line Measurements

  1. 1.

    Off-line cell counting: trypan blue solution (1∶2 dilution of the stock solution [1.8 g NaCl and 1.0 g trypan blue in 100 mL Milli-Q-water, 0.22 µm filtered] with PBS), Fuchs-Rosenthal chamber.

     
  2. 2.

    Off-line measurement of basic metabolites: glucose, lactate, glutamine, ammonium, glutamate, sodium, and potassium using either a Bioprofile 100 Plus (Nova Biomedical) or a YSI model 2700 or YSI model 7100 Biochemistry Analyzer (Yellow Springs Instruments) and a Vitros DT60-II (Ortho Clinical Diagnostics) with corresponding consumables.

     
  3. 3.

    On-line measurements: pO2 (InPro 6100/120/S/N, Mettler Toledo), pH (gel electrode 405-DPAS-SC-K8S/120, Mettler Toledo), and temperature (resistance thermometer PT100, JUMO MK Juchheim GmbH & Co.) sensors coupled to monitoring and control system (PCS7, Siemens).

     

3 Methods

Cell-culture-derived influenza vaccines are typically produced in a biphasic process, which comprises cell growth and virus replication. For adherent MDCK cells, the production process starts with the inoculation in T-flasks. After a scale-up into taller roller bottles, the cells are washed, trypsinized, harvested, and transferred into a vessel containing cell growth medium. The inoculation density is adjusted to cell numbers in the range 1–2 × 105 cells/mL at a concentration of microcarriers of about 2 g/L Cytodex 1. After a growth phase of 3–5 d, cells form a dense monolayer and cell numbers have increased four- to sixfold to 0.8–1.2 × 106 cells/mL.

In the next step spent growth medium is withdrawn and cells are washed several times with PBS before adding a serum-free medium containing low levels of trypsin to facilitate infection of cells. Virus seed is added at a low multiplicity of infection (MOI). The culture conditions are maintained at the same levels as before. Influenza is a lytic virus, and maximum cytopathic effect (CPE) occurs typically after 48–72 h with the detachment of the cells from the microcarriers and the release of virus into the medium. Eventually, more than 90% of the cells are in the supernatant and the demand for oxygen ceases, indicating cell death. For the manufacturing of dead vaccines, the bioreactor harvest is clarified into a new vessel and the virus is chemically inactivated by using chemicals such as β-propiolactone, binary ethyleneimine, or formaldehyde. Afterwards the harvest is transferred into a second inactivation vessel for a total inactivation time of 24–48 h at 37°C. The inactivated antigen is clarified through depth filters (optional step) and held at 4°C pending testing by quality control (QC). The final testing includes sterility, innocuity, typing, and a hemagglutination assay as a measure for virus yield. Eventually the inactivated harvest is concentrated and purified by several downstream processing methods before adjuvanting, blending, and filling takes place.

3.1 Cell Culture

  1. 1.

    Adherent MDCK cells are grown at 37°C in CGM in static T-flasks (5% CO2) up to RBs (passaged every 4–7 d, when confluent). RB cultures (850 cm2) are inoculated with approx 1.3 × 107 cells (1.5 × 104 cells/cm2) and grown for 7 d in 250 mL CGM. When fully confluent (1.0–1.4 × 108 cells or 1.2–1.6 × 105 cells/cm2) the cells are washed three times with PBS (without Ca2+/Mg2+) and detached by exposure to 0.05% trypsin/0.02% EDTA (10 mL, about 30 min). The trypsin activity is stopped by addition of an equal volume of FCS to the trypsin/cell solution. The cell suspension is used to inoculate a 5-L bioreactor with microcarriers in CGM (seeFig. 1; seeNotes 1 and 4).

     
  2. 2.

    For serum-free media, detachment with lower trypsin concentrations or only EDTA and shorter incubation times might be necessary. Trypsin activity is stopped by addition of medium or by adding PBS and washing of the cells.

