Cell-based influenza vaccine: current production, halal status assessment, and recommendations towards Islamic-compliant manufacturing

ABSTRACT

Influenza virus is a life-threatening pathogen that infects millions of people every year, with annual mortality in the hundreds of thousands. The scenario for controlling infection has worsened with increasing numbers of vaccine hesitancy cases reported worldwide due to objections on safety, religious and other grounds. Uses of haram (impermissible) and mashbooh (doubtful) ingredients in vaccine production has raised doubts among Muslim consumers and consequently stimulated serious vaccine hesitancy. To address this major problem, we have reviewed and recommended some alternatives appropriate for manufacturing cell-based influenza vaccine which comply with Islamic laws and consumers’ needs. Intensive assessments of current influenza vaccine production in both scientific and Islamic views have led to the identification of four main ingredients deemed impermissible in novel sharia-compliant (approved by Islamic laws) vaccine manufacturing. Only some of these impermissible components could be replaced with halal (permissible) alternatives, while others remain impermissible due to unavailability and unsuitability.

KEYWORDS:

• Cell-based influenza vaccine
• vaccine hesitancy
• impermissible ingredient
• sharia-compliant vaccine manufacturing
• halal pharmaceutical
• Islamic law

Introduction

A vaccine is a biopharmaceutical product which is used to prevent infection for a communicable disease throughout the world. It contains antigenic substances which can activate the immune system in the body to protect against a certain disease. Vaccination, which initially started in England, began to be implemented globally as part of a community immunization program in the 1800s.^1,2 The acceptance of vaccination around the world, however, has shown a declining trend recently due to vaccine hesitancy, particularly in Muslim countries like Malaysia, Afghanistan, Saudi Arabia, and Pakistan.^3,4 Vaccine hesitancy and rejection among Muslims is based on assumptions and beliefs that such a practice and the ingredients used are “doubtful (mashbooh)”, and they would prefer to abstain to maintain their sanctity.

In Islamic laws, the consumption of dead meat (carcasses), blood, and swine flesh which has been summoned with the name other than Allah are strictly prohibited and classified as al-najasat (ritually impure, sing. najs) and therefore haram (impermissible) unless in darurat (dire conditions) as stated in the Quran (al-Ma’idah: 3).⁵ Outside of dire conditions, those things cannot be eaten, drunk, or used for medicinal purposes as proscribed by the Prophet Muhammad in the hadith (prophetic traditions), “Allah has not placed a cure for your diseases in things that He has forbidden for you.”⁶

A vaccine, however, is permissible for Muslims’ consumption, even if some of its ingredients are made of porcine or other impermissible substances, based on the Islamic concept al-darurat tubih al-mahzurat (dire necessity renders the impermissible to be permissible).⁶ The opinion, however, varies among scholars from different legal schools, who have distinctive opinions in defining the exact meaning of darurat. Some scholars classify vaccinations as a dire need, while some of them consider it otherwise because of the unguaranteed effects of vaccines in preventing and curing diseases. To solve the intricate arguments and, at the same time, to avoid any destructive consequences, sharia-compliant or generally known as halal (permissible) vaccines that are purely free of any ritually impure ingredients are needed.

The production of halal vaccines could be enabled through the establishment of sharia-compliant vaccine manufacturing facilities according to Islamic jurisprudence. Fundamentally, sharia-compliant means any procedures and/or materials that obey sharia laws, the commands of Allah concerning the conduct of human beings who are accountable.⁷ The establishment of the sharia-compliant vaccine manufacturing facilities are expected to fulfill halal vaccine demands for more than 1.8 billion Muslim consumers around the world with an estimated market value USD 50 USD billion.⁸

Influenza vaccine is one of the most common vaccine taken annually to prevent influenza infection which cause three to 5 million cases of severe infection and approximately 300–650 thousand death worldwide every year.⁹ Yearly vaccination against the disease has been recommended in most countries in the world. The application of influenza vaccine is very important for Muslim community as well, particularly for pilgrimage purposes in Saudi Arabia, since respiratory illnesses related to influenza have been reported to be the most common disease infecting pilgrims every year.¹⁰

To fulfil the yearly demands of influenza vaccine, the manufacturing method of the influenza vaccine has been shifting from egg-based to cell-based. In the cell-based vaccine production, various types of impermissible ingredients employed which incite serious concerns among Muslim consumers. In current publications, there are very minimal studies conducted to analyze the halal status of ingredients used in the cell-based vaccine production. Therefore, this study was conducted with objectives: 1) to review the current production of cell-based influenza vaccine, 2) to identify the impermissible ingredients utilized in the current production, and 3) to search for suitable alternatives of the impermissible ingredients which are applicable for use in sharia-compliant cell-based influenza vaccine manufacturing. The goal of this paper is to serve as a useful and reliable reference to initiate and accelerate the research and development (R&D) of sharia-compliant manufacturing, especially for the production of a halal influenza vaccine, which is considered as the communal obligation (fard kifayah) of Muslims.

