Industrial Scale Suspension Culture of Living Cells || Industrial Large Scale of Suspension Culture of Insect Cells

Summary Box: Characteristics of Production System

Productionsystems, cells,and products

Expression systems type of cells Type of cells (Spodoptera frugiperda Sf-9 and Sf-21, Trichoplusia ni Tn-5,Bombyx mori Bm-5 and BmN, Drosophila Schneider 2 (S2) cells) andexpression systems (insect cell–baculovirus system and stable insect celllines for transient or continuous protein production)

Typical products preferred for theproduction of

Biopharmaceuticals, vaccines, recombinant glycoproteins, andbiopesticides

Special products Gene delivery vectors for human gene therapy, recombinant glycoproteins,insect stem cells

System maturityProducts on the market

Systems maturity (low-to-high) and products on market (Flublok1, Porcilis1

PVC, Ingelvac1 CircoFLEXTM, Provenge1, Porcilis Pesti1, Circumvent1 PVC,Cervarix1)

Characteristicsaffectingproduct quality

Secretion, product location,proteolysis

Secretion (yes), product location (extracellular, membrane and intracellular)

Post-translational modifications asdisulfide bonds, glycosylation,protein multimers, product size

PTM (protein folding, proteolytic cleavage and glycosylation), proteinmultimers (VLPs and capsomeres), and product size (proteins> 50 kDa)

System robustness, solubilityissues, endotoxins

Systems robustness (small-to-large scale), Solubility issues(e.g. insoluble proteins) and endotoxins (yes – requires removal in DSP)

Systemscharacteristicsconcerninggrowth andproductivity

Maximum specific growth rate,mmax (h�1)

0.029� 0.039

Maximum dry biomassconcentration, xmax (g l�1)

0.6� 1.8

Maximum specific productproduction rate, qP (g g�1 h�1)

0.007� 3.9� 10�3

Maximum volumetric productproduction rate, rP (g l�1 h�1)

0.004� 6.944� 10�3

Maximum product titers in cp,max

(g l�1)0.025� 500� 10�3

Specific maintenance, ms

(g g�1 h�1)—

Yield coefficients YX/S and YP/S

(g g�1)—

Energy and carbon sourceSpecial nutritional requirements

Carbon (organic compounds), oxygen, nitrogen (salts, amino acids,proteins), phosphorous (salts), and carbon dioxide (not required)

Maximum oxygen uptake rate,OURmax (mmol l�1 h�1)

0.0002–6.25

Heat production rate (W m�3) —

Typical duration from inoculation ofa production culture to harvest (d)

3–5

Systems shear sensitivity Sensitive to shear stress generated during oxygen supply and by bubbleentrainment during agitation in bioreactor culture systems

Cost andperformanceaspects

Preferred bioreactor designEquipment standard and typicalproduction scale

Preferred bioreactor design (stirred-tank bioreactors, single-use/disposablebioreactors, rotating wall vessels and wave bioreactors), Equipmentstandard (pH, DO, and OD monitoring devices, mass flow for gas-in andgas-out) and typical production scale (0.5� 2000 l)

Most important production costdrivers of production culture

Production cost associated with medium, bioreactors, purification process,possibility of contaminations and instrumentation to control and monitorthe production process

Process development cost aspectsDuration of process development

Very dependent on product complexity

Key strengths of system Insect cells grow in serum- and protein-free media, IC/BEVS is a lyticsystem, “plug and play” manufacturing platform

Key weaknesses of system IC/BEVS is a lytic system

Issues to be addressed in the future Improvement of production cycle and yields, alternative baculoviruspromoters, development of a fed-batch fermentation process and of a definedgrowth medium, viral and host modifications, co-expression of chaperones

348

10Industrial Large Scale of Suspension Culture of Insect CellsAnt�onio Rold~ao, Manon Cox, Paula Alves, Manuel Carrondo, and Tiago Vicente

Abstract

Since the first reported work on insect cells by Goldschmidt in 1915, morethan 600 insect cell lines have been established. With the generation of thefirst recombinant baculovirus in the late 1980s, the application of insect celllines for expression of heterologous gene products grew exponentially. Today,the insect cell–baculovirus system is well accepted as a universal manufactur-ing platform, as demonstrated by the number of approved veterinary andhuman vaccines. In addition, insect cells are an inexpensive, safe and efficientalternative to human and animal-derived cell lines for studying host–pathogeninteractions, cell metabolism, or cellular and humoral immunity, as well as forthe production of gene delivery vectors for human gene therapy, for example,modified baculoviruses and mammalian vectors generated via adeno-associatedvirus. In the longer term, the comprehensive understanding of the regulationand fate of insect stem cells may enable the use of insect cells as a model orga-nism for the development of novel therapies for human diseases. In this chap-ter, the potential of insect cells is thoroughly discussed, from the basicconcepts in cell culture to their requirements in terms of up- and down-streamprocessing as well as the regulatory hurdles.

10.1History

Early work on insect cells dates back to 1915 when Goldschmidt cultured youngspermatocytes of the Lepidoptera moth Samia cecropia in hemolymph hangingdrops [1]. However, similar to many other a posteriori studies, the unsuitability ofthe medium used rendered impossible the subculturing of spermatogenic cells formore than 3 weeks. The following decades registered fruitless attempts to developa synthetic medium that could sustain the cultivation of insect cells for extendedperiods of time. The first successful development of such medium was provided

Industrial Scale Suspension Culture of Living Cells, First Edition.Edited by Hans-Peter Meyer and Diego R. Schmidhalter.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

349

in 1956 when Wyatt cultivated ovarian tissue from Lepidoptera Bombyx mori larvaein a hemolymph-based synthetic medium [2]. In 1962, the first continuous insectcell line was established [3]. Using a modified version of Wyatt’s syntheticmedium containing ten additional vitamins, Grace was able to maintain cellsfrom the pupal tissues of the emperor gum moth Opodiphthera (former Antheraea)eucalypti, a lepidopteran insect in culture. Since then, more than 600 insect celllines have been established, the majority being derived from the order Lepidop-tera, for example, Spodoptera frugiperda, Trichoplusia ni, and Bombyx mori cell lines,and Diptera, for example, Drosophila cell lines [4].

The initial motivation for establishing a continuous insect cell line was to studyinsect virus pathology and the in vitro production of insect viruses as biopesticidesfor controlling agricultural pests. Host–virus interaction studies enhanced the dis-covery of efficient virus-based biopesticides, the most prominent example beingthe baculoviruses, an insect pathogen member of the Baculoviridae family of largerod-shaped viruses. The development of the first insect cell line would pave theway for the production of insect viruses in vitro rather than in vivo (in larvae), mak-ing the overall process simpler, cheaper, and less tedious. With the advances inmolecular biology and the generation of the first recombinant baculovirus in thelate 1980s, the potential of insect cell lines for expression of heterologous geneproducts using baculovirus vectors became obvious. Smith and co-authors, in1983, were the first to report the construction of a recombinant Autographa cali-fornica nuclear polyhedrosis virus (AcNPV) for the expression of human beta inter-feron in Sf-21 insect cells (Sf¼Spodoptera frugiperda cells) [5]. Today, it is wellestablished that gene products from almost any organism (prokaryotic or eukary-otic) and any cellular location (intracellular, extracellular, or membrane) can beexpressed using an insect cell/baculovirus expression vector system (IC/BEVS).

