Innovations in cell culture-based influenza vaccine manufacturing – from static cultures to high cell density cultivations

ABSTRACT

Influenza remains a serious global health concern, causing significant morbidity and mortality each year. Vaccination is crucial to mitigate its impact, but requires rapid and efficient manufacturing strategies to handle timing and supply. Traditionally relying on egg-based production, the field has witnessed a paradigm shift toward cell culture-based methods offering enhanced flexibility, scalability, and process safety. This review provides a concise overview of available cell substrates and technological advancements. We summarize crucial steps toward process intensification – from roller bottle production to dynamic cultures on carriers and from suspension cultures in batch mode to high cell density perfusion using various cell retention devices. Moreover, we compare single-use and conventional systems and address challenges including defective interfering particles. Taken together, we describe the current state-of-the-art in cell culture-based influenza virus production to sustainably meet vaccine demands, guarantee a timely supply, and keep up with the challenges of seasonal epidemics and global pandemics.

Introduction

Respiratory diseases, such as influenza, remain a serious global health as well as economic burden. Several vaccination modalities for influenza including inactivated (IIV), live attenuated (LAIV), and recombinant (RV) vaccines are licensed and constitute the cornerstone of global influenza prevention strategies. Additionally, nucleic acid-based platform technologies are under development. Typically, influenza vaccines are designed as a tri- or quadrivalent composition of one/two influenza A and B virus (IAV, IBV) strains in accordance with the annual recommendations of international health organizations. Production is traditionally based on a process where target strains are injected into fertilized hen eggs, allowing them to replicate. This method, though widely used (approximately 84.5% of global IIV production capacity),¹ is time-consuming, susceptible to egg supply shortages and struggles with waste disposal. In contrast, cell-based production offers faster time lines, an improved antigenic match and potency, better scalability and is not reliant on egg availability,^2–6 Thus, there has been a growing interest in transitioning toward cell culture-based approaches, driven by the need for more efficient and flexible manufacturing platforms to meet the global demand and combat shortages.

The early development of cell culture-based influenza vaccines dates back to the mid-20th century, when more and more continuous cell lines became available and some innovations were already implemented for the production of animal vaccines. But it wasn’t until the 21st century that significant advancements in bioprocessing technologies enabled their widespread adoption for human vaccine use. Static cell cultures, initially used for small-scale production, provided a foundation for subsequent innovations in bioreactor design, media formulation, and process optimization. Inherent limitations of static cultures, including low cell density batch processing and hurdles for efficient scale-up, provoked the exploration of dynamic culture systems to enhance productivity and scalability. Parallel advancements in adaptation of cell lines to suspension growth, establishment of designer cell lines and improvements in media formulation, ultimately resulted in high-yield production systems, and allowed for further process optimization and intensification measures to enhance productivity, scalability, and process robustness. An overview over technological advancements and their applications for IAV cell-culture-based processes using the example of MDCK cells is shown in Figure 1. By implementing high cell density (HCD) cultivation strategies, limitations of classic batch processes could be overcome. In particular, the implementation of fed-batch and perfusion strategies presented several engineering challenges that have been effectively addressed in recent years.

Figure 1.

Technological advancements in cell culture-based influenza virus production processes using MDCK cells. Traditional egg-based processes were available since the 1940s (red arrow). Starting from the initial isolation of MDCK cells from the kidney of a healthy cocker spaniel by Madin and Darby in 1958, various options for cell culture-based production were established. Advances in medium development and the adaptation of MDCK cells to suspension growth have opened the possibility for establishment of more efficient and intensified processes. For perfusion processes relying on suspension cell lines, a bioreactor system is coupled to a cell retention device that can either be membrane-based or an inclined settler (gravity) or acoustic filter. Since the turn of the millennium, MDCK cell-based processes were licensed for the production of human influenza vaccines (blue arrows). BR: bioreactor; CSTR: continuous stirred tank reactor.

In the following, we describe critical steps and innovations that may encourage further process development establishing upstream processes for influenza virus production with improved process efficiency, vaccine efficacy, and reduced production costs. We cover the identification and selection of suitable host cell lines, early manufacturing modalities using static cultures, the transition to suspension cultures, and options for process intensifications involving both adherent and suspension cell lines. We explore the underlying principles of technological advancements, and the challenges associated with each approach. Moreover, we discuss the applicability of single-use (SU) over conventional stainless steel (SS) technologies. By understanding new modalities in development and manufacturing, we can identify opportunities for further optimization and innovation to meet the ever-rising vaccine demands and increase global accessibility.

The focus of this review will be on mammalian and avian cell culture-based influenza virus production; plant and insect cells are not considered. Certainly, it is important to acknowledge that optimized upstream processing is only one aspect of vaccine manufacturing. The downstream processing, blending, and formulation stages within the vaccine production process are equally crucial. While the significance of the whole process is recognized, this aspect will not be discussed further in this context.

Cell culture-based influenza virus production

Key to the development of a cell culture-based influenza virus production process is the selection of an appropriate host cell line, which can support efficient virus replication and meets regulatory requirements. A cell line generally comes with a recommended growth medium, supporting either adherent or suspension cell growth, but adaptations and optimizations are possible from there. The composition is pivotal, ensuring a balance of nutrients and essential factors that allow rapid cell growth with doubling times of less than 30 h as well as high viral replication.⁷ Traditionally, cells are cultivated in serum-containing medium (SCM) as it provides all the essential growth factors and nutrients. However, serum is prone to introduce batch-to-batch variability and nowadays animal component-free processes are desired to minimize the risk of contaminations with adventitious agents.^8–11 Thus, modern options including serum-free (SFM) or chemically defined media (CDM) are preferred as they offer a more safe and consistent performance. However, achieving comparable cell growth and virus yields in these media requires careful optimization of formulation and supplementation with growth factors. With more than 40 different components, medium design becomes an art of its own and developing medium for one specific cell line and target virus is mostly out of scope. Therefore, available “off-the-shelf” media are used and combined even if they are not fully optimized for the intended process. Depending on the composition and the cell’s metabolism this can lead to undesired secondary by-products, such as ammonium and lactate, being accumulated to concentrations that adversely impact cell growth and virus production.^12,13 Moreover, the cell line, cultivation mode, and system dictate the optimal design space for various process parameters that should be tightly controlled throughout the process. General considerations on the appropriate choice for process parameters and intensification strategies for cell culture-based virus production were summarized previously by our group.^14,15 For influenza, temperature is typically maintained between 32°C and 37°C, pH between 7.2 and 7.6, and dissolved oxygen concentration above 30%. At the time of infection, seed virus is typically added along with a protease, such as trypsin, to enhance viral entry into the cell. The concentration and activity of the protease must be carefully adjusted to ensure optimal virus replication without causing excessive protein digestion, resulting in cell detachment in adherent cell cultures or even cell death. Equally important is the preparation of the seed virus, taking into account potential adaptation to the specific cell line and ensuring a high ratio of infectious to noninfectious particles. While it is well known that virus propagation in embryonated chicken eggs can result in mutations in the antigen-encoding genes that can adversely impact vaccine effectiveness, no cell-specific adaptive mutations have been observed in a sequence analysis study using MDCK cells.¹⁶ Here, the seed viruses passaged in MDCK cells were representative of those isolated in humans. Typically, the number of seed virus passages is limited to five. While cell-specific adaptations require 2–3 steps, there is still room for scale-up. Nevertheless, we clearly see a need for continuous monitoring of the genetic stability of seed viruses during large-scale production via sequencing and antigenic characterization. A very efficient and reliable tool for monitoring glycan composition from seed virus to final harvest and even downstream purification is CGE-LIF (capillary gel electrophoresis with laser induced fluorescent detection).¹⁷