Methods

The overview of the current production of a cell-based influenza vaccine was summarized based on inputs gathered from industrial visits to Biomedical Research Center, Northwest Minzu University, Lanzhou and Guan Jie Biotechnology Co. Ltd., Jilin, China. Additional information relevant to the production process were searched from Google and Google Scholar databases using search terms [cell-based influenza vaccine], [upstream process for influenza vaccine production], [downstream production of vaccine], [purification of vaccine], [formulation of vaccine], and [vaccine component]. The specific names of vaccine components were also searched to identify its origins and constituents.

The halal status assessment for the cell-based influenza vaccine production was conducted following guidelines published in Malaysian Standard for Halal Pharmaceuticals (MS 2424:2019).⁷ Additional opinions of scholars and sharia laws relevant with the context were searched from Google and Google Scholar databases using search terms [halal pharmaceutical], [halal and haram in biotechnology], [halal and haram in food], and [vaccine component and Islamic view]. All search terms used also were searched in the Malay language to diverse related information. In this study, only religiously impermissible ingredients were reviewed in details and their alternatives were suggested based on Islamic jurisprudences.

Results

Current production of a cell-based influenza vaccine

The current production of an inactivated cell-based influenza vaccine involves at least four stages of complex processes starting with upstream processes and followed by downstream processes. The downstream processes comprise of separation and purification processes, virus inactivation process, and vaccine formulation, fill and finish processes. Figure 1 depicts typical process units involved throughout the production of a cell-based influenza vaccine.

Figure 1. Generic production process of a cell-based influenza vaccine. 1) The upstream process involves replication of host cells grown in a bioreactor containing formulated growth media. Sufficient host cells are then inoculated with influenza virus with trypsin addition to assist internalization of the virus into the host cells. 2) After a few days of incubation, the culture solution containing influenza virus undergoes separation and purification processes using various types of centrifuge and filtration units. 3) Subsequently, the purified virus is exposed to formaldehyde to inactivate its virulence. 4) The aseptically filtered inactivated influenza virus is later formulated with excipients before being packaged as a powdered or liquid influenza vaccine

In upstream processes of the cell-based influenza vaccine production, a dog-kidney-derived cell line, Madin-Darby Canine Kidney (MDCK), has been widely used as a substrate for influenza virus propagation.¹¹ The MDCK cells can be grown either adherently on microcarriers or in suspension using serum-containing or serum-free media.^12–14 Cultivation of suspension MDCK cells in serum-free media is currently favored due to lower labor and processing costs and is proven to be effective enough for high-yield production of influenza vaccine.¹² Antibiotics such as neomycin, streptomycin, polymyxin B, and amphotericin B might be added to the culture media to prevent bacterial contamination during the manufacturing process.^15,16 The MDCK cells are cultivated in the culture media until their concentration adequate for influenza virus inoculation. Prior to influenza virus inoculation, trypsin is added to facilitate effective internalization of influenza virus into MDCK cells.¹⁷ The culture is then incubated for a few days to propagate a sufficient amount of influenza virus before being harvested.

The harvested influenza viruses are subsequently recovered from mixed culture media through downstream processes. In general, influenza viruses can be separated and purified through combination of various methods, such as precipitation, flocculation, centrifugation, extraction, microfiltration, ultrafiltration, and by using bead-based or membrane-based chromatography.^18,19 In common practices, mixed culture media containing influenza viruses are initially centrifuged to separate solid and highly dense wastes such as microcarriers, host cells, cell debris, organelles, and insoluble impurities from the mixture.¹⁸ After that, the resulting supernatant is further purified through repeated membrane filtration and ultracentrifugation. Ultrafiltration is the preferred and most economical method to concentrate and purify viruses, as it effectively and efficiently separates viruses from soluble biopolymers such as proteins, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and lipids.¹⁹ Besides that, nuclease treatment using benzonase and additional polishing steps using chromatography unit operations may also be added to enhance the purity of recovered influenza viruses from nucleic acid residuals and contaminations, as suggested by Wolff and Reichl.¹⁹