The widespread implementation of IC/BEVS for recombinant protein expres-sion is related to the versatility and robustness of the system. Baculovirus vectormanipulation is simple, thus providing a fast route from gene cloning to productexpression. The recently approved Flublok1 hemagglutinin-based influenza vac-cine (Protein Sciences, USA) [6] is a success story of how IC/BEVS can sharplyshorten the manufacturing process of biologics in a commercial bio-pharmaceutical setting. Owing to its high transgene load capacity and self-expan-sion nature, baculoviruses are excellent tools for the expression of multimericprotein complexes such as virus like particles (VLPs) in a rather controlled, highyielding, and simple manner [7–9]. For different applications as vaccine candi-dates or tools for biologics, there is industrial interest in using the IC/BEVS plat-form, especially where multi-protein expression is a requirement for the morecomplex constructs: an example is the developed rePAX1 assembly platform forsingle baculovirus co-expression in insect cells [7,10]. Thereby, stable cell linedevelopment and transfection of multiple plasmids required to introduce theessential transgenes are not necessary. Recombinant baculovirus stocks can bereadily amplified to high titers. Since baculoviruses are not infectious to verte-brates, the IC/BEVS is a safe gene expression system. IC/BEVS can sustain highexpression levels of functional heterologous proteins, typically higher than

350 10 Industrial Large Scale of Suspension Culture of Insect Cells

mammalian cells and, when optimized, of the same order of magnitude as yeastand bacteria (E. coli) (Figure 10.1). The process scale-up is straightforward as theinsect cells grow robustly in suspension cultures. The inability of insect cells toproduce proteins with complex mammalian-like glycosylation and sialylation [11]may represent a caveat for the production of particular proteins, such as erythro-poietin. Recent studies have, however, demonstrated that “mammalianized” insectcell lines can be a reality. In one of these pioneering studies, insect cells wereengineered with mammalian genes responsible for the N-glycosylation pathwayand thereafter evaluated regarding their ability to grow and express glycosylatedproteins [12]. In a second example, BmFDL (N-linked glycan-specific b-N-acetyl-glucosaminidase) (Bm¼Bombyx mori cells) was suppressed using a soaking RNAinterference (RNAi) sensitive cell line, BmN4-SID1, thus enabling the expressionof proteins with uniform N-linked glycan of GlcNAc2Man3GlcNAc2 in insect cells[13]. A third example is the expression of respiratory syncytial virus fusion surfaceglycoprotein in Sf-9 insect cells [14].

Simple molecular biology tools for insect cells transformations and baculovirus-based expression vectors have made insect cells an inexpensive, safe, and efficientalternative to human and animal derived cell lines for studying host–pathogeninteractions, cell metabolism, or cellular and humoral immunity, as well as for theproduction of gene delivery vectors, such as modified baculoviruses [48] andmammalian vectors generated via adeno-associated virus (AAV) [49], for humangene therapy. In the longer term, further advances in molecular and biochemicaltechnologies will enable a comprehensive understanding of the regulation andfate of insect stem cells, thus potentially making insect cells an important modelorganism for the development of new therapies for human diseases [50].

10.2Concepts in Insect Cell Culture

10.2.1Cell Types, Expression Systems, and Products

The number of insect cell lines established exceeds 600, the majority beingderived from the order Lepidoptera (moths and butterflies) and Diptera (flies andmosquitoes) [4]. Among the Lepidoptera order, Spodoptera frugiperda Sf-9 and Sf-21, Trichoplusia ni Tn-5 (Tn¼Trichoplusia ni cells), and Bombyx mori Bm-5 andBmN are the insect cell lines most widely used in research and technologicalapplications. The insect cell line from the Diptera order receiving largest interestis the Drosophila Schneider 2 (S2) cell line.

The first insect cell line to be extensively characterized was Sf-21 (formerlynamed IPLBSF21-AE), a cell line derived from the pupal ovarian tissue of the fallarmyworm Spodoptera frugiperda [51]. This cell line is used, for example, to pro-duce a prostate cancer vaccine commercialized under the name Provenge1 byDendreon (Seattle, USA) (Table 10.1). The Sf-9 cell line is a clonal isolate of Sf-21

10.2 Concepts in Insect Cell Culture 351

[70]. These two cell lines share common features such as the ability to grow wellin monolayer and suspension cultures, and amenability to serum-free medium.Sf-21 and Sf-9 cells are susceptible to Autographa californica multiple nuclear poly-hedrosis virus (AcMNPV) infection and are therefore good candidate cells for thegeneration of high-titer baculovirus stocks and expression of recombinant protein.The small size and regular shape makes them ideal for the formation of mono-layers (cell propagation) and visualization of plaques (virus titration). Sf-21 cellsare often used for cell propagation and virus titration. Sf-9 cells are commonlyused for isolation of recombinant clones, generation of high-titer viral stocks, andexpression of recombinant proteins (Figure 10.1). In recent years, Sf-9 cells havebeen used to generate an insect cell line with the necessary cellular machinery toexpress proteins with “human-like” glycosylation patterns. In one of the mostrecent examples, Mabashi-Asazuma and coworkers constructed a Sf-9 derivedinsect cell line, SfSWT-6, which was able to express high levels of recombinantsialylated glycoproteins [71].

The Trichoplusia ni Tn-5 (known as High FiveTM) cell line was isolated from theeggs of the cabbage looper, Trichoplusia ni [16]. It is susceptible to AcMNPV infec-tion and thus, similarly to Sf-21 and Sf-9 cells, it is widely used for the productionof recombinant proteins. This cell line is currently used to produce a vaccine toprevent cervical lesions associated with cervical cancer caused by human papillo-maviruss (HPV), commercialized under the name Cervarix1 by GlaxoSmithKline(London, UK) (see details below and in Table 10.1). High FiveTM cells duplicate inless than 24 h, grow well in monolayer and suspension cultures, and are amenableto serum-free medium. Contrarily to Sf cells, they form irregular monolayers andplaques, making it difficult to identify infection foci and thus estimate viral titers.High FiveTM cells are considered a better host for expression and secretion ofrecombinant proteins than Sf cell lines, commonly yielding 5–10-fold higher titers[71]; conversely, Sf-9 cells are better suited to carry out viral proteins assembly, andthus to produce viruses or virus-like particles (VLPs). Noteworthy is also the devel-opment of expression of recombinant “human-like” glycosylated proteins no lon-ger limited to mammalian cells [72].

The main advantage of the three insect cell lines described above is their abilityto grow in serum- and protein-free media such as SF-900TM II/III or ExpressFive1 (Invitrogen, CA, USA) and HyClone1 (Thermo Fisher Scientific, MA,USA). These media do not contain serum (fetal bovine serum), which not onlydecreases media cost but also facilitates regulatory processes and simplifies thepurification process. In addition, it eliminates issues related with the use ofserum, for example, variation in the concentrations of growth factors, hormones,endotoxins, and hemoglobin from batch to batch [73].Bombyx mori Bm-5 and BmN cell lines were isolated from the ovarian tissue of

the commercial variety “Kolar Gold” of silkworm, Bombyx mori [3]. Bm-5 and BmNcells are commonly used in functional [74,75], host–virus interaction [76,77] andcharacterization [78] studies, and in recombinant protein production as they arereadily infected with AcMNPV. Bombyx mori cells can be constitutively engineeredto express recombinant proteins in a continuous mode. For example, a stable


Bm-5 cell line was successfully constructed, Bm5-hGM-CSF, and used for expres-sion and secretion of the human granulocyte macrophage colony-stimulating fac-tor (hGM-CSF) [79]. Despite their obvious advantages for continuous proteinexpression when compared to lytic insect cell-based processes, non-lytic systemsdo not seem to have progressed towards industrial applications as they have limi-tations such as low protein yields, long times for the establishment of a stable cellline, and high process cost.

The Drosophila S2 cell line was isolated from late stage (20–24 h old) Drosophilamelanogaster embryos [80]. S2 cells are able to grow in monolayer and suspensioncultures at room temperature and ambient CO2. Drosophila S2 cell lines can betransformed either transiently (transfected with the recombinant expression vectoralone) or permanently (transfected with the recombinant expression vector plus aselection vector) to express recombinant proteins without becoming lytic. A down-side of Drosophila cell lines is the presence of retrovirus-like particles and reversetranscriptase activity [81]. The most well known and studied virus related to trans-posable element D found in Drosophila is the retrotransposon called gypsy [82],whose expression is thought to protect the host cell from infection by retrovirusesor baculoviruses sharing a related env protein. Although infectious for Drosophila[83,84], the infectivity of gypsy appears to be very limited [84,85]. Nonetheless,extensive, comprehensive characterization of Drosophila cell lines is mandatory toguarantee product safety. Major applications of S2 cells, besides protein produc-tion, relate to host–virus interaction [86], cell physiology [87], and biochemical andmolecular studies [88,89].