The optimal harvest for influenza is usually between 36 and 72 h post-infection time when viral titers reach their peak. However, this may vary with the cell line, process parameters, and virus strain used. Especially for influenza virus production, the seed virus input plays a pivotal role not only for the speed of replication but also for the emergence of defective interfering particles (DIPs). Thus, the multiplicity of infection (MOI), usually between 10 and 10⁻⁵, needs to be carefully considered with regard to the objective of the study.¹⁸

In the following, we outline the current state-of-the-art in cell culture-based influenza virus production, first focusing on cell line selection, then elaborating on process strategy options and finally, discussing the choice of (single-use) cultivation systems.

Adherent and suspension cells for influenza virus production

Currently, there are no universally applicable solutions or platform processes for cell culture-based virus production. This limitation primarily arises from the restricted number of host species that viruses can productively infect, a property known as host range.¹⁹ Furthermore, virus yields for different host cell lines can differ dramatically and cell lines might not qualify for vaccine production due to high requirements regarding safety of vaccines, i.e. tumorigenicity and adventitious agents.^8–11 Accordingly, historically established, yet often underperforming cell lines are in wide use, necessitating systematic cell line screening of host cells approved by the regulatory authorities for vaccine production to select the ideal candidate.^20–22 Identification is a labor-intensive challenge and involves navigating through several hurdles including medium choice, cultivation mode and process conditions. Moreover, the commercial and even academic use of potential cell lines is often restricted due to intellectual property and licensing issues, which may also limit screening options. Besides virus yield and process productivity, considerations should include the antigenic match and immunogenicity of the vaccine product, as several studies could show distinct differences in dependence of the production system (eggs or specific cell line).^6,16,23–26 Criteria for selection of optimal host cell lines have been previously delineated,⁷ and suitable candidates for influenza production have undergone thorough examination in prior reviews.^27,28 The use of several traditional or designer cell lines of various origins such as MDCK, Vero, HEK293, AGE1.CR, PER.C6, EB66, CAP, DuckCelt-T17, or PBG.PK2.1 cells have been evaluated so far for influenza virus production.^29–38 (Tables 2 and 3). Despite multiple benefits of those candidate host cell lines, only a limited number have been qualified and used in the manufacturing of cell culture-based influenza vaccines including MDCK and Vero cells for IIV and LAIV as well as Sf9-derived cells for RV (Table 1). The fact that the industry’s proprietary data and the decision-making processes behind this selective use are not disclosed to the public also limits our understanding of why certain cell lines are ultimately selected for vaccine production. From an academic point of view, we can assume hurdles including regulatory approval, established production protocols, first-on-the-market decisions, and cost-effectiveness.

Table 2.

Overview on MDCK cell lines considered for IAV production.

Name	Adh/sus	Original source	Derived from	Adaptations/changes	Media type	Media (antibiotic use ±)	Ref.
MDCK	adh	ECACC	#84121903		SCM/SFM	GMEM/FCS – Episerf (-)	74
MDCK-PB2	adh	ECACC	#84121903	insertion of IAV PB2 gene	SCM	GMEM, 1% pep., 10% serum ()	75
MDCK.SUS2	sus	ECACC	#84121903	weaning medium, >20 weeks	CDM	SMIF 8 (-)	58
MDCK.Xe.E	sus	ECACC	MDCK.SUS2	weaning 180 d, >50 passages	SFM/CDM	DrivingM (-)/Xeno (-)	30,76
MDCK.S8.E	sus	ECACC	MDCK.Xe.E		CDM	SMIF 8 (-)	76
MDCK-PB2(sus)	sus	ECACC	MDCK.Xe.E	insertion of IAV PB2 gene	CDM	Xeno (-)	77,78
MDCK.Xe.A	sus	ATCC	CCL-34	adaptation 4 passages	CDM	Xeno (-)	79
MDCK.S8.A	sus	ATCC	MDCK.Xe.A		CDM	SMIF 8 (-)	76
MDCK C59/C113	sus	ATCC	CCL-34	adaptation and single-cell cloning	CDM	MDXK (-)	63
MDCK 9B91E4	adh	ATCC	CCL-34	single-cell cloning	SFM	MedImmune (-)	64
MDCKad	adh	ATCC	CCL-34		SCM/SFM	MEM, serum (+)	59
MDCK-SF	adh	ATCC	CCL-34	adaptation to SFM, 7–12 passages	SFM	UltraMDCK (+)	59
MDCK-SFS	sus	ATCC	CCL-34		SFM	SFM4BHK21 (+)	59
MDCK-SIAT1	adh	UK		SIAT1 transfected	SCM	DMEM 10% serum (-)	80
hCK	adh			overexpressing α-2,6-sialoglycans	SCM	MEM 10% serum (+)	62
IRF7-/- MDCK	adh	ATCC	CCL-34	IRF7 knock-out	SCM	MEM 10% serum (+)	81
MDCK H1	adh	ATCC	CCL-34	single-cell cloning	SCM	DMEM 10% serum (-)	67,73
NIID-MDCK	adh	ATCC	CCL-34	gradually reducing serum	SFM	Optipro SFM (-)	82
MDCK-QIV	adh	ATCC	CCL-34	monoclonal	SCM	DMEM 4% serum (-)	66
MDCK-M60	adh	ATCC	CCL-34	cell pool	SCM	DMEM 10% serum (-)	65
MDCK-CL23	adh	ATCC	CCL-34	monoclonal, non-tumorigenic	SCM	DMEM 10% serum ()	65
MDCK 33,016-PF	sus	DSM ACC2219			SFM	Iscove (-)	40,41,83
MDCK-siat7e	sus	ATCC	CCL-34	siat7e transfection	SCM	DMEM 10% serum (-)	57,84,85
MDCK	sus	ATCC	CCL-34	adaptation to SFM	SFM	MDCK-SFM2/Ex-cell MDCK (-)	29
sMDCK	sus	ATCC	CCL-34	serial adaptation	SFM	BalanCD Simple MDCK (-)	86–88
MDCK-XF04/XF06	sus	ATCC	CCL-34	adapted from CCL-34 CL23 or M60	SFM	Lanzhou inhouse ()	65
MDCK Sky1023/10234/3851	sus	ATCC	CCL-34	adaptation to CDM	SFM	ExCell MDCK, UltraMDCK, VP-SFM ()	45

Grey: Cells handled in the authors laboratory.