For producing an inactivated influenza vaccine, the purified influenza viruses are then treated with inactivating agents such as formaldehyde, β-propiolactone and glutaraldehyde to render it nonvirulent.^15,16 Formaldehyde is the most common inactivating agent used in vaccine production; however, it is known to be carcinogenic to humans.^15,16 To ensure the safety of influenza vaccine produced, complete removal of formaldehyde from the solution using ultrafiltration following the inactivation process must be held effectively prior to formulation with excipients. Additional chemical treatments to split influenza viruses may also be added after the inactivation process to produce more purified inactivated influenza vaccine.¹⁶

In the last stage of influenza vaccine production, highly purified inactivated influenza viruses are formulated with excipients to improve the immunogenicity and stability of the produced influenza vaccines.²⁰ The excipients may be comprised of one or more of the following components: adjuvant(s), stabilizer(s), surfactant(s), buffering agent(s), diluents (e.g., saline solution, sterile water, or protein-based fluids), and preservative(s).²¹ Currently, adjuvants made of aluminum salts and squalene-based oil-in-water emulsions (e.g., MF59 and AS03) are widely used in influenza vaccine formulation and are proven to be effective at enhancing the immunogenicity of vaccine antigens.²² Furthermore, the addition of surfactants (e.g., polysorbate, tween, pluronic) and stabilizers either from sugar-based (e.g., sucrose, trehalose, dextrose, mannitol, sorbitol), protein-based (e.g., human albumin, gelatine), or amino acid-based stabilizers (e.g., arginine, glycine, glutamate) can prevent the rapid degradation of protein antigens caused by mechanical stresses and temperature changes, especially during the drying process and vaccine transportation, distribution, and storage.^20,21,23

The formulated influenza vaccine can be produced in either liquid or powdered form. The liquid influenza vaccine is generated directly after the formulation process and filtered aseptically before being packaged in a single-vial vaccine. On the other hand, to generate the powdered influenza vaccine, the formulated solution must undergo aseptic freeze-drying using cooling agents such as dry ice vapor, liquid nitrogen, or ethanol²³ to make it powdered before being packaged in two individual vials containing lyophilized vaccine and vaccine diluent, respectively. Preservatives such as phenol, 2-phenoxyethanol, and thimerosal (disuse in certain countries) may be added to the liquid diluent or vaccine to prevent bacterial and fungal contamination.^15,16

Impermissible ingredients used in influenza vaccine production and their alternatives based on Islamic jurisprudence

Analyses of current cell-based influenza vaccine production indicated some serious concerns regarding the state of ingredients and unit operations used during the manufacturing process. According to the authors’ judgments based on Islamic rules, most of the unit operations used in influenza vaccine production seem to be free of any impermissible ingredients, except for membrane separation and purification units such as microfiltration, ultrafiltration, nanofiltration, and chromatography units. Those operations units are probably made of, coated with, or linked to gelatin,^24,25 heparin,²⁶ or animal bone charcoal that originate from non-halal animals (e.g., pigs and non-slaughtered cows).²⁷ However, reviews undertaken by Cassano and Basile²⁸ and Wolff and Reichl¹⁹ showed that most of the membrane filters or resins used in vaccine production are made of natural polymers (e.g., cellulose, agarose, dextran), synthetic polymers (e.g., polysulfone, polyethersulfone (PES), polytetrafluoroethylene (PTFE)), ceramics other than bone charcoal (e.g., silica, glass, alumina), or sintered metal.

Therefore, the authors believe there is no serious concern or prohibition regarding the use of any unit operation in vaccine production, as sharia-compliant and contaminant-free membrane separation units with significantly good efficiency are available for use instead, but more precise investigations should be conducted to confirm this hypothesis. In terms of vaccine ingredients and excipients, there are four main types of ingredients that are clearly judged as najs, either completely haram or mashbooh according to Islamic laws. These ingredients exist in the form of host cells, growth media, proteolytic enzymes, and gelatin.