The major application of insect cell lines is the expression of heterologous pro-teins for human and veterinary use. Several insect cell-based systems are commer-cially available to aid the generation of recombinant baculoviruses and stableinsect cell lines for transient or continuous protein production. Insect cell-baculo-virus expression systems include (i) BacPAK from Clontech Laboratories, Inc.(CA, USA), (ii) Bac-to-BacTM, Bac-to-Bac1 HBM TOPO1, BaculoDirectTM andBac-N-BlueTM from Invitrogen (CA, USA), (iii) flashBACTM from Oxford Expres-sion Technologies Ltd (Oxford, UK), (iv) ProEasyTM, ProFoldTM, from AB Vector(San Diego, USA), (v) pTriEx from Novagen (WI, USA), and (vi) BaculoGoldTM

from BD Biosciences (CA, USA). These baculovirus kits/vectors are extensivelyused in combination with Spodoptera frugiperda Sf-9 and Sf-21, Trichoplusia niHigh FiveTM, and Bombyx mori Bm-5 and BmN cell lines (Table 10.1). The genera-tion of stable insect cell lines for transient or continuous heterologous proteinexpression can be achieved using (i) InsectDirectTM from EMD Millipore (MA,USA) or (ii) InsectSelectTM and DES1 from Invitrogen (CA, USA), for example.These non-lytic expression systems are commonly established in Bombyx moriBm-5 and Drosophila S2 cell lines (Table 10.1). The advantages and disadvantagesof all these methods will not be discussed; this information can be found in Refer-ences [90] and [91].

The most frequently used cell lines for large-scale production of recombinantproteins are Spodoptera frugiperda Sf-9 and Trichoplusia ni High FiveTM. Spodopterafrugiperda Sf-21, Bombyx mori Bm-5 and BmN, and Drosophila S2 cell lines are also


used but to a lesser extent. The first protein-based, commercial therapeutic pro-duced in insect cells was Porcilis Pesti1 (Merck, USA), a veterinary vaccine toprevent swine fever based on the E2 protein derived from the swine fever virus(Table 10.1). The second vaccine Circumvent1 PVC (B. Ingelheim, Germany) orPorcilis1 PCV (Merck, USA) is a subunit vaccine to prevent wasting disease inpigs and is based on the porcine circovirus type 2 ORF2 protein as antigen pro-duced in Sf-cells using recombinant baculovirus. The first human commercialprotein-based product generated in insect cells was the aforementioned vaccineCervarix (GlaxoSmithKline, UK) (Table 10.1). Cervarix is a bivalent HPV16 and 18L1 VLP-based vaccine produced in Trichoplusia ni High FiveTM cells using recom-binant baculovirus. Since VLPs are multimeric protein structures identical to thenative virus but devoid of any genetic material (non-infectious), VLP-based biolog-ics are regarded as safe. Many other protein-based therapeutic vaccines and bio-pharmaceuticals produced using the IC/BEVS are in development (Table 10.1 andreviews [92–95]).

In developmental biology, insect cells are widely used for studying host-cellphysiology and metabolism, for example, cellular and humoral immunity, insectviruses, for example, host–pathogen interactions, and microbial pathology, forexample, diagnosis of virus-related diseases. In addition, expressed proteins viathe IC/BEVS can be used for functional analyses or crystallography.

In biomedicine, one of the main applications of insect cell lines is the produc-tion of gene delivery vectors for human gene therapy. These vectors can be gener-ated using various methods. Since baculoviruses per se are potential mammalian-cell gene delivery vectors [48], insect cells can be used for virus replication. Inaddition, gene delivery vectors can be produced via AAV [49] by infecting Sf-9cells with three recombinant baculovirus vectors (Rep-baculovirus, VP-baculovi-rus, and AAV ITR vector genome baculovirus), which encode the main compo-nents of the recombinant AAV production machinery. An example of a genetherapy product based on AAV is the recently approved product Glybera1, agene therapy treatment that compensates for lipoprotein lipase deficiency, whichcan cause severe pancreatitis, commercialized by uniQure (Amsterdam, TheNetherlands). VLPs produced using IC/BEVS have proven to be efficient for invivo gene delivery [96,97]. Recently, in vitro production of gene delivery vectorshas been successfully attempted [98]. In a chemically defined environment ofMgCl2, CaCl2, and ATP (adenosine triphosphate), nuclear extracts from Sf-9 cellsinfected with baculoviruses encoding the simian virus 40 (SV40) major coat pro-tein, VP1, were able to package supercoiled plasmid DNA or RNAi sequences,generating SV40 pseudovirions and thus creating a potential gene deliverysystem.

As more molecular and biochemical tools become available, a comprehensiveunderstanding of the regulation and fate of insect stem cells will certainly beachieved, thus potentially making insect cells an important model organism forthe development of new therapies for human diseases [50].

Finally, insect cell lines have been used for decades for in vitro propagationof insect viruses to produce a biopesticide for the control of insect pests [99].


The most common insect cell line used for the manufacture of insect virus-basedinsecticides is Sf-9.

10.2.2Maintaining Insect Cells in Culture – Requirements of the Bioreactor Design

Insect cells have a round morphology, measuring between 10 and 20 mm in diam-eter (Table 10.2). For example, Sf-9 cells are smaller and more homogeneous thanHigh-Five cells. It is important to highlight that insect cells size is highly depen-dent on the DNA/genome content (M. Cox, personal communication) as well ason the culture conditions such as the medium, temperature, and shear stress [10].

The optimal temperature for insect cells cultivation is 27 �C [101] at which theypresent maximum growth rates (mmax) between 0.029 and 0.039 h�1 (duplicationtime of 18–24 h) and cell densities varying from 0.6 to 1.8 g l�1 (cell dry weight)(Table 10.2). Although insect cells can withstand temperature fluctuations between22 and 37 �C [39,102], cell growth and productivity are affected when temperaturesdeviate from the optimum. Below 27 �C, the specific growth rate is reduced as aconsequence of lower glucose and oxygen consumption. Above 27 �C, maximalcell density and viability are significantly decreased. Although temperatures otherthan 27 �C have been shown to negatively impact protein production [39], recentstudies demonstrate that insect cell capacity to express recombinant proteins canbe enhanced by lowering the normal culture temperature during the protein pro-duction phase [103,104]. By doing this, not only protein expression is sloweddown, which reduces significantly the impairment of the Golgi apparatus, butalso proteolytic activity is mitigated, thus increasing productivity [105,106].

The optimal pH of most insect cell cultures is around 6.0–6.4 [93]; and maydecrease slightly in infected cultures (Table 10.2). Outside this range, the growthrate, viability, and maximal cell density is negatively affected. The impact of pH onprotein production levels is not clear. Nonetheless, one can speculate that varia-tions in extracellular pH lead to significant alterations in intracellular pH, whichsubsequently negatively impacts the cell’s capacity to express heterologous pro-teins. The supply of carbon dioxide (CO2) for pH control is not required sincemost insect cell media are buffered with phosphates rather than carbonates ascompared to mammalian cell cultures.

Typical osmolarities of insect cell cultures are between 300 and 380 mOsm l�1,which is higher than mammalian cell lines. For this reason, insect cells are not assensitive to changes in osmolarity as mammalian cells [107]. Nonetheless, a signif-icant reduction in protein expression titers is observed when osmolarity increasesby 30 mOsm [108].

Insect cells media is normally rich in organic compounds (e.g. carbohydrates),amino acids, and salts (Table 10.2). Although not widely used today due to safety(presence of contaminants and adventitious agents), regulatory (longer time forcommercial approval), cost (more expensive), and process efficiency (complicatesdownstream processing) issues, supplementing the media with serum, for exam-ple, fetal bovine serum, can enhance cell growth and protein expression. Several


serum-free media are commercially available for insect cell culture [93]. Some ofthem contain yeast extract and lactalbumin hydrolysate while others are alreadyprotein-free (see Section 10.2.1 for examples). In-house developed media haveproven to be as efficient as commercial media for insect cell growth and produc-tion of recombinant proteins; moreover, their cost might be significantly reduced[111]. The main carbon source for insect cells is glucose. The nitrogen source forprotein synthesis during cell growth is provided by amino acids such as aspartate,asparagine, glutamate, glutamine, serine, and alanine [112,113].