Empty fields: no information available.

adh: adherent, sus: suspension

SFM: Serum free medium, SCM: serum containing medium, CDM: chemically defined medium, (+) antibiotic use, (-) no antibiotic use, () no information on antibiotic use.

Table 3.

Key characteristics of influenza A virus production in different cell lines.

Cells	Origin	adh/ sus	Media	Max. VCC	VCC at TOI	Max. HA (ref)	Max. HA	Max inf. titer	CSVY	CSVYi	process mode	Ref.
MDCK (ECACC)	canine	adh	GMEM/FCS	1.1E+07	6.2E+06	5248 HAU/100 µL	3.7	nd	13630		STR, B	92
MDCKad (ECACC)	canine	adh	SFM, Episerf	2.0E+06	1.2E+06	3.3 log10(HAU/100 µL)	3.3	1.20E+07	33255	10	wave, B	31
MDCK-PB2	canine	adh	GMEM, pepton, serum	na	na	na						75
MDCK.SUS2 (MDCK.S8.E)	canine	sus	SMIF 8	8.0E+06	2.3E+06	2.9 log10(HAU/100 µL)	2.9	7.60E+08	7000	330	wave, B	30, 58
MDCK.Xe.E	canine	sus	DrivingM	5.9E+07	4.6E+07	4.5 log10(HAU/100 µL)	4.5	9.40E+09	13600	206	SF, SP	30
MDCK.Xe.E	canine	sus	Xeno	8.0-9.0E+06	4.0E+06	3.1 log10(HAU/100 µL)	3.1	1.00E+08	8200	30	SF, B	76
MDCK.S8.E	canine	sus	SMIF 8	5.0-6.0E+06	2.0E+06	3.4 log10(HAU/100 µL)	3.4	1.00E+09	8200	230	SF, B	30
MDCK-PB2(sus)	canine	sus	Xeno	2.8E+07	2.8E+07	na	na	PFU 1.74E+09		61	STR, P	77, 78
MDCK.Xe.A	canine	sus	Xeno	5.1E+07	4.5E+07	4.4 log10(HAU/100 µL)	4.4	1.80E+10	11690	400	STR, P	103
MDCK.S8.A	canine	sus	SMIF 8	5.0-6.0E+06	2.0E+06	3.2 log10(HAU/100 µL)	3.2	9.00E+08	9500	160	SF, B	76
MDCK C59	canine	sus	MDXK	1.7E+07	4.3E+06	2.9 log10(HAU/100 µL)	2.9	5.60E+08	3784	130	ambr15, B	63
MDCK C113	canine	sus	MDXK	5.5E+06	2.9E+06	3.5 log10(HAU/100 µL)	3.5	1.00E+09	19413	350	ambr15, B	63
MDCK clone 9B91E4	canine	adh	Medimmune		1.0E+06	nd	#, nd	FFU 5.01E+08	nd	501	STR, B	64
MDCK-SF	canine	adh	UltraMDCK	1.3E+05	1.3E+05	8.2 log2(HAU/100 µL)	2.5	1.30E+08		1000	6Well, B	59
hCK	canine	adh	MEM, serum	nd	nd	nd	nd	PFU 1.00E+08	nd	nd		62
IRF7-/- MDCK	canine	adh	MEM, serum	nd	nd	16 HAU	1.2	nd			T25, B	81
MDCK clone H1	canine	adh	DMEM, serum	2.0E+06		210.75 HAU/100 µL	#, 3.2		35100	1530	T, B	67, 73
NIID-MDCK	canine	adh	Optipro SFM	nd	nd	nd	nd	PFU 3.98E+08	nd	nd	nd	82, 113
MDCK 33016-PF	canine	sus	Iscove	1.8E+07	1.8E+07	4096 HAU	3.6	nd			STR, P	40, 41
MDCK-siat7e	canine	sus	DMEM, serum	5.2E+06	1.5E+06	2048 HAU/50 µL	#, 3.6	1.00E+08		67	SF, B	114
MDCK-SFS	canine	sus	SFM4BHK21	2.2E+06	3.0E+05	8.5 log2(HAU/100 µL)	2.6	5.00E+08	nd	1667	SF, B	59
MDCK	canine	sus	MDCK-SFM2 or Ex-cell MDCK	6.0E+06	3.0E+06	3.9 log10(HAU/50µL)	#, 4.2	2.19E+10	51700	7300	STR, B	29
sMDCK	canine	sus	BalanCD Simple MDCK	1.8E+06	1.8E+06	512 HAU/50 µL	#, 3.0	1.00E+08		56	spinner	86-88
MDCK Sky1023/ 10234/ 3851	canine	sus	ExCell MDCK, UltraMDCK, VP-SFM	2.0E+06	2.0E+06	2048	#, 3.3	nd			spinner	45
Veroad	monkey	adh	SFM, Episerf	2.0E+06	1.0E+06	2.3 log10(HAU/100 µL)	2.3	1.20E+07	3990	12	wave, B	31, 38
AGE.CR	avian	sus	CDM	5.3E+07	2.6E+07	3.3 log10(HAU/100 µL)	3.3	1.78E+10	1428	683	STR, P	101
AGE1.CR.pIX	avian	sus	CDM	5.2E+07	4.3E+07	3.7 log10(HAU/100 µL)	3.7	8.80E+09	3059	205	OSB, P	102
EBx/EB66	avian	sus	CDM	1.6E+08	7.5E+06	nd	nd	1.90E+07	nd	3	STR, B	115–118
PBG.PK2.1	swine	sus	CDM	6.8E+07	4.6E+07	3.9 log10(HAU/100 µL)	3.9	3.20E+09	3929	70	STR, P	38
Cap	human	sus	CDM	3.3E+07	2.7E+07	3.7 log10(HAU/100 µL)	3.7	7.50E+09	4086	279	STR, P	36
HEK293SF_3F6	human	sus	HyQSFM4- Transfx293TM	1.0E+07	1.0E+07	3.0 log10(HAU/100 µL)	3.0	8.00E+09	3960	792	STR, P	32, 37
Vero.SUS	monkey	sus	PCD	3.4E+06	3.4E+06	2.6 log10(HAU/100 µL)	2.6	4.22E+07	nd	12	SF, B	p.c. I.B.
sVero	monkey	sus	SFM4-sVero	4.6E+06	na	nd	nd	nd	nd	nd	STR, P	49, 119
CCX.E10	avian	sus	SFM	8.5E+06	5.0E+06	nd	#, nd	FFA 1.00E+08	nd	200	Spinner, B	p.c.M.W., 120
DuckCelt-T17	avian	sus	Optipro SFM	1.1E+07	1.0E+06	1.8 log2(HAU/100 µL)	nd	3.20E+08	nd	320	TubeSpin, B	37, 121
PBS-12SF	avian	adh	Optipro SFM	nd	nd	36 HAU/50 µL	#, 1.9	3.39E+09	nd	nd	12 well, B	122
PER.C6	human	sus	Ex-Cell 525 medium	3.0E+06	3.0E+06	2100 HAU	#, 3.3	1.00E+10	nd	3333	STR, B	34, 87
aCHO-K1	hamster	adh	F12/ExCell 302	0.1E+06 cells/cm2			no titer	6.92E+01			B	91, 123
sCHO-K1	hamster	sus	ExCell 302	2.7E+06			no titer	5.13E+02			SF, B	123
aBHK-21	hamster	adh	MEM/Optipro	0.2E+06 cells/cm2			no titer/ 1.0	5.62E+02			B	91, 123
sBHK-21	hamster	sus	GMEM/VPSFM	5.0E+06			no titer	2.00E+03			Spinner, B	123