Host cells for virus production

To date, the MDCK cell line, which is derived from a dog kidney, had been used widely in the manufacturing of influenza vaccine as the most ideal host cell line for propagating the influenza virus.^11,29 In Islam, dogs are haram to be consumed because they are categorized as a carnivorous animal with claws and fangs.³⁰ All Sunni schools of thought except Maliki consider dog, in whole or in part, as najs, while Shafi’i classifies them precisely as wholly najs mughallazah (severely impure), which requires a special ritual cleaning procedure.³¹ Hence, the use of the MDCK cell line to generate vaccines is forbidden in Islam, as it cannot be eaten or injected into bodies and could implicate arduous treatments or ritual cleaning to ensure the “purity” of the production equipment and the end products based on the Islamic definition.

Even though separation and purification processes could efficiently sieve out enormous amounts of residual host cells, the end products usually would not be 100% free of residual host-cell proteins. Based on the requirements set by authorities such as the U.S. Food and Drug Administration (FDA)³² and the World Health Organization (WHO),³³ the maximum residual host-cell DNA in each dose of vaccine must be less than 10 ng, and, if possible, must be less than 10 pg. If vaccine manufacturers merely followed the standards set by the authorities, the vaccines we use today would probably contain a miniscule amount of najs, as the residuals cannot be eliminated completely. Concerns regarding the effects of residual host cells on human health have also been described in a few previous studies.^34–37

To address these problems, both in terms of safety and Islamic concerns, a better alternative host cell line, specifically for influenza vaccine manufacturing, must be chosen. Other than the MDCK cell line, various types of cell lines originated from a human (e.g., HEK293³⁸ and PER.C6³⁹), hamster (e.g., BHK-21⁴⁰), monkey (e.g., Vero¹⁴), avian (e.g., EB66,^41,42 AGE1.CR,⁴³ DuckCelt-T17,⁴⁴ and PBS-1⁴⁵), and insect (e.g., Sf9⁴⁶) have been tested as candidates for large-scale production of influenza virus. Considering the ethics, norms, and religious aspects, as listed in Table 1, the avian cell lines from either duck (EB66, AGE1.CR, or DuckCelt-T17) or chick embryo (PBS-1) seem to be the best options available to replace the MDCK cell line. Human-derived products or cell lines are totally rejected in Islam because the human is a pure and respected organism, and its exploitation is haram.⁴⁷

Table 1. Opinion of Islam on various types of host cells used for influenza virus propagation

Host Cell	Origin	Source	Opinion of Islam ^a
MDCK	Dog	Kidney ^b	Categorized as a carnivorous animal with fangs and claws, thus, haram to be consumed.
Vero	Monkey	Kidney ^b	Categorized as a wild animal with fangs and claws, thus, haram to be consumed
BHK-21	Hamster	Kidney ^b	Categorized as a dirty and disgusting animal, thus haram to be consumed
Sf9	Insect (fall armyworm)	Ovarian cell ^c	Categorized as a dirty and disgusting animal, thus haram to be consumed
HEK 293	Human	Embryonic kidney or neuronal cell ^b	Human is a pure creature that must be respected, thus haram to be consumed
PER.C6	Human	Embryonic retinoblasts ^b	Human is a pure creature that must be respected, thus haram to be consumed
EB66	Duck	Embryonic stem cell ^b	Categorized as a bird without fangs and claws, which is halal to be consumed only if slaughtered with the name of Allah. Otherwise, will be considered as carcasses which are haram to be consumed
AGE1.CR	Duck	Embryonic retina ^b	Categorized as a bird without fangs and claws, which is halal to be consumed only if slaughtered with the name of Allah. Otherwise, will be considered as carcasses, which are haram to be consumed
DuckCelt-T17	Duck	Embryo ^d	Categorized as a bird without fangs and claws, which is halal to be consumed only if slaughtered with the name of Allah. Otherwise, will be considered as carcasses, which are haram to be consumed
PBS-1	Chick	Embryo ^b	Categorized as a bird without fangs and claws, which are halal to be consumed only if slaughtered with the name of Allah. Otherwise, will be considered as carcasses, which are haram to be consumed