An important variable to consider/control when culturing insect cells is theaccumulation of CO2. As mentioned above, CO2 is not required for cell growth,but normally accumulates in the medium as a by-product of cellular growth. Athigh amounts (>24 mM), CO2 strongly inhibits cell growth and protein expres-sion but not cell viability [114–116]. The reason for such an effect is not yet fullyunderstood but it is thought that the dissociation of CO2 molecules into HCO3

�

and Hþ ions, in culture medium and cell cytoplasm, triggers a series of mecha-nisms with negative impact on cell metabolism such as oxidative stress, intra-cellular pH acidification, and medium osmolality variability [116,117].

Insect cells can be cultivated in anchored, for example, tissue culture flasks, orin suspension culture systems, for example, Erlenmeyer, shaker flasks, spinners,roller bottles, and bioreactors, to high cell densities (Table 10.2). Insect cells sub-culturing must be performed when cells reach mid-log phase of growth, around90% confluency (adherent), or 3–5� 106 cells ml�1 (suspension), and seeded to anew culture at a cell density of 2–5� 104 cells cm�2 (adherent) or 3–5� 105 cellsml�1 (suspension). They can be cultured indefinitely although for passagesbeyond the fiftieth cells begin to show morphological changes. Cells are easilyadapted to suspension cultures, either in media containing serum or in serum-free media, and are normally stored at �80 �C (working cell bank) or in liquidnitrogen (long-term storage).

Oxygen is a key nutrient for insect cell growth. Depending on the culture system(anchored and suspension), cultivation phase (cell growth or infection/productionphase), media, and cell line, the consumption of oxygen by insect cells (OUR –

oxygen uptake rate) varies significantly. In anchored systems and some suspensionculture systems (Erlenmeyer, shaker flasks, and spinners), the amount of oxygenavailable for cells to consume is limited to the volume of the headspace. An accu-rate estimation of the working volume and cell concentration to use based on OURis thus essential so that cells do not run into hypoxia conditions. In suspensionculture systems such as bioreactors, oxygen is supplied via sparging or surface aer-ation systems. The rate of oxygen supply (oxygen transfer rate – OTR) within bio-reactors should at least equal the OUR in order to provide cells with the necessaryamount of oxygen for their growth. This is normally met by increasing the agita-tion rate, enriching the sparged gas with oxygen, or by increasing the gas flow rate.The control of dissolved oxygen (DO) is essential to avoid oxygen limitation orexcess, inhibiting the synthesis of proteases or oxidative damage to proteins [118],and maximizing cell growth. During the growth phase, most insect cell lines arenot significantly affected by DO levels between 5% and 100% [100,119,120]. In


contrast, during the infection/production phase, different cell lines have showndifferent DO sensitivities; Sf-9 cells have been shown to be less sensitive to DOthan High FiveTM, Bm-5, and Sf-21 cells [93]. Notably, no apparent consensus existsregarding the DO values at which protein production in insect cells is maximized.Some studies constrain optimal expression yields to DO values around 25–30%[119,120], others at 50% [121], while others even report no impact of DO on pro-tein production [122].

Insect cells are sensitive to shear stress generated during oxygen supply and bybubble entrainment during agitation in bioreactor culture systems [123]. Theimpact of oxygen supply on insect cells can be minimized by selecting the appro-priate oxygen delivery system: surface or sparged aeration. Surface aeration createsminimal stress to cells as oxygen is supplied through a bioreactor’s headspace (nobubble formation). However, it requires a high surface area to volume ratio forefficient oxygen supply, thus rendering difficult its large-scale application (>500 l)[124]. Sparged aeration is the most widely used system for oxygen supply in small-and large-scale bioreactors. Using an air inlet pipe, oxygen is delivered directly tothe culture medium, thus making O2 molecules accessible to cells in a faster andmore efficient way. An important parameter to control is the size of the bubblesgenerated during sparging. It must be tuned in such a way that oxygen is suppliedin sufficient amounts, cells are not negatively affected, and gaseous metabolicbyproducts, for example, CO2, are efficiently removed. Small bubbles provide bet-ter oxygen transfer than large bubbles. However, the damage inflicted to the cellsis markedly more severe. The agitation rate must be carefully selected to minimizethe hydrodynamic shear stress to cells while providing adequate mixing to keepcells in suspension, distribute nutrients homogeneously, and supply enough oxy-gen to cells. Low agitation rates induce negligible shear stress but impair cellulargrowth as oxygen is not supplied at high enough amounts. On the other hand,high agitation rates promote better oxygen supply due to increased mass transferarea and bubble break-up. The downside is that vortex and foam formation aresignificantly enhanced, negatively affecting cell viability and growth. To circumventthis, addition of non-ionic copolymers, for example, Pluronic1 F-68, is essentialsince they reduce the culture medium surface tension impeding the attachment ofcells to bubbles [125] and interact with the cell membrane, increasing its rigidityand its resistance to hydrodynamic forces. If one wants to rank some insect celllines regarding their capacity to tolerate shear stress, High-Five cells appear as themost tolerant, followed by Sf-9, and finally Sf-21. Interestingly, this order is main-tained on looking at tolerance towards pH and osmotic stress [70,102,126].

Insect cell culture in bioreactors can be performed in four basic modes of opera-tion: batch, fed-batch, perfusion, and continuous cultures. In batch operation, therun starts with the inoculation procedure and ends with the harvesting of theproduct; addition or removal of media does not occur. Typical cell densities forbatch cultures are 1–14� 106 cell ml�1 (Table 10.2). Thus, the control of toxicproduct formation and nutrients consumption is essential to guarantee normalcell growth and protein expression. To avoid process limitations, fed-batch strate-gies can be used [127]. In fed-batch operation, the aim is to maintain the


concentration of key nutrients constant while limiting the accumulation of toxiccompounds so that cells can be maintained in culture for longer periods of time,thus increasing cell concentration and viability, and subsequently final titers. Thisis normally achieved through selective addition of nutrients and/or amino acidsvia complex, robust control systems that in most cases are none-existent and needto be developed in-house. Although extremely high cell densities can be reached(57� 106 cell ml�1) [128] (Table 10.2), the scaled-up process is expensive since cul-ture medium is inefficiently used. In addition, fed-batch operation may requirethe use of larger bioreactors to accommodate the volume of media added duringthe run. In perfusion mode, cells are retained within the bioreactor while replen-ishing the media and removing toxic by-products. This enhances cellular growthand protein expression [129] but requires large volumes of media as well as cell–medium separation devices that negatively impact cellular growth rate, proteinexpression, and production cost. Continuous culture systems enfold shorter man-ufacturing throughput times than batch or fed-batch models as the number ofprocessing steps (from raw materials to finished goods) is significantly lower.However, continuous bioreactors operated for long periods of time are highly sus-ceptible to contamination by microorganisms, for example, yeast and bacteria.

Several types of bioreactors are available for insect cell culture, for example,stirred-tank bioreactors, rotating wall vessels, and wave bioreactors [130]. Thestirred-tank bioreactor is the most widely used type of bioreactor. It consists ofa cylindrical shaped vessel with a radial flow impeller, for example, flat-bladeand Rushton, or axial flow impeller, for example, marine, to maintain cells insuspension and a homogeneous oxygen concentration throughout the bio-reactor. Air supply, mainly oxygen, is achieved via surface or sparged aerationsystems. A rotating wall vessel is a cylindrical type vessel similar to stirred-tankbioreactors but rotating around a horizontal axis. This system was optimized toproduce a laminar flow and thus reduce significantly the shear stress to cells inculture [131,132]. Wave bioreactors consist of a flexible plastic chambermounted on a rocking platform that generates waves [133]. The waves areresponsible for keeping cells in suspension as well as for appropriate and uni-form distribution of oxygen and nutrients. Air, O2 supply is provided through asterile filter. This is a blade- and bubble-free system and thus generates lowshear stress to cells in culture. Wave bioreactors have proven to be efficient forrecombinant protein production using the IC/BEVS [134].