Grey: MDCK cells used by authors, yellow: MDCK cells used by others, blue: other cell lines, adh: adherent, sus: suspension.

SFM: serum free medium, SCM: serum containing medium, CDM: chemically defined medium.

Max. VCC: maximum viable cell concentration described for this cell line, VCC at TOI: viable cell concentration at time of infection for the given virus yield.

Virus titer values mainly for IAV H1N1 A/PR/8/34 or for H1N1 A/California/07/2009, H1N1 A/New Caledonia/20/99, H7N9 A/Taiwan/1/2017, B/Massachusetts/2/2012, or H9N2 A/chicken/Guangxi/SIC6/2013: indicated by # in max HA column.

Max. HA: maximum HA values as given in the reference or calculated to log10HAU/100 µL, HAU: HA units.

Max. inf. titer as TCID50/mL (Tissue culture infectious dose 50) or PFU/mL and FFU/mL (plaque forming units/focus forming units) if stated otherwise.

CSVY: cell-specific virus yield (virions/cell) (conversion of HA to virions as stated in the respective reference), CSVYi: cell-specific infectious virus yield (inf. virions/cell), italic: calculated based on max. VCC of the respective run, other CSVYs calculated based on VCC at TOI.

Cultivation mode: cultivation vessel and cultivation mode used production of virus titers given; STR: stirred tank bioreactor, OSB: orbitally shaken bioreactor, SF: shake flask, T: T flask, B: batch, P: perfusion.

nd: not done, na: not applicable, no titer: no titer could be measured, empty fields: no values given.

p. c. I.B.: personal communication with I. Behrendt (MPI Magdeburg), p.c. M.W.: personal communication with M. Woschek (Nuvonis).

Table 1.

Summary of licensed animal cell culture-based influenza vaccines.

License year	Cell line	Product name	Manufacturer	Type	Culture conditions	Active	Ref.
2001	MDCK	Influvac	Solvay Pharmaceuticals	IIV	adh, carrier	no	39
2007	MDCK	Optaflu, Flucelvax	Novartis, Seqirus	IIV	sus, SFM	yes	40,41
2009	Vero	CELVAPAN	Baxter International	IIV	adh, SFM	no	42,43
2013	Sf9	FluBlok	Protein Sciences, Sanofi	RV	sus	yes	44
2015	MDCK	SKYCellflu	SK Chemicals/Bioscience	IIV	sus, SFM	yes	45

Product name shows one example but not all products.

SFM: serum free medium, sus: suspension cell line, adh: adherent cell line.

Type refers to the modality being an inactivated (IIV) or recombinant vaccine (RV).

In a head-to-head comparison, Genzel et al. elucidated the question whether adherent MDCK or Vero cells are the most suitable cell substrate for influenza vaccine production.³¹ Although similar virus titers could be obtained for both in the supernatant under optimized small-scale conditions (roller bottles, T-flasks), MDCK cells surpassed in bioreactors on microcarriers (wave bioreactor and stirred tank bioreactor (STR)). With advanced media development and cell line engineering, a variety of suspension MDCK cells are now available, offering great advantages for bioprocess operation and scalability (Table 2). In contrast, the development of Vero suspension cells presents a more challenging task that has engaged multiple research groups for over three decades.^46–49 Here, a maximum cell concentration of up to 8 × 10⁶ cells/mL was reported so far; however, this was only reached in a shake flask cultivation over 13 d with three full medium replacements; otherwise about 2 × 10⁶ cells/mL were achieved in batch and 6.8 × 10⁶ cells/mL in 3 L bioreactor perfusion culture.⁴⁹ Moreover, doubling times exceeding 40 h and the frequent formation of aggregates remain significant issues, thereby limiting the options for efficient or intensified processes. Thus, MDCK cells are mostly dominating industrial manufacturing (Table 1).