^aBased on sharia laws as stated in Standards Malaysia.⁷

^bInformation extracted from Genzel.⁴¹

^cInformation extracted from Cox and Anderson.⁴⁶

^dInformation extracted from Petiot et al.⁴⁴

In the Islamic perspective, a duck or chick slaughtered in the name of Allah (otherwise defined in this review as a non-slaughtered animal) is considered as halal meat and absolutely permissible for consumption by Muslims.⁴⁸ Although the established avian cell lines are not extracted from a religiously slaughtered duck or chick and deemed as carcasses, the authors have confidence that the selection is the next closest option to halal ingredients. In term of production suitability, avian cell lines such as PBS-12SF, DuckCelt-T17, EB66, and AGE1.CR are known to be free of adventitious agents, can proliferate to high densities in suspension forms using serum-free media in various production modes, are highly permissive to a broad range of influenza strains and are capable of producing a high yield of influenza virus efficiently.^42–45 In brief, avian cell lines of either duck or chick origin have great potential to be used as alternative substrates for the production of sharia-compliant influenza vaccines.

Growth media for host-cell consumption

To propagate a sufficient amount of influenza virus for vaccine production, high densities of host cells must be obtained first. Like other living organisms, the viability and growth rate of host cells depends directly on the supplemented nutrients. Serum-containing media is the most common and effective synthetic media used for cultivating high densities of assorted cell culture types. This media involves the addition of mammalian serum usually extracted from human, bovine, or equine blood, which is then mixed with established basal media.^49–51 Serum is a naturally complex, ill-defined, and varied biological component rich in active substances such as hormones, growth factors, binding and transport proteins, attachment and spreading factors, natural fatty acids, lipids, amino acids, vitamins, and trace elements necessary for the survival and growth of cell and tissue cultures.^50–52

As stated in the Quran, blood is strictly forbidden for consumption by Muslims,⁵ thus, the application of blood in vaccine production should be avoided. Uses of human-derived and animal-derived components, especially serum extracted from bovine fetuses, incites not only religious concerns but also ethical and safety concerns.^53,54 For example, draining of blood from bovine fetuses using cardiac puncture causes severe suffering to the fetuses, and those practices are absolutely inhumane and against ethical procedures.^52,53 In terms of safety, the transmission of variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) between species through infectious proteins or prions causes serious apprehension among researchers, manufacturers, and consumers.^52,54

Fortunately, with advancements in life sciences, the numbers of animal components used in cell cultures are gradually being reduced, and a few types of serum-free media are already applicable for certain cell types. Although the use of serum-free media could eliminate supplementation with ill-defined serum, some essential proteins and hormones originating from human or animal sources such as insulin, transferrin, and albumin are still needed to maintain healthy and stable cell growth.^49,50 Surprisingly, chemically defined media which categorized under serum-free media and generally claimed as animal-component-free media also may actually contain recombinant proteins, which are often categorized as non-animal components.⁵⁴ According to sharia laws, recombinant proteins or any genetically modified products expressed using DNA, RNA, plasmid or any related materials derived from human, forbidden animals (e.g., porcine), or non-slaughtered animals (e.g., bovine) are stringently forbidden and regarded as najs unless halal materials are used instead.^55,56

Apart from that, the basic components of synthetic media known as basal media, such as Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI 1640), which are usually supplemented with serum or serum-substitutes, typically constitute at least 13 essential amino acids, 8 water-soluble vitamins, 6 inorganic salts, and glucose to support lasting cell growth.⁵⁷ Toward the establishment of serum-free media, more complex basal media containing wider types of amino acids, vitamins, minerals, and metabolites are used to compensate essential components of serum.⁵⁸ Aligned with safety and religious concerns raised by Ermis,⁵⁹ Fletcher and Harris⁵⁴ and Karahalil,⁶⁰ amino acids used in basal media most likely contain animal-derived residues resulting from amino acid extraction from animal tissues, enzymatic processes, or microbial fermentation.⁶¹ Some of the amino acids can be synthesized chemically, but most of the organic components exist abundantly in animal tissues or are naturally synthesized by microorganisms presumably fed with animal-sourced media during the fermentation process. Vitamins such as myo-inositol,⁶² riboflavin,⁶³ and pantothenic acid⁶⁴ may also be produced similarly to amino acids, thus similar concerns should be evaluated.

Disappointingly, limited access to information due to proprietary issues and ambiguous origins of components constituting commercialized cell culture media impede comprehensive evaluations and selection of sharia-compliant growth media. Until now, the authors believed that each of the established growth media available today, including so-called animal-component-free media, still contained at least one component as fine as DNA that originated from humans, forbidden animals, or non-slaughtered animals, which are considered as impermissible ingredients in Islam. Besides that, purified blood-derived components like albumin, transferrin, and hydrocortisone⁴⁹ may still be in use in most of the growth media to imitate the innate growth environment of cells and tissues, which contradict sharia laws that prohibit the utilization of najs including blood, carrion, and pus.³⁰ Additional opinions of Islam regarding various origins of growth media components are further described in Table 2.