     

3.2 Cultivation Methods/Cell Growth

  1. 1.

    Cultivation in roller bottles: As a typical split ratio from T175-flask to RB 1∶2.4 is taken; 6–8 RBs are needed for a 5-L stirred tank bioreactor; a parallel T75-flask should be kept as reference and control for the microcarrier cultivation; RB rolling speed is typically set to 0.66 rpm (seeNote 5).

     
  2. 2.

    Cultivation on microcarriers: The microcarriers are hydrated in PBS according to the manufacturer’s instructions, autoclaved, and added to the medium. For serum-free conditions: additional preconditioning in serum-free medium at 4°C overnight before addition to the medium in the corresponding final concentration might be needed. Typical microcarrier concentrations are 2 g/L (seeNote 6).

     
  3. 3.

    Bioreactor settings: 37°C, 50 rpm, pH control (1 M NaOH) at pH 7.3, aeration at 40% pO2 by pulsed O2-aeration through a sparger; sterilization, monitoring, and control by a digital control system (PCS7, Siemens). For the washing step before infection, a dip tube (6 mm inner diameter) is inserted into the reactor top to remove the medium above the settled microcarriers (seeNote 7).

     
  4. 4.

    Wave bioreactor settings: 37°C, platform angle of 7°, rocking rate of 15 rocks per minute, and aeration with 2–5% CO2 mixed with air at 0.1 NL/min. For cultivation in a 2-L wave bioreactor, either serum-containing medium or serum-free medium (1 L), microcarriers (2 g/L), and cells (start cell concentration: 2 × 105 cells/mL) are added in a feed bottle, transferred into the cellbag and incubated for 1 h without rocking at 37°C for better attachment. Then the rocking is started, and cells are grown to confluency on the microcarriers (seeNote 8).

     

3.3 Virus Infection

Washing steps (typical dilution is about 1∶2000 to remove spent cell growth medium containing serum) and medium exchange:

  1. 1.

    5-L stirred tank bioreactor with dip tube: Remaining volume after removal of the supernatant is approx 400–500 mL; washing is done at least three times by addition of up to 4 L PBS. Virus and trypsin are added to 4.5 L VMM and transferred into the bioreactor via a steam sterilized addition port.

     
  2. 2.

    Wave bioreactor: The cellbag is taken under a laminar flow hood. After settling of the microcarriers, the medium is removed and the remaining suspension is washed three times with 1 L PBS. Virus and trypsin is added to 1 L VMM that is pumped into the cellbag.

     

Virus seed is added at a low MOI, typically in the range 0.001–0.01 based on TCID50/mL, depending on optimal yield of the corresponding virus subtype (seeNotes 913).

3.4 Virus Harvest, Clarification, and Inactivation

  1. 1.

    Harvesting, clarification: Cultivation broth is directly filtered through a 5-µm depth filter when coming from microcarrier cultivations. Then either a 1-µm Polyfil II depth filter or a 0.65-µm Flotrex AP depth filter is used, and the filtrate is inactivated. In an optional step the inactivated broth is filtered through a 0.45-µm polysulfone membrane filter (Memtrex AP, CMMP94, GE Infrastructure).

     
  2. 2.

    Binary ethyleneimine (BEI) inactivation: The filtrate is inactivated chemically by the addition of 1.5 mM BEI (14) (3.2 mL of a stock solution from 0.41 g 2-bromoethylamine hydrobromide in 4 mL NaOH is added together with 53 mL HEPES buffer to 1 L cultivation broth). After short incubation the reaction is transferred to a new vessel and incubated for 24 h at 37°C. (2-Bromoethylamine hydrobromide and BEI are very toxic; butyl rubber gloves must be used and waste has to be treated correspondingly; 2% citric acid should be used for neutralization.)

     
  3. 3.