Over the last decade, increased cost pressures during large-scale manufacturinghave led to game-changing stimuli in upstream processing. The biopharmaceuticalindustry, including the many contract manufacturing organizations (CMOs),started to shift their bioreactor equipment to single-use, disposable formats. Anincreasing number of disposable bioreactors are in use, both in R&D and manu-facturing stages. These bioreactors are well suited for multi-product facilities asthey reduce significantly the likelihood of cross-contamination and eliminate clean-ing validation activities. Nevertheless, they are limited at present to “pilot” scales(<2000 l). With the increase on volumetric yields due to cell line and bioreactiondevelopment, such bioreactors incrementally became the preferred choice in


comparison to stainless steel stirred-tank bioreactors. Table 10.3 focuses on differ-ent state-of-the-art single use bioreactors being used or developed for large-scalecell cultures, including references for further reading and case studies.

10.2.3Insect Cell Metabolism: A Brief Overview

A comprehensive understanding of insect cell metabolism under distinct cultureconditions, for example, cell growth and infection/production phase, is critical forthe design of robust processes that could maximize cell growth and product for-mation (recombinant proteins, virus-based pesticides, or insect cell-virus basedproducts) simultaneously.

The preferred carbon source of insect cells is glucose [138]. Insect cells can alsometabolize five-carbon sugars (fructose) and disaccharides (sucrose, maltose, and tre-halose), the latter after hydrolysis. Fructose and trehalose are only consumed afterglucose is depleted from the media [11,139]. Sucrose is consumed in both cellgrowth and infection/production phases, although more in the later phase [140].

Amino acids fulfill the nitrogen requirements of insect cells, being essential forthe production of energy and biosynthesis [112,113]. Since insect cells cannot syn-thesize most of the amino acids by themselves [141], their supplementation to themedia is essential to guarantee cell growth and, if applicable, protein expression.Although the recipe of most commercial insect cell media is not disclosed, it islikely that it includes a complex mixture of amino acids. The most essential aminoacids for cell growth and protein expression in Sf-9 and High FiveTM cell lines areglutamine, asparagine, aspartic acid, glutamate, arginine, methionine, and serine[112]. Within this list, glutamine, asparagine, and aspartic acid are the amino acidswith higher consumption rates; the remaining amino acids are consumed to alesser extent.

Insect cells are extremely efficient in adapting their metabolism to changesin media properties. For example, when sensing glucose-limiting concentra-tions, insect cells rapidly change their metabolism and start to consume aminoacids, mainly glutamine, for energy generation [140]. Briefly, glutamine is firstconverted into glutamate and further on into a-ketoglutarate, which feeds thetricarboxylic acid (TCA) cycle for sustaining cellular growth [142]. The maindrawback in this two-step process is the production of ammonia, a by-productwhose accumulation significantly reduces the cell growth rate [143] (see belowmore details). If glutamine becomes limiting, asparagine and aspartic acid areused as carbon source [107]. Once all energy sources are exhausted, cell apo-ptosis takes over and cells start to die, some faster (Sf-9) than others (HighFiveTM – consume lactate to maintain viability) [93]. An alternative pathway forenergy generation in glucose-limited conditions could be the consumption ofTCA intermediates. However, insect cells are well known not to consumethem during cell growth or infection/production phases [140]. Under excessiveamounts of glucose, lactate is accumulated to high amounts in High FiveTM

cells (10–20 mM) while in Sf-9 cells no lactate accumulation is observed


[107,140,144]. The accumulated lactate in High FiveTM cells is exported to themedium, leading to a decrease in extracellular pH and the formation of cellclumps [111]. Under oxygen-limiting conditions and excessive glucose, lactateis generated independently of the cell line. Cell growth is not significantlyarrested until concentrations of lactate reach 8–10 mM [145,148]. After glucosedepletion, insect cells can use lactate as a carbon source [147].

As mentioned above, the accumulation of ammonia as a by-product of gluta-mine metabolism (glucose-limited conditions) significantly reduces cell growth[143]. The underlying mechanism of ammonia toxicity to insect cells has not yetbeen clarified but, if similar to mammalian cells, it involves (i) increase in extrac-ellular pH due to export of ammonia out of the cells; (ii) mitochondria acidifica-tion due to an increase in proton concentration as result of the outflow ofammonia from the mitochondrial matrix to the cytoplasm; (iii) cytoplasm acidifi-cation in consequence of several cycles of transport of ammonia from cytoplasmto extracellular medium and transport of ammonium from environment to cyto-plasm; and (iv) changes in the potassium gradient as a result of the competitionbetween potassium and ammonium cations for the Naþ/Kþ-ATPase transporters[148,149]. The concentrations of ammonia at which insect cell growth is signifi-cantly impaired are 10–30 mM [107,113,149]. Noteworthy, Sf-9 cells are able to pro-duce alanine instead of ammonia, a non-toxic by-product that serves as anammonia sink to detoxify the medium [150].

Other nutritional requirements of critical importance for cellular growth and/orproduct formation are lipids, cholesterol, and vitamins. Since insect cells areunable to synthesize, desaturate, and elongate fatty acids, supplementation of cul-ture media with lipids is essential to avoid cell degeneration and formation ofdefective interfering particles (non-infectious viruses) [150,151]. The supplementa-tion of cholesterol and vitamins is also required (insect cells cannot produce them)as they play major roles in membrane formation and regulation of key metabolicenzymes [152].

10.2.4A Bottom-Up Approach for Industrial Insect Cell-Based Cultures

Although bacteria and yeast systems generally achieve higher productivity levels,the IC/BEVS allows us to generate hundreds of mg l�1 of recombinant proteinproducts such as single or multiple proteins, multimeric protein complexes, forexample, VLPs, naked capsids, or enveloped particles [95] (Figure 10.1). In addi-tion, the IC/BEVS has been used as a workhorse expression system for recombi-nant protein production requiring post-translational modifications (PTMs) [153];insect-cell-produced proteins can undergo most PTMs (folding, proteolytic cleav-age, glycosylation, disulfide bond formation, and secretion) (Table 10.2). The maincaveat of insect cells was until recently their inability to express proteins thatrequire complex N-glycosylation for their function due to the lack of N-acetyl-glu-cosaminyltransferases. Instead, expressed proteins had high mannose or trun-cated structures, containing two to six mannose (Man) residues linked to the core


[11]. This was overcome by using insect cell lines that have been engineered withmammalian genes responsible for the N-glycosylation pathway. In one of the mostrecent examples, Mabashi-Asazuma and coworkers constructed a Sf-9 derivedinsect cell line, SfSWT-6, which was able to express high levels of recombinantsialylated glycoproteins [71]. These “humanized”-insect cell lines might be neces-sary in some applications and can prove to be a good compromise before switch-ing to a mammalian expression system, typically at an expense of a lower productyield, even if the same vector is used to deliver the transgene DNA to the hostcells [154].

10.2.4.1 Upstream Process Development Strategies

The Expression System: Lytic or Non-Lytic Insect cells are very useful for expres-sion of recombinant proteins in large quantities. High expression levels are com-monly achieved using IC/BEVS and are explained by the ability of the virus toshut off the transcription of early host genes and the consequent allocation of thecellular transcriptional and translational apparatus for the expression of the heter-ologous gene(s). The main drawback is its lytic nature. Since cell lysis occurs bythe end of the cultivation, the release of proteases may negatively impact the quan-tity and quality of the recombinant protein. In some cases one may benefit fromthese proteases for the removal of certain “contaminants.” In addition, the lyticnature of this system may complicate the purification steps following harvesting(see Section 10.2.4.2 for details).

Defective baculoviruses tend to be generated in viral stocks passed over manytimes. These so-called defective interfering particles may replicate and alter theoutcome of the infection process when co-infecting insect cells together with anon-defective recombinant virus [155].

Finally, as seen before, viruses are generally engineered with a late promoterthat becomes active only at the end of the infection; hence, the full expressionpotential of the vector may not always be exploited.

The deletion of the proteases chi-A and v-cath included in the rePAX1 assemblyplatform (Redbiotec, Switzerland) to open the secretory pathway and keeping thecell membrane intact for a longer time period after infection opens the door forimproved production of secreted proteins, especially enveloped VLPs.