The role of MDCK cells for influenza virus production

As early as 1958, MDCK cells were isolated from the kidney of a healthy cocker spaniel by SH Madin and NB Darby, and a few years later deposited at the American Type Culture Collection (ATCC).⁵⁰ Being very well suited as a model for polarized cells rendered the MDCK cell line as one of the most extensively studied and well-characterized epithelial cell lines for cell physiological and virological investigations. Leading research institutes worldwide have employed adherent MDCK cells for the isolation and replication of human influenza viruses, primarily for epidemiological studies and diagnostic purposes.⁵¹ Besides this, MDCK cells are also dominating cell culture-based influenza vaccine manufacturing (Table 1), as they are permissive to all influenza subtypes and produce various strains to high titers. Thus, efforts to utilize MDCK cells for human influenza vaccine manufacturing instead of eggs have been ongoing since the end of the last millennium, resulting in extensive safety analysis to rule out concerns regarding tumorigenicity and oncogenicity.^39,52–56

Moreover, academic and industrial endeavors have been made to enhance cell-specific virus yields (CSVY) and overall productivity (Figure 1). Such efforts encompass the adaptation to suspension growth instead of adhering the cells to microcarriers or flask surfaces, which could be achieved by transfecting adherent MDCK cells with human siat7e gene or following advanced medium development.^29,57–59 Furthermore, strategies to enhance cell concentrations, real-time monitoring of virus infection dynamics for timely virus harvest, development of multiscale models for improved process understanding or genetic manipulation to obtain a cell line expressing mainly human influenza receptors were applied.^60–62 Finally, the selection of high-yield cell clones possesses significant potential for enhancing productivity in the upstream manufacturing process.^52,63–67 This seems to be particularly relevant, as MDCK cells display a vast cell-to-cell heterogeneity with significant differences in morphological, electro-physiological, and biochemical properties, carbohydrate representation,^68–70 and most importantly in virus production capacities. Here, studies at the single-cell level could elucidate up to roughly 1,000-fold differences in progeny virus yields and intracellular viral RNA levels.⁷¹ Efforts to uncover the main drivers behind this heterogeneity could not yield a simplistic answer yet.⁷² However, recent transcriptomic analyses suggested the balance between suppression of host cell functions to divert cellular resources and sustainment of sufficient activities for virus replication as a relevant factor.⁷³ Owing to those intensive efforts by multiple academic and industrial research groups, several MDCK cell lines with different properties became available, where most of these cell lines originate from the lineage CCL-34 deposited at the ATCC (Table 2).

Processes involving adherent cell lines

Suspension cells are nowadays favored over anchorage-dependent cells in many bioprocessing contexts for their ability to support high cell density growth, ease of scale-up, flexibility, cost-effectiveness, and options for process control. However, suspension cells are not always available and for some cell lines it remains a challenge to adapt or engineer them to support suspension growth. As processes involving such anchorage-dependent cells are always limited by the available surface area, extensive efforts were made in the past to optimize production for maximal efficiency. This can either be determined by the surface area of T-flasks, roller bottles, cell factories, or within a fixed-bed reactor or the number of microcarriers used. Some of those static cell culture systems are not only limited in their surface area but also lack process control options. Although proper medium mixing and gas diffusion can be achieved in, e.g., roller bottles, control of crucial parameters such as pH and pO2 is not given.⁸⁹ However, virus production can easily result in high titers with minimal operator interactions. In previous studies, a process involving adherent MDCK cells in roller bottles yielded 1.0 × 10⁹ PFU/ml of A/PR/8/34 (H1N1).⁹⁰ Attempts to optimize such a static production process by using pyruvate instead of glutamine in the culture medium to bypass ammonia accumulation did not result in significantly higher yields.⁹¹ In contrast, implementation of carrier technologies could already help to mitigate surface limitations and gain more process control while having a smaller footprint. With MDCK cells in batch mode, cell densities of 3.2 × 10⁶ cells/mL have been reached with microcarrier concentrations of 2 g/L, yielding a maximum virus titer of 2.9 log₁₀(HAU/100 µL) in SFM (A/PR/8/34 (H1N1)).⁹² Implementation of a wave bioreactor allowed for higher cell densities and better attachment compared to stirred tank bioreactors (2 g/L Cytodex 1, 2 L scale), however, maximum titers remained in the range of 3.3 log₁₀(HAU/100 µL) (A/PR/8/34 (H1N1)).³¹ Using a repeated fed-batch strategy, HCDs with 11.2 × 10⁶ cells/mL for 12.5 g/L Cytodex 1 could be achieved, yielding 2.8 log₁₀(HAU/100 µL). In perfusion, even higher total virus titers of 3.3 log₁₀(HAU/100 µL) could be reached at a maximum cell concentration of 5.5 × 10⁶ cells/mL (10.5 g/L Cytodex 1) (Table 3).⁹² Although microcarrier cultures display multiple benefits over static cultures, shear force, the need for sterilization and cleaning procedures can limit their use. Moreover, not all cells show good attachment when cultivated in SFM. Alternatively, in packed-bed bioreactors cells are immobilized on a loosely packed matrix within the bioreactor, which provides a large surface area, high oxygen transfer, and availability as single-use option. Using a 30 mL small-scale model, Sun et al. reported cell numbers of 3.2 × 10¹⁰ cells delivering maximum virus titers of 2.9 log₁₀(HAU/100 μL) and 7.8 × 10⁷ TCID₅₀/mL 3 d after infection (MDCK cells, A/New Caledonia/20/99 (H1N1)).⁹³ Moreover, implementation of hollow fiber (HF) bioreactors may be a promising approach when using adherent cells for virus production. Those systems are composed of a cartridge with hundreds of capillaries, wherein the extra-capillary space cells are grown and virus is produced, while nutrients and oxygen are constantly supplied via the intra-capillary space. Here, a maximum total virus titer 3.53 log₁₀(HAU/100 μL) could be reached with a single harvest step, which corresponds to a cell-specific virus yield (CSVY) of 1,604–3,209 virions/cell (1–2 × 10⁹ cells/mL in the extra-capillary space, A/PR/8/34 (H1N1)). Using multiple harvest steps and adjusted conditions, even higher CSVYs of 4,055–8,110 virions/cell could be reached.⁹⁴ Recent developments such as single-use fixed-bed bioreactor systems (scale-X, Univercells or iCELLis, Pall) allow to overcome several of the previously described limitations of static cultures. Those systems provide growth surfaces ranging from 2 to 600 m², offering scalable processes with automated process control (pH, DO, liquid flow rates) while functioning as prefilter retaining cell debris and impurities. For instance, Berrie et al. reported up to 1 × 10¹⁰ cells for a 10 m² unit and titer improvements of two to four orders of magnitudes for vesicular stomatitis virus production using Vero cells in a scale-X bioreactor compared to flasks and cell factories,⁹⁵ however, no data are available for influenza yet.

Processes involving suspension cell lines

The availability of suspension cells as hosts for virus production provided many new possibilities for process development and scale-up. To meet the rising demand for sustainable and cost-efficient processes, there has been a shift toward developing intensified and integrated production methods emerged. Therefore, scale-down models for high throughput screenings and process optimization as well as quasi-continuous production systems with a small footprint for large-scale manufacturing now became available. Generation of suspension cell lines can either be achieved by adaptation to an appropriate medium or via genetic engineering. Modern media even allow suspension growth of MDCK cells to high cell concentrations.^29,30,63 However, not with every medium single-cell growth was directly supported and cells formed clusters or aggregates.⁵⁸ In the following, we summarize such process optimization and intensification strategies for influenza virus production demonstrating the potential of these approaches.