    β-Propiolactone inactivation: The filtrate is inactivated chemically by the addition of 3 mM β-propiolactone. Addition of 6.4 mL from the stock solution (0.254 mL β-propiolactone and 7.75 mL PBS) to 53 mL 30 mM HEPES buffer and 1 L cultivation broth); the pH of the reaction is stabilized with HEPES buffer (pH 7.5 as recommended by Budowsky et al. (15,16). After short incubation the reaction is transferred to a new vessel and incubated for 24 h at 37°C. (β-Propiolactone is very toxic; butyl rubber gloves must be used, and waste has to be treated correspondingly; 2% citric acid can be used for neutralization.)

     
  4. 4.

    After clarification and inactivation, the filtrate is stored at 4°C until further processing.

     
  5. 5.

    Sterility test: From each sample two sterility tests are prepared by addition of about 2.5 mL per CASO medium flask and incubation at 37°C. Sterility is confirmed after a minimum of 14 d of incubation with negative result.

     
  6. 6.

    Innocuity assay: From the inactivated broth 1 mL is added to a T75 flask (confluently grown with MDCK cells) in VMM medium. After incubation for 3 d at 37°C the HA titer is determined and 1 mL of the supernatant of the first T75 flask is added to a second T75 flask (confluently grown with MDCK cells) in VMM medium. After incubation for 3 d at 37°C the HA is determined. The titer for the first flask must not exceed a HA corresponding to the dilution factor. For the second flask 0.0 log HA units per 100 µL indicate the successful inactivation.

     
  7. 7.

    For release of inactivated harvests for downstream processing, further testing might be required, for example, innocuity and typing, depending on the corresponding dossiers of the manufactures.

     

3.5 Virus Quantitation

  1. 1.

    HA: Titration of influenza virus by hemagglutination is based on the method described by Mahy and Kangro (17). Serial double dilutions of the test samples (100 µL) are made in round-bottomed 96-well microtiter plates containing 100 µL PBS. Each sample is measured in duplicate. When choosing the improved assay, two rows are needed for one sample as in the second row the sample is analyzed from a 1∶20.5 predilution for higher precision. For the standard assay only one row per sample is needed. To each well 100 µL of a chicken red blood cell solution (2 × 107 red blood cells/mL) is added and incubated for 60–90 min at room temperature. The last dilution showing complete hemagglutination is taken as the end point and is expressed as log HA units per test volume (100 µL). For photometric evaluation the plates are scanned with a plate photometer measuring extinction at 700 nm. A Boltzmann sigmoid is fitted to each data set (from one sample), and the dilution at the point of inflection (one of the parameters) is defined as the end point of the titration. The inverse of the dilution is defined as the volumetric HA activity with units 1 HAU (per 100 µL). An internal standard is used to compensate fluctuations caused by the varying quality of chicken erythrocytes (seeFig. 2A; seeNotes 14, 16, and 17).

     
  2. 2.

    For active virus titration (TCID50) 10-fold serial dilutions of the culture supernatants are prepared in VMM with addition of gentamycin (1% v/v). Prior to inoculation, the cells are washed three times with 100 µL PBS per well. To each well of a 96-well plate (confluently grown MDCK cells), 100 µL of the diluted culture supernatants is added to inoculate (eight replicates per dilution). After 1 d at 37°C, 5% CO2 100 µL of VMM with gentamycin is added to each well, and the plate is subsequently incubated for another day at 37°C, 5% CO2. The plate is washed once with PBS and 100 µL of ice-cold acetone solution (80%) is added to each well for fixation (30 min, 0°C). Then the plate is washed three times with filtered PBS before addition of the primary antibody (40 µL per well of a 1∶5 dilution [filtered PBS] of the pretreated primary antibody). After 60-min incubation (37°C) with pretreated primary antibody, the plate is washed three times with filtered PBS and the secondary antibody (40 µL per well of a 1∶500 dilution with filtered PBS) is added. The plate is washed three times with filtered PBS after 60-min incubation (37°C) and a final volume of 100 µL PBS is added before fluorescence microscopy. The titers of infectivity are calculated from eight replicates according to the method of Spearman-Kärber (seeFig. 2A; seeNotes 1517).

