Considerations in Scale-Up of Viral Vaccine Production
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V ACCINES SECTION TWO Considerations in Scale-Up of Viral Vaccine Production by William G. Whitford and Alain Fairbank n 28 June 2011, the Food and Steve Pettit (director of cell culture Agriculture Organization of development at InVitria) told us. “We the United Nations declared have been working with CDC [US O the Rinderpest cattle plague Centers for Disease Control and virus to be the second troublesome Prevention] and vaccine manufacturers virus (after smallpox) that humans have to help them eliminate animal eradicated from the Earth (1). Such components from vaccine achievements herald exciting times manufacturing.” both for classical vaccinology and for Similar pressures exist for many new and developing technologies. chemically defined and low-protein Here we consider scaling up of vaccines designs, but this is to a lesser degree. and related hybrid, targeted, and Such advanced SFM formulation conjugated viral therapeutics that are features have been accomplished for Fluorescent image of confluent Vero cells on made through animal cell culture. The a SoloHill microcarrier; DAPI-stained nuclei most cell lines used in other areas of vaccine industry is now moving from appear blue, and actin filaments stained bioproduction. But a number of virus production in platforms based on whole with phalloidin-ALEXA 488 appear green. and vaccine production formats still SOLOHILL ENGINEERING (WWW.SOLOHILL.COM) animals and primary tissues (e.g., require proteins, hydrolysates, animal embryonated chicken eggs) to cultured- products, or other media components cell–based production (2). This platforms for vaccine manufacture. for optimum performance, which transition began over 50 years ago with More recent developments in this prohibits such designations. polio vaccine development. It is still in arena include the introduction of Modern SFM, feed cocktails, and progress today, as evidenced by Baxter entirely new cultured cell lines and the supplements provide for such required International’s recent European rapid adoption of large-scale single-use functions as cell nutrition, shear-force introduction of influenza vaccine technologies (Figure 1), protection, cell banking, and virus produced by Vero cell culture. And the replication in many cell lines and soon-to-open Novartis plant in Holly CELL CULTURE MEDIA processes. But individual clone- Springs, NC, one of the first producing Selection and optimization of cell selected or primary cell characteristics flu vaccine by cell culture in the United culture media is important to any require optimization or even redesign States. production-reactor format or cell-line of current formulations. For even well- Scale-up of cell-culture-based platform for a number of safety, established cell lines, manufacturers’ vaccine manufacturing was first process, economic, and regulatory desire for increased production accomplished 60 years ago in reasons. The general direction for all efficiency often demands adherent-cell cultures using large glass platforms is toward use of serum-free customization of feeds and final flasks. That seminal achievement media (SFM) and animal-derived- processes. Growth media for cells eventually gave way to arrays of single- component–free (ADCF) used in vaccine and related therapeutic use roller bottles, which remain in formulations, and the pressure for this applications begin with basal significant use today. Along with the transition is somewhat greater for formulations often developed for each newer platforms of stacked array flasks human than for veterinary vaccines. cell line in consideration of other and macro- and microcarrier materials, “Many vaccines are still purposes. In many cases, however, roller bottles complement fed-batch manufactured using bovine serum or special media requirements are suspension culture as popular other animal-derived components,” imposed by such diverse sources as 16 BioProcess International 9(8)s SEPTEMBER 2011 SUPPLEMENT Figure 1: Animal-cell bioreactor development; (LEFT ) world’s first commercial “cell culture factory” • large-scale culture circa 1981 (New Brunswick Scientific); (RIGHT ) scalable, automated, single-use bioreactor used in • the particular production format vaccine manufacturing circa 2011 (Finesse Solutions) involved • the particular virus being cultured • the degree of product understanding and characterization accomplished to date • product-specific safety and regulatory constraints • the relationship between differential cell mass generation and viral product accumulation. More than a dozen adherent or suspension-adapted cell lines are used by vaccine manufacturers, including both mammalian and insect (e.g., Spodoptera frugiperda Sf9) cells. Mammalian cell