To circumvent the lytic nature of the IC/BEVS, it is nowadays possible to tran-siently or stably transfect the expression vector into the chromosomal DNA ofinsect cells for subsequent protein expression [156]. A key milestone in the gener-ation of such insect cell lines is to rapidly screen and identify the appropriateexpression vector and/or system for target protein production. Recent years haveseen tremendous efforts in this area. Rapid, high-level protein expression andpurification from insect cells can now be achieved using commercially availablekits [157] (see Section 10.2.1 for details). New expression vectors have also beendeveloped to allow efficient and cost-effective parallel cloning and thus screeningof different protein constructs, tags, and expression hosts [158]. Without doubt,interest in these non-lytic systems has been growing as they commonly lead to


high protein yields and fast expression of recombinant proteins. However, sincethese non-lytic systems require an additional screening step of selecting viableclones [159], industrial implementation is still questionable.

The Insect Cell Media: from Design of Experiments to Metabolic Flux AnalysisThe design of an insect cell medium that can sustain efficiently cell growth andprotein expression via baculovirus infection is quite complex, involving the adjust-ment of numerous interacting components and their concentrations. Traditionalmethods were extremely time-consuming as one had to perform several rounds oftitrations of each individual medium component, while keeping all other compo-nents constant at original levels, until the optimum is achieved. At each step ofthis iterative procedure, cell growth and protein expression has to be assessed andthe process revised accordingly. The use of design of experiment (DoE) statisticalmethods allowed the development of insect cell media in a more rational manner.Since DoE takes into account the interaction between different media compo-nents, the number of conditions to test is reduced and the time required formedia development is shortened. Ultimately, this may lead to substantial cost sav-ings while maintaining high cell densities and product yields [111]. With the dawnof systems biology tools and its “omics” technologies, for example, metabolomicsand proteomics, it is now possible to have comprehensive knowledge of how hostcell physiology changes in response to virus infection and protein production, andthus it is possible to design in a more efficient way an insect cell medium withbeneficial properties for both cell growth and protein expression. For example,metabolic flux analysis approaches have been used to explore the central metabo-lism of Sf-9 [140,160], providing useful information for bridging cells metabolic/energetic state with improved cell culture performance (protein expression orvirus propagation).

The Macro-environment: Optimal Bioprocess Parameters Protein expression inthe insect cell baculovirus expression system is highly dependent on processparameters such as the multiplicity of infection (MOI – number of virus per cell),time of harvest (TOH), and cell culture at infection (CCI). The optimal MOI isdependent on the target product (single or multiple protein expression, secretedor non-secreted), the production strategy (single- or co-infection), and the viraltiter. For single protein expression, 0.01<MOIs (virus/cell)< 1 normally inducehigher volumetric protein productivities than MOIs> 1 as a consequence ofhigher infected cell concentrations [161]. In theory, this difference could be bal-anced with the increase in CCI. However, cell specific productivity decreases asCCI increases due to the “cell density effect” [140]. For multiple protein expres-sion, the self-assembly of protein complexes is highly correlated with the produc-tion strategy; thus, several combinations of MOI and CCI may maximize productformation. The main advantage of low MOIs is that they require low concentra-tions of viruses. The major downside is the action of proteases; because the overallprocess is slow (requires several rounds of infection), the product of interest isexposed to proteases for extended periods of time, which compromises yields and


product quality. The addition of anti-proteases is then critical to alleviate productdegradation. On the other hand, high MOIs require large viral stocks and potenti-ate the selection of fast-replicative defective viruses [155]. Interestingly, commer-cial manufacturing of recombinant proteins relies on MOIs> 1 as they guarantee100%, synchronous infection, thus narrowing the time-frame at which peak pro-duction (TOH) is achieved and potentially reducing operating costs.

Although previously suggested to be between 72 and 120 h post-infection (40% and70% of cell viability) [120], the optimal TOH is highly protein specific and dependenton how sensitive the heterologous protein is for cleavage. The selection of optimalTOH is of special relevance if the production of complex products such as envelopedvirus-like particles with labile surface target proteins (as in a vaccine) is intended.With the increase of cell death post infection, protein degradation is enhanced anddownstream processing becomes more difficult as contaminant proteins (host andviral) are released to the extracellular medium along with the product.

Systems Biology Tools for Production Optimization The aim of systems biologyand its “omics” technologies, for example, genomics, transcriptomics, proteomics,metabolomics, and fluxomics, is to provide a comprehensive analysis of the sys-tem, from genes to proteins, so that product yields or productivities can be opti-mized. Although extensively studied, insect cell-based systems still suffer fromthe current unavailability of systems-level tools such as those presented above,thereby hampering the understanding of the dynamics of host cell response tobaculovirus during protein expression. This is mainly due to the non-existence ofinsect cells genome sequences and the scarcity of curated databases [162]. None-theless, a series of recent studies have enriched our knowledge of this complexsystem. For example, microarray analysis was performed to analyze the impact ofbaculovirus infection on host gene expression in Sf-21 cells [163]. Results indicatethat most genes were down-regulated upon infection, including genes related toprotein expression and trafficking in the endoplasmatic reticulum (ER) and Golgi,with the exception of a small number of up-regulated genes, for example, heatshock protein 70 s. This is considered the first study providing a comprehensivehost transcriptome overview of Sf-21 cells during baculovirus infection. The firstcomparative quantitative proteomic analysis of the response of Sf-9 cells to baculo-virus infection was provided by Carinhas and coworkers in 2011 [164]. Using thestable isotope labeling by amino acids in cell culture technique, they were able toidentify key proteins with significantly different expression levels upon infection.These are connected to energy metabolism (PDH-E3 and ALDH), ER and oxida-tive stress (chaperone ERp57 and polypeptide transporter SRP57). Both PDH-E3and ALDH were up-regulated, thus indicating an increased metabolic flux in thecentral carbon metabolism during baculovirus infection. The chaperone and trans-porter were both down-regulated, thus corroborating the idea that baculovirusesare able to take control of host cell machinery upon infection, regulating and con-trolling its use for protein expression. More recently, next-generation sequencinghas been used to investigate the impact on gene expression levels following bacu-lovirus infection of Bm-5 cells [165]. Results show that gene sets related to energy


metabolism, ubiquitin–proteosome pathways, transcription, and translation aredifferentially expressed. This data is being further used to construct a bio-molecular network of interactions reflecting the impact of virus infection on hostcell metabolism.

10.2.4.2 Downstream Process Development StrategiesThe downstream processing (DSP) of IC/BEVS-based products is typically a chal-lenging task as it depends on several factors: (i) product complexity, for example,single or multiple proteins, multimeric protein complexes, and viral particles;(ii) morphology and structure of the product, for example, size and molecularweight; (iii) nature of the product (extracellular or associated with cellular compo-nents); (iv) bioreaction medium; and (v) stability of the bioproduct, among others(Figure 10.2). The DSP should be accomplished with as few steps as possible andbe based on simple but efficient unit operations compatible with the requiredquality requirements.

In fundamental research, IC/BEVS-generated proteins typically include His-tagsallowing the use of immobilized metal affinity chromatography (IMAC) [166].This affinity chromatographic process facilitates simple and rapid purification of agiven preparation for further downstream biochemical analysis. Typically, highpurities are obtained when using an affinity process, as the selectivity for the His-tagged protein is high. However, specifically with the insect cell derived bioreac-tion bulks, proteases and also the baculovirus vectors used for infection also showaffinity to the metal-charged IMAC matrix [103]. Such a drawback thus requiresprocess development if high purity is necessary for a certain application. In addi-tion, IMAC is rather expensive at industrial scales due to matrix costs and chem-icals used; moreover, the use of coordination metals, such as Ni, poses someregulatory concerns.

In pre-clinical and clinical applications, the downstream processing strategy hasto accommodate several requirements and constraints so that product purity,potency, and consistency are met. In addition, it must enclose high yields, shortthroughput times, and low overall cost for the product to be commercially compet-itive [167]. To achieve this, process scalability and robustness is of critical impor-tance [168,169].

A clear trend observed in bioprocess engineering is the development of fullyscalable unit operation steps that can be easily scaled-up in later stages toallow straightforward technology transfer to current GMP (good manufacturingpractice) facilities. In the specific case of VLPs or viruses the classical, labora-tory-scale purification based on gradient ultracentrifugation methods are pro-gressively being replaced with scalable processes such as microfiltration,tangential flow filtration, and chromatography [170,171]. Within a given prod-uct development program, ultracentrifugation-based procedures, which arenon-scalable and labor-intensive, remain key tools to prepare reference materi-als for early stage product function/biological integrity screening as well asbenchmark material for analytics serving quality control both up- and down-stream. In some cases, even up to Phase IIa/b clinical trial batches of


enveloped VLP vaccine candidates, ultracentrifugation-based methods are stillan integral part of DSP.