     
Fig. 2.

Virus titer during equine influenza virus production in Madin-Darby canine kidney cells (0–363 h) in roller bottles (MOI = 1). Cells have been cultivated in serum containing medium for 91 h before infection. Profiles of (A) virus titer in log HA units/100 µL (■), virus titer as TCID50 (□); (B) virus titer in log HA units/100 µL (■), virus titer as TCID50 (□), glutamate (●), and total cell numbers in supernatant (▲).

3.6 On-Line and Off-Line Measurements

  1. 1.

    Cell counting is done according to standard procedures. For cell counting from microcarriers, it has to be checked if all carriers are empty after trypsinization. Especially, confluent microcarriers tend to agglomerate, which influences sampling. When counting virus-containing samples, all material has to be inactivated afterwards.

     
  2. 2.

    Basic metabolites like glucose, lactate, glutamine, ammonium, and glutamate can be measured using biosensors (e.g., a Bioprofile 100 Plus or a YSI model 2700 or YSI model). Depending on sample volume and concentration range, one of the two can be more advantageous (seeTable 1; seeNote 18). An on-line measurement of glutamate might be a fast means to follow virus production (seeFig. 2B). Samples can be measured directly or after storage at −70°C. Virus samples should be heat inactivated for 3 min at 80°C.

     
  3. 3.

    During the first part of the process (0–99 h, “cell growth”) on-line data for a typical cultivation show an increased frequency of pure oxygen pulses because of an increasing number of MDCK cells attaching and actively growing on microcarriers (seeFig. 4, p. 471). Cells grow exponentially at the beginning. However, a linear increase in cell numbers is finally observed because of limitations of the available space on microcarriers resulting in contact inhibition. When fully confluent, a maximum cell number of 1.2 × 106 cells/mL is obtained. After washing and the addition of fresh VMM together with virus seed, the aeration pulse frequency slowly decreases and oxygen consumption finally stops completely at about 123 h when all cells are dead after virus replication (99–140 h, “virus production”). The increase of the pO2 signal to about 165% at the end of the cultivation results from the diffusion of oxygen from the headspace of the bioreactor into the supernatant. (Because of sparging with pure oxygen, the partial pressure of oxygen in the headspace is higher than in air.)

     
Table 1

Comparison of Validation Results for Bioprofile 100 Plus and YSI 7200/Vitros

 

Glucose

Lactate

Glutamine

Glutamate

Ammonia

 

Bpc

YSI

Bpc

YSI

Bpc

YSI

Bpc

YSI

Bpc

Vitros

Validated range (mM)

1.1–41.1

2.9–27.8

2.3–27.0

3.4–23.6

0.2–2.6

0.2–2.6

0.2–2.6

0.05–1.6

0.2–5.2

0.03–0.35

Linear?

Yes

Yes

Yes

Yes

No

Yes

Yes

Yes

Yes

Yes

XBa

2.72

0.89

2.13L

1.35

0.17

0.25

0.23

0.05

0.4

0.1

Slope

1.09

0.98

0.92

0.95

1.15

1.06

1.15

1

0.92

0.96

Medium

PBS

Water

GMEM

Water

GMEM

Water

GMEM

Water

GMEM

GMEM

SDb

1.3

0.6

1.5

1

1.2

1.7

1.6

1.8

1.5

5.3

aLimit of quantitation (mM) (definition: XB = 10 sXO)

bRelative standard deviation of the method in %. Degree of freedom 7 (eight measurements for upper and lower limit), one-point detection for the calibration curve.

cBioprofile 100 Plus.

PBS, Phosphate-buffered saline; GMEM, Glasgow minimum essential medium.

Fig. 4.