lines include Vero (based on African green monkey Figure 2: Adherent cell vaccine production formats; (A) Roller bottles come with standard or kidney cells), Madin Darby canine expanded surfaces and a range of surface areas; pictured are Thermo Scientific Nunc In Vitro PETG brand (Thermo Fisher Scientific). (B) Stacked-array flasks have been optimized for industrial-scale kidney (MDCK) cells, baby hamster production of vaccines; pictured is a Thermo Scientific Nunc 10-tray Cell Factory system (Thermo kidney 21 (BHK-21) cells, and chick Fisher Scientific). (C) Microcarriers have surfaces that support cell attachment and growth; pictured embryo fibroblast (CEF) cells. All are cells on Hillex II microcarriers (SoloHill Engineering). (D) Hollow-fiber bioreactors provide scalable, such lines were established and can be continuous-processing virus production; pictured is a FiberCell Duet system (FiberCell Systems Inc.). cultured in a basal medium with an added animal serum. Concerns specific to large-scale and production formats include foaming propensity and remedies; minimum culture seeding density; medium formulation richness and the ability to support concentrated feeds; the molecular weight of active components (e.g., in perfusion applications); and downstream process facility. Virus- specific and product understanding considerations are numerous and include the fact that some virally infected cultures display a heightened or unique metabolite requirement and that some viruses require special culture media activities such as enzyme activation of virion components. Recent product and process characterization approaches are revealing product heterogeneity in such properties as virus membrane lipid constituents and capsid protein optimization include economic (e.g., amino acids) or special sources of glycomoieties induced by culture considerations such as production cellular energy (e.g., adenosine procedures and media formulations efficiency and cost containment, as triphosphase [ATP]) appear not to be (3). Ambient culture medium has been well as the recent popularity of obligate factors for competent virus demonstrated to influence such outsourcing elements of process replication. However, along with such production parameters as time of development to experienced vendors physiological consequences of infection infection, multiplicity of infection and with deep biomanufacturing as virus-induced host-cell defense and time of harvest. In the vaccine experience. apoptosis, they have proven to be industry, primary drivers in process With some notable exceptions (4, 5), important events to consider in development and materials differential metabolic precursor supply optimization of most virus yields. So 18 BioProcess International 9(8)s SEPTEMBER 2011 SUPPLEMENT Figure 3: Vero-based production in Figure 4: Viral vaccine production history; (LEFT ) Elsie Ward tends cultures designed by Jonas Salk microcarriers; seed train of Vero cells cultured with roller drums containing tube cultures of poliovirus in primary monkey kidney tissue circa 1951 on Hillex II micocarrier beads (SoloHill (University of Pittsburgh); (RIGHT ) Thermo Scientific HyClone 100-L Single Use Bioreactor (SUB) Engineering) capable of pandemic response vaccine production using monkey kidney cell (Vero) culture in serum-free Thermo Scientific HyClone SFM4 MegaVir media circa 2011 (Thermo Fisher Scientific) 60 Passage 3 (10-L bioreactor) 50 Passage 1 2 (0.2-L spinner) Passage 2 40 (2-L bioreactor) Ni/cm 30 4 20 × 10 10 0 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 6 7 Days in Culture Table 1: Comparing egg-based influenza production with Vero-cell–based production using Hillex II microcarriers in Thermo Scientific HyClone SFM4MegaVir media (SoloHill Engineering) Figure 5: Smallpox vaccine production history; (LEFT ) lymph harvest from calf in competent Vaccinia production circa 1882; (CENTER ) scarification of calf in competent Vaccinia production circa Production Panama H1N1 Titers 1981; (RIGHT ) robotic cell culture in MVA Vaccinia production (IDT Biologika) circa 2011 System (log10 TCID50/mL) Egg 7.8 Vero: Serum- 7.9 containing Vero: Serum-free 8.0 ADCF virus and vaccine manufacturers have spent considerable effort in optimization of SFM for production, concerns. In many cases, very similar array of 5-L glass (Povitsky) flasks. and a number of commercially production media support viral vaccine Roller bottle use was originally available SFM have been developed for production from infection-based, developed at Johns Hopkins many established cell lines as well as stably transfected or transduced cell University for growing large quantities for some of the newer and/or lines. But some transient-transfection of adherent cells. In addition to proprietary ones such as Vivalis’s EB66 or -transduced platforms require