Simple and straight-forward: batch culture

For virus production in batch mode, a finite quantity of culture medium is added to a bioreactor and cells are grown to a desired cell concentration. Following infection with a seed virus, viral replication is initiated and when titers reach their maximum, the production cycle is ended by a harvest step. After cleaning and sterilization, the bioreactor can be prepared for the next batch operation. Most industrial vaccine production processes are still operated in batch mode, as setup and maintenance are comparably easy and production of independent batches helps to mitigate risks in safety and release of lots. Unfortunately, data of such industry processes are not comprehensively available to public.^52,64,66 Exemplarily, there are, however, academic descriptions at laboratory scale of potential processes for influenza vaccine productions involving suspension HEK-293 or MDCK cultures.^79,96 Here, Bissinger et al. showed good performance with an integrated IAV production process, where MDCK suspension cells were cultivated in a CDM.⁷⁹ Very high titers of 3.6 log₁₀(HAU/100 µl) could be achieved using fast‐growing MDCK cells at concentrations up to 9.5 × 10⁶ cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L STR. Upstream production capacity was estimated to be 600 doses/L. Assuming a total recovery of 50% during downstream processing, approximately 300 monovalent vaccine doses (15 µg/dose) per liter of bioreactor harvest in 4–5 d could be obtained.⁷⁹ In contrast, only about 10.4 doses/L of monovalent vaccine can be achieved using roller bottles and about 3–4 eggs are required for a tri- or quadrivalent vaccine dose depending on the manufacturer and strain.^6,90

A first step towards process intensification: fed-batch culture

While batch processes are easy to set up and to maintain, they can be limiting in times of a pandemic with increased need of output and speed. Then, a more efficient use of bioreactor capacity and thus intensification becomes essential.

Applying a fed-batch approach, involving an initial growth phase followed by stepwise addition of fresh medium, can dilute accumulated ammonia and lactate (virus production and cell growth inhibiting metabolites) and replenish consumed nutrients, thus boosting cell concentrations and leading to higher virus yields and specific virus productivity. In fed-batch mode, the production of A/PR/8/34 (H1N1) in HEK293SF cells resulted in a 2.7-fold higher space-time-yield (STY).⁹⁷ However, in a study involving PBG.PK2.1 cells, applying a fed-batch strategy could not outcompete results from a batch process (Table 3).³⁸ Yet, regardless of the dilution step, operation in fed-batch mode can still result in the accumulation of metabolic side products such as lactate and ammonia to inhibitory levels, especially when concentrated feeds are used and thereby hinder virus production.

Next level of process intensification: perfusion culture

Truly continuous processes pose challenges for lytic virus production (see 2.4.4). Thus, the implementation of perfusion strategies has emerged as a fundamental advancement in influenza virus production to intensify processes by enhancing cell concentration, virus yields, and overall efficiency. To achieve HCDs and high productivity, cells have to be provided with an optimal metabolic environment. This can be achieved through a constant supply of fresh medium while simultaneously removing an equivalent volume of spent medium. This exchange does not only provide nutrients for cell growth but also allows the partial removal of adverse metabolic by-products (e.g. lactate and ammonia) or protease inhibitors, carbon dioxide produced by the cells and bicarbonate from the base added for pH control.⁹⁸

In contrast to truly continuous cultures, retaining cells within the cultivation system is a critical technological aspect, typically facilitated by membranes, sedimentation, or centrifugation. Literature by Chotteau et al. and Göbel et al. provide a comprehensive overview of available technologies.^14,98 Process control can be achieved by manual intervention or automation.⁹⁹ For the latter, real-time monitoring of cell growth and critical phases of viral production for instance using capacitance is highly useful to achieve precise control and optimization of the culture conditions (e.g. dynamic feed regime or harvesting time point).¹⁰⁰

Semi-perfusion cultivations offer the easiest approach to assess whether cells can be grown to HCDs at small scale. Here, cells are grown in shake flasks and a certain amount of medium is regularly exchanged by centrifugation typically guided by a cell-specific perfusion rate.⁹⁹ With this mode of cultivation, Bissinger et al. reached MDCK cell concentrations of up to 60 × 10⁶ cells/mL in a CDM.³⁰ However, the highest titers were reached when cells were infected with A/PR8/34 (H1N1) at 40 × 10⁶ cells/mL, resulting in total virus titer of 4.2 log₁₀(HAU/100 µL), which corresponded to an accumulated titer of almost 4.5 log₁₀ (HAU/100 µL) and infectious titer of almost 10¹⁰ TCID₅₀/mL³⁰ (Table 3). For bioreactor operations, maintaining cell sterility and continuity through offline centrifugation poses challenges and demands a substantial investment, particularly at large scales. For scale-up using bioreactors, membrane-based systems for cell retention, such as spin-filters, tangential flow filtration (TFF), or alternating tangential flow filtration (ATF), are usually considered in industry. Here, ATF and TFF mode are more preferable, as tangential flow in contrast to orthogonal flow of the cell broth to the filter surface prevents membrane fouling. Various reports using ATF or TFF systems for the production of influenza viruses in suspension MDCK, AGE1.CR, CAP, and PBG.PK2.1 are available that highlight improved virus production in comparison to batch production (Table 3; Figure 2).^38,101–104

Figure 2.

Comparison of available cultivation systems for influenza vaccine manufacturing in adherent or suspension cells. STR: stirred-tank bioreactor; OSB: orbitally shaken bioreactor (single use); Wave bioreactor (single-use); semi-perfusion in shake flask; ATF: alternating tangential flow filtration; TFF: tangential flow filtration; TFDF: tangential flow depth filtration; AS: acoustic settler; IS: inclined settler; HFBR: hollow-fiber bioreactor; semi-continuous: two-stage semi-continuous shake flask cultivation system; CSTR: two-stage continuous stirred-tank bioreactor cultivation system.

Using PBG.PK2.1 cells, Gränicher et al. reached cell concentrations of up to 50 × 10⁶ cells/mL in an ATF cultivation and a maximum HA titer of 3.93 log₁₀(HAU/100 µL); however, CSVY was lower than those obtained with an optimized batch or fed-batch cultivation.³⁸ In contrast, at a maximum MDCK cell concentration of 45 × 10⁶ cells/mL Wu et al. could show very high titers of 4.42 log₁₀(HAU/100 µL), which corresponded to an accumulated titer of 4.5 log₁₀(HAU/100 mL) and 1.8 × 10¹⁰ TCID₅₀/mL in ATF mode (A/PR8/34 (H1N1)).¹⁰³ From this cultivation, very high yields could be determined: CSVY of 11,690 virions/cell; STY of 8.0 × 10¹³ virions/L/d; volumetric productivity: 1.02 × 10¹³ virions/L/d.