The natural tendency of single proteins to adopt different stable quaternarystructures poses concerns to the DSP, especially when the desired product pos-sesses a given biological activity that is dependent on the conformation of the pro-tein, for example, an epitope, or as a vaccine candidate based on a VLP. In thelatter case, the sought product should best mimic the original virus. At the end ofthe day, there can be many product-derived entities in the bioreaction bulk thatcan be themselves detrimental and must be removed during DSP as if they wereany other process-related impurity [167]. The DSP strategy has to be developedsuch that it addresses removal of the so-called product-derived impurities, whichare common in VLP systems, besides removal of the process-derived impurities(host-cell protein, host-cell DNA, and baculoviruses) [95,167].

One of the critical drawbacks of the IC/BEVS is the co-production of recombi-nant baculoviruses (themselves, enveloped viruses) during the infection process.For vaccine candidate products, baculoviruses have shown adjuvant activity in T-cell based immune responses; thus, if not removed they might induce effects thatrender analysis of VLP-based immunologic responses difficult [172]. DSPbecomes even more complex, for cases when the product is an enveloped VLPwith similar size to that of the baculovirus, as both virus particles – the VLPs andthe baculoviruses – are composed of a lipid envelope from the same host cell ori-gin. A challenging process development screening different combinations of chro-matographic and density-based unit separation processes is needed to leverage thesmall biophysical differences between both particles. An alternative strategy is towork upstream in the process to minimize baculovirus generation; for example, inHigh FiveTM cells baculovirus expansion is typically attenuated after infection asopposed to Sf-9 cells [105].

From a different standpoint, control of process conditions such as ionic strengthand pH may play a crucial role in avoiding product precipitation, flocculation, oraggregation. Therefore, an initial buffer screening should be performed as early aspossible during process development in order to narrow down the range of opti-mal buffers to choose from. This allows a knowledge based development of chro-matographic steps since parameter settings and operating conditions greatlyimpact process performance, more precisely, recovery yield, purity, and productconcentration [167].

Engineering Challenges in DSP DSP optimization relies still mainly on empiricalinformation coming from trial-and-error experiments. In most product systems,very little knowledge is available, especially with regard to its biophysical-chemicalproperties. These are paramount for a more rational process modeling and optimi-zation. Ideally, as soon as a critical amount of process and product understanding isgathered, DSP unit operations are combined and implemented with the support ofcost-effective optimization strategies based on scaled-down approaches and model-ing. An example is the design of new adsorption materials for adsorptive unit oper-ations where in silico mathematical models can be used to determine operating


conditions optimizing product yield and productivity [167] (Figure 10.2). Nonethe-less, despite the advantages of modeling for assisting DSP design, a great deal ofinformation is necessary, in particular the stability data of the product. Oftentimes,this largely restricts the usable design space – for example, pH and salt concentra-tion in chromatography – during screening experiments and scale-up testing.

Quality Control Methods for Product/Process Analysis and Optimization Availa-bility of analytical methods plays a crucial and often limiting role in the develop-ment of complex IC/BEVS-based products, for example, a VLP-based vaccine.Different assays need to be developed and implemented for best characteriza-tion of the complex products. Specifically for clinical products, product identity,quality, titer, purity, and consistency throughout the manufacturing process isrequired.

The list of quality control methods can indeed become rather extensive, andincreases as the number of proteins expressed increases, for example, in a multi-protein VLP [168]. The following methods are used: SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) (one- or two-dimensional for proteinprofiling), Western blotting (for protein identity matching), direct, indirect, andsandwich enzyme-linked immunosorbent assays (ELISAs) (for physical virus par-ticle quantification), transmission or scanning electron microscopy (for particlevisualization), total protein assessment, total endotoxin assessment, total DNAassessment, glycoprotein analysis and/or N- or C-terminal protein sequencing(for exposed, essential protein identity matching), mass spectrometry (for proteinidentity matching), capillary zone electrophoresis (for enhanced particle or proteinsize and molecular weight measurement), size exclusion chromatography, HPLC(high-performance liquid chromatography), dynamic light scattering (DLS) (forsize and f-potential measurements), surface plasmon resonance (sorption kineticsof product or impurities), baculovirus infective virus titer, and real-time quantita-tive PCR (polymerase chain reaction). This extensive list of QC methods illustratesthe challenges and complexity accompanying purification of IC/BEVS-derivedcomplex biopharmaceuticals.

Case Study: A Typical DSP Strategy of an IC/BEVS-derived VLP The first DSP stepdepends on whether the VLPs are secreted to the extracellular medium. If the VLPis not efficiently secreted, as is for instance the case for the HPV-VLP [173], celllysis or another extraction step is mandatory prior to clarification [168]. In thecase of enveloped VLPs, such as for instance influenza VLPs, which undergo anatural budding process from the host cell membrane, clarification follows as afirst step. Protein capsids eventually still entrapped inside host cells at the time ofharvest, that is, incomplete VLPs, should be removed immediately to avoid furtherload of product-derived impurities to the subsequent DSP steps [7].

Disposable depth-filtration methods, sometimes following a prior decanting stepthat reduces the cell biomass load, are used with very encouraging yields in manyapplications [171,174–176]. This step is scalable as the number and size of car-tridges can be adjusted.


Host-cell protein, host cell DNA, proteases, and baculoviruses are the major pro-cess-derived impurities present in an IC/BEVS-derived bioreaction. These areremoved or shed by consecutive concentration and purification steps [167]. In a thirdand last phase, a polishing step reduces any remaining trace impurities, for example,low molecular weight host-cell proteins, to acceptable values as defined for the spe-cific product. Evidently, this depends highly on the final application [177].

Ultrafiltration, diafiltration using tangential flow filtration membranes (cassettesor hollow fiber units), and chromatography are the most widely used unit opera-tions in concentration and purification steps, respectively. Most used chromato-graphic techniques are based on ion exchange, for example, sulfopropyl andquaternary amines, affinity (including IMAC), and hydrophobic interaction [167].Chromatography is typically performed using either packed-bed columns withbead-based matrix or with porous matrices, membrane adsorbers, or monoliths[176]. The former are specially suited for single proteins or protein complexes,with molecular weight lower than 500 kDa; the latter are suitable for large biophar-maceuticals, particularly if a capture step is envisioned [178]. The accessibility ofthe large particles to the matrix surface is dramatically increased and thus it allowsfor effective matrix capacity utilization.

10.3Regulatory Hurdles for Insect Derived Human Products

IC/BEVS is generally considered a safe production system, with limited growthpotential for adventitious agents. The recent product approvals of Cervarix andFlublok have certainly paved the way to reduce regulatory hurdles relating to thisrelatively young cell substrate.

Insect cells can be grown in the absence of fetal bovine serum and other animalderived ingredients, significantly reducing the chances of introducing an adventi-tious agent during manufacturing [179–181]. The likelihood of insect cells servingas a host for vertebrate viruses or the likelihood of vertebrates serving as a host forinsect viruses is further reduced by genetic distance between insects and verte-brates. Many insect viruses described to date exhibit a relatively narrow host rangewith only a small number of viruses capable of amplifying in both insects and verte-brates [182,183]. These insect viruses along with some tick viruses that can alsoamplify in vertebrates are informally referred to as arboviruses, reflecting theirarthropod-borne origin, and have closely co-evolved with the hematophagous arthro-pods and the vertebrate hosts upon which they feed [182]. The susceptibility of Sf-9cells to arbovirus infection is reported to be very low; the St. Louis encephalitis virusis the only arbovirus tested to date that could produce a persistent, productive, andcytopathic infection [184]. Menzel and Rohrmann [185] described the presence oferrantivirus (retrovirus) sequences in two insect cell lines, including Sf cells [185].

The recent progress in using novel animal cell lines as substrates for the pro-duction of biologicals has led to the re-evaluation of existing criteria used for


evaluating the acceptability of such cell lines. Improvements to existing criteria fordetermining the acceptability of novel cell substrates as well as development ofnew criteria have recently been the focus of regulatory agencies [186]. There arethree general issues with new cell substrates: intact cells, residual cellular compo-nents, and the adventitious agents.