On-line data from a typical run during influenza virus production in Madin-Darby canine kidney cells on microcarriers in a 5-L stirred bioreactor (medium exchange and virus addition at 99 h): control at 40% pO2 value by pulsed O2-aeration, process monitoring, and control by Siemens PCS7 system.

4 Notes

  1. 1.

    A general overview on regulations, directives, and guidelines for the production of pharmaceuticals can be found in the The Orange Guide (18). For the production of vaccines for human and veterinary use, the corresponding Pharmacopoeias such as EP or USP (19,20) are relevant. In addition, there are several documents giving guidance for the use of cell substrates for production of biologicals and viral safety, for example, by the Food and Drug Administration (21,22). The main aspects of research and development of vaccines are thoroughly described by Gregersen (23).

     
  2. 2.

    All media for production should preferably not contain any ingredients known to cause toxic, allergic, or other undesirable reactions in humans. Other compounds such as pH indicators or approved antibiotics should also be avoided or kept at the lowest effective concentration. Poorly defined components like peptone, serum, and soy hydrolysate should be thoroughly tested for product stability as well as for support of cell growth (especially on microcarriers) and virus yields. Additionally, the influence of culture medium ingredients such as antifoaming agents (pluronic) and detergents on downstream processing and analytics should be verified.

     
  3. 3.

    If possible, approved reference standards should be used, e.g., from the National Institute for Biological Standards and Control or the World Health Organization. For in-house standards, stability over time must be carefully monitored. Changing from one batch of reference standard to the next should be done with a time overlap for thorough parallel testing.

     
  4. 4.

    When using suspension cells, scale-up into larger production volumes is more easily achieved (seeFig. 1). For primary cells, production scale depends on the cell material. Predominantly, bioreactors are started directly from the harvested cell suspension (seeFig. 1).

     
  5. 5.

    For RBs 0.25 rpm might be required for serum-free media with poor cell attachment. Especially for serum-free media, the type of RB and surface treatment, for example, plasma-treated polystyrol or polyethylene terephthalate, might be important for cell attachment. Therefore, different suppliers should be checked.

     
  6. 6.

    The initial number of viable cells and attachment phase to the microcarriers are crucial; otherwise, cell recovery is poor, resulting in reduced cell and virus yields. Especially in wave cultivation, attachment was improved by discontinuous rocking at the beginning of the process. Typically, the microcarriers differ slightly in size.

     
  7. 7.

    During the washing steps pH control and aeration has to be modified to avoid foaming. Air bubbles take microcarriers to the liquid surface, leading to losses in cell and virus yield.

     
  8. 8.

    CO2 and pH control could be added to the wave bioreactor setting. For sampling the rocking has to be stopped.

     
  9. 9.

    Washing steps are needed to remove the serum from the CGM because the trypsin activity needed for infection is inhibited by high protein concentrations. However, these washing steps are time-consuming and complicated and involve additional contamination risks. The use of serum-free media might allow using the same medium for cell growth and virus replication. Washing steps could be avoided, and the process would be simplified from a two-step process to a one-step process. Prerequisite is that no substrate limitations or inhibitions by metabolites occur. Further development can go into the direction of perfusion systems and higher cell densities. Viruses that replicate only in dividing cells have to be added directly at the beginning of cell growth; this results in single phasic (adherent) processes (seeFig. 1).

     
  10. 10.

    Infection can be performed at different MOI (e.g., from 0.0001 to 1). Typically, all MOI result in the same maximum HA titer, only with a different delay between HA increase and maximum titer. As the production of virus working seeds is time-consuming and expensive for industrial processes, lower MOI are preferred over faster virus replication times as long as the same maximum virus yields are reached.

     
  11. 11.

    The time point of infection should be optimized. Usually, cells are infected toward the end of their growth phase when maximum cell numbers are reached. In addition, the concentration of potential inhibitors of cell growth and virus replication like ammonia and lactate should not be too high. For processes without pH regulation, such as RBs or wave bioreactors, the decrease of the pH value resulting from lactate formation should not exceed 6.8.