For an avian cell line (AGE1.CR.pIX) with a much lower CSVY, Vazquez-Ramirez et al. (2019) proposed a hybrid fed-batch/perfusion strategy with an ATF system.¹⁰⁴ With this combination strategy, titers of 3.3 log₁₀(HAU/100 μL) were reached, which represented a 5-fold increase compared to the reference perfusion only cultivation and 7-fold compared to a conventional batch process. Despite elevated medium consumption, the hybrid strategy achieved a volumetric productivity similar to the batch process (5.4 × 10¹¹ vs. 6.5 × 10¹¹ virions/L/d). Together, this demonstrated the potential of integrating different cultivation strategies to achieve optimal virus yields and process efficiency.

Nevertheless, conventional membrane-based cell retention faces challenges in virus production due to the lytic nature, large size (up to 360 nm for vaccinia viruses), and distinctive surface properties of viruses. Apoptosis and cell lysis following virus infection shorten process time but increase impurity levels as cell debris, proteins, and host cell DNA accumulate, resulting in common drawbacks including membrane clogging and undesired virus accumulation within the bioreactor. Although influenza virus particles are only between 80 and 120 nm in diameter, several studies observed virus retention and membrane clogging when using an ATF membrane with cutoffs of 50 kDa, 0.2 µm, and 0.5 µm.^101,103,104

More recently, membrane-based technologies became available that allow for continuous virus harvesting, which could improve CSVYs and product quality due to a shorter residence time inside the bioreactor. For instance, Hein et al. set up an automated perfusion process for the continuous production and harvesting of IAV DIPs using a tubular membrane (VHU) with about 10 µm pore size in ATF mode.⁷⁸ DIP production was initiated at a cell concentration of about 20 × 10⁶ cells/mL. While cells were effectively retained in the bioreactor, 100% of the produced virus particles passed through the membrane and no fouling was observed.⁷⁸ In a control process using a regular HF membrane (pore size 0.2 µm) most IAV DIPs were retained in the production vessel. Tangential flow depth filtration (TFDF) technology (Repligen) might equally be an option. Here, cells are efficiently retained using a membrane with a pore size of 2–5 µM in TFF mode, while simultaneous depth filtration allows to harvest virus particles through the filter. With a TFDF fed batch/perfusion setup for the production of IAV in HEK293SF cells, Silva et al. could recently show virus production and efficient harvest through the membrane without facing issues with virus retention, membrane clogging, or increased shear stress.⁹⁷ We also obtained promising results for IAV production in MDCK cells using this technology in our lab; however, data are not published yet. TFDF modules are scalable and commercially available from pilot to 2,000 L scale and could push process development for vaccines toward integrated continuous downstream processing.

Another option to overcome issues with membrane fouling and virus retention is cell retention devices relying on gravity or acoustic waves and both have been applied for influenza virus production.^32,105,106 Acoustic and inclined settlers both allow for continuous virus harvesting, preferentially retain viable cells, and lower shear stress is applied in comparison to membrane-based systems, which is especially critical as infected cells tend to be more sensitive to shear.^106,107

Using an acoustic settler, Petiot et al. obtained promising results using HEK293 cells at concentrations up to 18 × 10⁶ cells/mL.³² In comparison to a batch process, perfusion mode titers were higher by almost one order of magnitude. Following, Granicher et al. (2020) compared the performance of an acoustic settler in AGE1.CR.pIX cell cultivations at concentrations between 25 and 50 × 10⁶ cells/mL to an ATF cultivation.¹⁰⁶ Owing to the ability to harvest the virus continuously, at least 1.5-fold increase in CSVY and an up to 3-fold higher volumetric productivity were achieved. Those results again highlight the importance of continuous virus harvest for influenza production processes and show the potential of acoustic settler applications. However, devices are mainly established at laboratory scale and less common in commercial manufacturing. Similarly, Coronel and Gränicher et al. reached HCDs of up to 50 × 10⁶ AGE1.CR.pIX cells/mL using an inclined settler.¹⁰⁵ Continuously harvesting the virus resulted in a CSVY of 3474 virions/cell, which corresponded to a 4.7-fold increase compared to an ATF control cultivation and also volumetric virus productivity was 2.2 times higher. However, the limitation of this device resides in the perfusion rate capacity, lack of off-the-shelf solutions at industrial scale, high residence time, and temperature gradients. Collectively, all discussed findings emphasize the complex advancements of perfusion strategies for influenza virus production as they provide clearly improved metabolic environments for cell growth and virus production phase (Table 3). Multiple highly potent options for cell retention are available with each one having distinct advantages and disadvantages, influencing factors such as shear stress, scalability, and ease of implementation. Yet, choosing a bioreactor system and coupling it to a suitable cell retention device is a critical consideration in designing an efficient and robust perfusion process, as virus yields can highly benefit if the device allows for continuous virus harvesting (Figure 2).

Challenges in intensified virus production: continuous culture

In recent years, there has been a growing interest in utilizing continuous manufacturing approaches over batch mode for various biopharmaceutical applications. Such an operation at a steady state can allow for a prolonged production phase while potentially leading to increased productivity and efficiency. For the production of recombinant proteins or non-lytic viruses, this can be attained through a chemostat process, wherein uniform flow rates are maintained for both the feed and the product harvest, ensuring a consistent working volume and nutrient supply. On the contrary, many viruses as influenza are lytic, necessitating the separation of cell growth and virus propagation in a cascade of distinct vessels to avoid the effects of apoptosis and cell lysis. Such a cascade can be realized in a two-stage continuous stirred tank reactor (CSTR) system at its simplest case. From a primary STR dedicated to cell growth, cells are continuously transferred into a secondary STR that is exclusively used for infection and virus propagation.