The International Conference on Harmonization document Q5A (2005) and theUS Food and Drug Administration’s (FDA) 2010 guidance “Characterization andQualification of Cell Substrates and Other Biological Materials Used in theProduction of Viral Vaccines for Infectious Disease Indications” provide advice tomanufacturers for the qualification testing of cell lines. Additional testing may berequired based on product-specific comments from the FDA. Examples of addi-tional testing are described elsewhere [6] and may include the following: (i) trans-mission electron microscopic examination of stressed cells to assess the presenceof viral particles; (ii) adventitious virus detections by using consensus-degeneratehybrid oligonucleotide primers strategy [187]; (iii) development of specific PCRscreen for Tn-5 cell line nodavirus described by Li et al. [188]; (iv) a quantitativePCR-enhanced reverse transcriptase assay (Q-PERT) was used to compare reversetranscriptase (RT) activity between early production and late production cellsand to rule out increased production of retroviral-like particles due to the manu-facturing process; and (v) end of production cell samples were determined notto contain infectious retrovirus particles when tested in a cellular co-cultivationassay [55].

Finally, extensive co-cultivation studies were performed to rule out that theRT activity detected in insect cell media was not related to the presence of aninfectious virus. These studies included Q-PERT assay data of baculovirus-infected or uninfected insect cell culture supernatant co-cultured with humanA549, HEK 293 (HEK¼human embryonic kidney cell), Raji, RD (human rhab-domyosarcoma cells) and PBMC (peripheral blood mononuclear cells) andcanine MDCK (Madin–Darby canine kidney epithelial cells) for 11 passages. Inaddition the cell pellets of A549, HEK 293, Raji, RD, PBMC, and MDCK cellsthat were co-cultured with baculovirus-infected or uninfected insect cell culturesupernatant were subjected to a nested PCR to confirm that errantivirussequence (Sf-37) DNA described in Reference [185] does not enter or amplifyin the selected mammalian cells.

The body of evidence required to convince regulators that a cell substrate issafe and suitable for its use depends on the risk/benefit evaluation. For thecase of a prophylactic vaccine that will be given to many healthy people moredata will be required as compared to a therapeutic vaccine or a gene therapyproduct where the “patient” is suffering from a severe disease. The power ofthe IC/BEVS manufacturing technology is that the same cell substrate andsame baculovirus master bank is used for the manufacturing of any product;therefore, once the approval hurdle is cleared for one product the next prod-uct questions will be product/process specific. These questions will then focuson the ability of the downstream process for a specific product to clear

10.3 Regulatory Hurdles for Insect Derived Human Products 375

adventitious agents (viral clearance) and contaminants from the cell substrate(DNA and host-cell proteins).

10.3.1Case Study: Flublok1 Regulatory History

Protein Sciences submitted a Biologics License Application to the US FDA forcommercial production and marketing of Flublok for the prevention of seasonalinfluenza, using the accelerated approval pathway (21 Code of Federal RegulationsPart 601, Subpart E – Accelerated Approval of Biological Products for Serious orLife-Threatening Illnesses) on 17 April 2008. Prior to that Flublok was grantedFast Track Product status on 11 December 2006 for addressing an unmet medical-need (those with egg-allergies).

Protein Sciences underwent first Pre-Approval Inspection in its Meriden, Con-necticut facility on 7–11 July 2008. A Form 483 was issued on 11 July 2008 with16 GMP observations.

Protein Sciences subsequently received a Complete Response (CR) Letter onAugust 29, 2008 containing 12 CMC (Chemistry, Manufacturing, and Controls)comments, 12 Clinical/Statistical Comments, and 4 Pharm/Tox comments. Pro-tein Sciences completed the response to this CR letter including the completestudy report, with clinical endpoint efficacy results for Study PSC04, and the com-plete study report for Study PSC06 on 28 April 2009 to support traditionalapproval by the end of April, 2009. Protein Sciences received notice on 29 May2009 that the submission was considered complete, Class 2 response to the actionletter, and that the user fee goal date is 28 October 2009. During the review pro-cess Protein Sciences received and responded to a number of Informationrequests. Protein Sciences had a second Pre-Approval Inspection (PAI) from 19 to22 October 2009 resulting in a Form 483 with seven GMP observations. On 19November 2009 a VRBPAC (Vaccine and Related Biological Products AdvisoryCommittee) meeting was held to discuss the safety and efficacy of Flublok. TheCommittee voted 9 : 2 that Protein Sciences proved efficacy of Flublok in subjectsaged 18–49; the efficacy vote for 50–64 was 5 in favor versus 6 against; and inadults 65 and older 2 in favor versus 9 against. The Committee further voted 5 infavor versus 6 against that Protein Sciences had proven safety in all ages. An addi-tional non-formal vote was obtained for the age group 18–49 and the Committeestated that the safety database of �2500 adults in this age range was adequate.Protein Sciences received a second CR letter in January of 2010 with eight remain-ing CMC questions primarily relating to process consistency and cell substrateissues. In July 2012 Protein Sciences submitted final data on the Flublok manufac-turing process, that is, cell substrate, to the FDA, covering the remaining out-standing questions. As a result, the FDA restarted the review clock, whichrequired a decision on Flublok licensure not later than 16 January 2013 (the socalled “PDUFA date”). Protein Sciences had a third PAI from 5 to 9 November2012 resulting in a Form 483 with three minor GMP observations and receivedapproval for Flublok use in adults 18–49 years of age on 16 January 2013.


10.4What Comes Next?

IC/BEVS has gained acceptance as a universal manufacturing platform with theapproval of various veterinary and human vaccines, and most recently the recom-binant hemagglutinin influenza vaccine named Flublok. In addition, the approvalof Glybera demonstrates the suitability of the technology to produce the AAV genetherapy vectors. The key advantage of this manufacturing platform is that a uni-versal “plug and play” process may be used for producing a broad range of prod-ucts while offering the potential for low manufacturing costs. Large-scalefermentation facilities previously established for the manufacturing of bio-technology products that may have become obsolete due to yield improvementcould be deployed for the manufacturing of IC/BEVS derived products.

10.4.1Improvements in Production Cycle and Yields

Alternative baculovirus promoters, such as the p10/p6.9 chimeric promoter [189],are being evaluated from a yield improvement perspective using the Dcathepsin-/chitinase-negative AcMNPV bacmid [190].

Ongoing process improvement efforts include the development of a fed-batchfermentation process and the development of a defined growth medium. In thecurrent influenza production process the insect cells are infected at a density of2–2.5� 106 cells ml�1. The development of a fed-batch process for hemagglutinin(HA) will be aimed at increasing the cells density at infection to 8–10� 106 cellsml�1 as previously described [128,191] without reducing specific productivity. Asimple single shot feed-strategy resulted in a twofold increase of HA production[192]. A semi-continuous fed-batch system was described by van Lier et al. [193].Further improvements in cell culture will be aimed at establishing a continuousfed-batch process for which 40-fold improvements in antibody production inmammalian cells was reported [194].

Yield improvement has also frequently been reported as a result of changingthe cell culture media. Additions of plant hydrolysates, other growth and pro-duction enhancing factors, and control of proteolysis have been reviewed byIkonomou et al. [11] and offer promising areas for yield improvement. Specifi-cally, adding the plant hydrolysate, Hypep 1510, to an insect cell culturedoubled expression of a reporter gene [195], and simple changes in pH mayalso offer great benefit [196].

Viral and host modifications can improve cell survival and production of heterol-ogous proteins. Modifications to the host insect cell line, for example, by includingthe anti-apoptotic gene Bcl-2, may limit the cytopathic effects of the baculovirus andmay result in enhancement of expression as well as was reported quite recently forSindbis virus in a mammalian cell line [197]. Co-expression of chaperones may alsobe a promising prospect for the efficient production of recombinant secretory pro-teins in insect cells as was recently reported by for instance Kato et al. [198].

10.4 What Comes Next? 377

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Industrial Scale Suspension Culture of Living Cells || Industrial Large Scale of Suspension Culture of Insect Cells