     
  12. 12.

    Trypsin activity can vary from batch to batch. If trypsin activity in VMM is too low, virus infection can be incomplete, resulting in reduced yield. Also, a delay in virus replication and an extension of harvest time is possible.

     
  13. 13.

    Cell numbers in the supernatant after infection should typically increase with time as the cells go into apoptosis and detach. At the end of the infection phase, most cells detach from their growth surface. However, because of the different cultivation conditions in the RB, stirred tank bioreactor, or wave bioreactor, not all cells detached can be found in the supernatant. In the wave bioreactor cells seem to disintegrate faster than in RBs and are no longer countable.

     
  14. 14.

    The time required for one HA assay is about 2–3 h. The detection limit is 0.3 log HA units/100 µL, which corresponds to about 4.1 × 107 virions/mL, assuming that the number of erythrocytes is proportional to the number of virus particles. The assay was validated with a dilution error for standard HA test: ± 0.3 log HA units/100 µL. For an improved assay, a resolution of ± 0.15 log HA units/100 µL with an error of ± 0.06 (95% confidence interval) can be obtained. To compare different HA data from other labs, the concentration of the chicken red blood cell solution should be defined. We have tested different assay volumes and obtained similar results; thus comparison between laboratories is possible. The number of virus particles is correlated with the number of chicken red blood cells, with a 1∶1 ratio. For accurate mass balances the number of chicken cells should be exactly determined. Maximum value depends on virus strain, cell line, and cultivation conditions.

     
  15. 15.

    TCID50 assays take about 1 wk; one sample requires one microtiter plate; good reproducibility and quality of data can only be obtained when performed by highly trained personal; detection limit: 102.5 viruses/mL, dilution error: ± 0.3 log. Maximum value depends on virus strain, cell line, and cultivation conditions.

     
  16. 16.

    Other virus-quantification methods can be used for further details on the process, such as plaque-forming units (pfu), egg infectious dose (EID50), laser scanning and electron microscopy, laser light scattering, real-time polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay. All assays have to be adapted to the corresponding medium, cell, and virus type.

     
  17. 17.

    To determine the optimal time point of virus harvest during the process, neither pfu nor TCID50 analysis are adequate because analysis time is too long. For routine monitoring, HA assay is the best choice. Glutamate concentration in the cell culture medium, cell numbers, or lactate dehydrogenase activity give additional on-process information about the infection status of cells (seeFig. 2B). Analysis on optimal harvest time for either live attenuated or inactive vaccines can be supported by mathematical models (24) and virus stability studies under process conditions (seeFigs. 2A and 3).

     
  18. 18.

    The Bioprofile biosensor was developed for blood sample analysis. Therefore, calibration curves are needed for cell culture media. The slopes in Table 1 indicate where the Bioprofile biosensor overestimates compared to the YSI measurement. Each change of the service pack requires a new calibration curve. The enzyme membranes used in the biosensors vary in stability and quality, which must be monitored.

     
Fig. 3.

Dynamics of equine influenza A virus replication. Experimental data (◆ TCID50; ▲ HA) and simulation data (—) according to a mathematical model of virus replication. Optimal time for harvesting of live virus is about 20 h postinfection when infectivity peaks. Optimal time for harvesting for inactivated vaccines is about 25–40 h postinfection when HA achieves its maximum. Note: Dynamics strongly depends on virus subtype and multiplicity of infection.

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Yvonne Genzel
    • 1
  • Udo Reichl
    • 1
    • 2
    • 3
  1. 1.Max Planck Institute for Dynamics of Complex Technical SystemsMagdeburgGermany
  2. 2.Otto-von-Guericke-Universität MagdeburgMagdeburgGermany
  3. 3.Institute for Bioprocess TechnologyMagdeburgGermany

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