Initial attempts by Frensing et al. to continuously produce IAV in AGE1.CR cells in a CSTR system could not meet the expected benefits.¹⁰⁸ Instead, production resulted in periodic oscillations in virus titers, noticeably decreasing the volumetric productivity compared to batch cultivations. Such fluctuations are described by the “von Magnus effect” and could be attributed to the emergence of DIPs.¹⁰⁸ DIPs themselves are non-replicating, yet compete and interfere with the replication of infectious influenza virus particles, which can severely limit the usability of continuous processes for the production of IAV or other DIP producing viruses. However, with the development of a tubular plug-flow bioreactor system, Tapia et al. could circumvent those limitations and show IAV production with stable titers in AGE1.CR.pIX and MDCK cells.¹⁰⁹ The system was built of a CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor used for virus propagation. With that, titer oscillations were omitted and an about 2-fold STY compared to typical batch cultures was achieved.¹⁰⁹

However, DIPs are not always considered as an unwanted byproduct in IAV production, as they also show potential to serve as a prophylactic and therapeutic antiviral.¹¹⁰ To further explore DIP evolution, mathematical modeling was applied to describe experimental results of two CSTR cultivations at different residence times in the secondary STR.¹¹¹ Moreover, Pelz et al. recently used a simplified, semi-continuous setup to mimic continuous virus propagation at small scale. Instead of cascading STRs, two distinct shake flasks were used for suspension MDCK cell propagation and IAV production.¹¹² Over a cultivation period of 21 d, again strong periodic oscillations in virus titers were observed due to the competitive dynamic interaction of DIPs and infectious particles. Following, next-generation sequencing was employed to deepen the understanding on DIP formation and identify promising candidates for antiviral therapy.¹¹² Moreover, these studies demonstrated the importance of DIPs also in seed virus production for influenza.¹⁸

Although continuous production may promise substantial reductions in footprint and manufacturing costs compared to batch processing, further limitations comprise the required high qualification level for technical staff and the increasing risk of contaminations due to the complexity of the setup and the prolonged process time. Thus, continuous influenza production cannot necessarily outweigh the advantages of a batch process that offers great flexibility and control (Figure 2).

Single-use technology for influenza virus production

Implementation of SU technologies progressively gained popularity in the biopharmaceutical industry and the pandemic COVID-19 situation has even accelerated the steadily growing demand for SU devices in bioprocessing. According to a recent survey, operations using SU equipment in the clinical upstream processing increased from 38.6% in 2014 to 62.9% in 2022.¹²⁴ Nowadays, SU options are commercially available for several kinds of bioreactor operations, yet, deciding whether SU or SS technologies should be used for vaccine production involves a careful balance of advantages and challenges (Figure 2). Due to their low turnaround time, SU systems offer great flexibility, rapid scalability, and increased production rates, making them particularly suited for responding to diverse market demands and adjusting production campaigns efficiently. Moreover, such systems are a key factor in expanding the global production capacities as they can be easily set up in less developed areas due to lower capital investment, engineering, and operational costs. However, the pandemic has shown that especially supply and sterilization of SU equipment was limited when demand increased. In contrast, SS reactors, known for durability, safety, and suitability for long-term generation, are favored for predictable, large-scale production. The question of sustainability and leachables of SU versus SS is still discussed controversially. Likewise, some of the SU equipment is provided by novel companies that not always remain on the market. However, especially vaccine manufacturing processes need to sustain production for many years and fall-back options need to be provided. Thus, quality and availability of SU equipment need to be guaranteed to allow long-term supply and production of the respective vaccine. Any changes in the process require demonstration of adequate product quality. Finally, challenges in scaling and adapting SS systems during clinical and commercial manufacturing can limit flexibility and responsiveness to changing market demands.^124,125

For influenza virus production, several single-use systems have been evaluated so far, including HF, packed-bed,⁹³ orbitally shaken, oscillating, and stirred bioreactor systems.^{63,64,94,102,126,127} More SU bioreactor systems used in biopharmaceutical production and their associated challenges have for instance been reviewed in literature.^128,129 The design of the SU systems varies a lot with respect to the mass transfer and mixing principle, scale and application area being research and development or manufacturing. In recent years, SU microbioreactor systems as the ambr systems from Sartorius (15 mL wv) or Erbi Breez by Merck (2 mL wv, perfusion) have gained popularity in early development. In our lab, promising results were achieved when screening multiple monoclonal MDCK cell lines for influenza virus production at microbioreactor-scale using an ambr15 system.⁶³ Running up to 48 SU vessels in parallel under controlled conditions with automated sampling allows to rapidly generate data on multiple process variations.

Implementing SU systems for production processes in perfusion mode adds another layer of advantages. For instance, Coronel et al. investigated IAV production in a SU orbitally shaken bioreactor coupled to membrane-based cell retention devices. In a comparative analysis, perfusion strategies employing ATF or TFF systems could clearly outcompete the reference batch process.¹⁰² Setup of the system was easily achieved due to the simple bag design without coupling or stirrer, suitable for shear-sensitive cells and also oxygen demands of the AGE1.CR.pIX cells could be met even at HCDs. Moreover, the system is suitable for scale-up, as commercially available for working volumes between 1.5 L up to 2,500 L (Kühner). Many other suppliers also offer SU systems up to 6,000 L (e.g. ABEC).

Taken together, SU systems can offer flexible, fully scalable, plug-and-play bioreactor operations, and especially efficient perfusion processes give a perfect fit with the limited volume capacity of disposable equipment. They are already commonly used for the manufacturing of recombinant proteins; we see similar potential in the field of cell culture-based vaccine production.

Outlook and concluding remarks

In the last two decades, many technological advancements have been made to establish and intensify processes for cell culture-based influenza vaccine manufacturing. A shift from traditional egg-based production toward cell culture methods seems more and more inevitable, especially with the option for quasi-continuous processes, aiming to improve efficiency and to reduce costs. However, remaining challenges include optimizing cell lines and media for higher virus yields, implementation of advanced online monitoring tools, and adapting downstream processing to new modalities. Achieving robust and scalable perfusion strategies with high virus productivities at small footprints may be crucial to meet the ever-rising vaccine demand. However, the choice between batch and continuous modes for vaccine production depends on several factors, including the scale of production, cost-effectiveness, process control, product quality, and regulatory considerations. In some cases, a robust batch process with a high performing cell line can still outcompete a perfusion process and additionally persuades with an easier setup and less demanding engineering and operational tasks. Furthermore, the industry is actively exploring the integration of SU technologies in both areas, development and manufacturing. Here again, a combination of technological progress and intensified processes can result in efficient upstream processes that can progressively be done using smaller equipment, which favors implementation of SU technologies besides the enhanced flexibility and reduced contamination risks. As advancements in cell culture-based influenza vaccine production progress, addressing these challenges will be pivotal for realizing the full potential of this approach and finally take over from egg-based production. This includes refining process intensification strategies, establishing platform processes, ensuring product consistency, and optimizing the entire bioprocessing workflow to meet the demands of large-scale vaccine production and rapid response to emerging influenza strains.

Funding Statement

The author(s) reported that there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization: T.Z., Y.G.

Methodology/investigation: T.Z., Y.G.

Writing – original draft: T.Z.

Writing – review and editing: T.Z., U.R., Y.G.

Supervision: Y.G. and U.R.

All authors agree to be accountable for all aspects of the work.