Table 2. Components of growth media and their corresponding opinion of Islam

Category	Example ^a	Opinion of Islam ^b
Human-derived product	Insulin, transferrin, albumin, xeno-free media, etc.	Haram for consumption as human parts/derivatives are pure materials that must be respected and not being exploited or commercialized.
Animal-derived product	Amino acid, lipid (cholesterol, steroid, fatty acid), growth factor, hydrolyzate (animal tissues, milk), hormones, carrier protein, etc.	Halal if sourced from halal and religiously slaughtered animals (chick, bovine, fish) Haram if sourced from non-halal animals (e.g., porcine, wild animals, amphibians) or non-slaughtered animals (e.g., bovine)
Plant-derived product	Hydrolyzate (soy, wheat, rice), glucose, maltose, vitamins, lipid, etc.	All plant-based products except those from poisonous, intoxicant, or hazardous plants are halal to be consumed.
Blood-derived product	Serum, plasma, platelet-derived product, albumin, etc.	Haram as blood is considered as filth material which stringently forbidden for any consumption.
Chemical synthesis product	Inorganic salts (CuSO4, KCl CaCl2,), trace elements, selenite, sodium bicarbonate, etc.	All chemical-based or chemical synthesized products except poisonous, intoxicant, or hazardous chemicals are halal to be consumed.
Fermentation/Microbial product	Amino acid, vitamins, yeast hydrolyzate, glutathione, etc.	Halal if the materials (e.g., growth media) and microbes used in the fermentation are sourced from halal resources.
Recombinant product	Insulin, transferrin, etc.	Recombinant products expressed using genetic materials (e.g., plasmid, DNA, RNA) originated from halal and slaughtered animals or microorganism are considered as halal. Otherwise, will be considered as najs which are haram for consumption.

^aThe examples were extracted from Yao and Asayama.⁴⁹

^bBased on sharia laws as stated in Standards Malaysia.⁷

As there are no alternative growth media that are 100% free of impermissible ingredients and compliant with sharia laws, chemically defined and animal-component-free media is the best option available for selection. However, the suitability of growth media for host cells must be considered. Auspiciously, avian cell lines such as PBS-12SF, DuckCelt-T17, EB66, and AGE1.CR were successfully adapted to grow in serum-free media.^42–45 If those cell lines can be adapted in chemically defined media without supplementation with any animal-derived components or consist solely of plant-based ingredients, a better alternative growth media usable for sharia-compliant vaccine processing could be created in the future.

Proteolytic enzymes

In cell culture technology, most naturally isolated cells or primary cells tend to grow adherently by forming protein bridges on the surface of culture flasks or microcarriers. Disruption of the protein bridges using proteolytic enzymes, also called proteases, is thus necessary to detach and dissociate the cells during passaging. Trypsin, a serine protease that specifically cleaves the peptide bonds at the C-terminal end of positively charged lysine and arginine side chains, has been proven to detach and dissociate cells effectively from culture flasks and microcarriers.⁶⁵

Beside been used solely as a cell detachment agent, trypsin is also useful for mediating efficient propagation of influenza virus in host cells. The addition of trypsin prior to influenza virus infection could assist the internalization process of influenza virus into the host cells through cleavage of the virion surface glycoprotein known as hemagglutinin (HA) precursor HA0 into HA1 and HA2 subunits.¹⁷ The presence of trypsin also interferes in the pathogen defense mechanisms of host cells, which significantly promotes high titer production of influenza virus.⁶⁶ Although trypsin plays important roles in the successful production of influenza vaccines, its origin is frequently disputed mainly by Muslims. Most of the commercialized trypsin applicable to cell culture studies and vaccine productions today were extracted from porcine pancreas,^4,55 and some of it originated from bovine pancreas.⁶⁵ The use of porcine trypsin in sharia-compliant processing is clearly forbidden by Islamic laws, while the use of bovine trypsin is only permissible if it has been extracted from a religiously slaughtered bovine as explained in Table 3.

Table 3. Opinion of Islam on different types of commercialized proteolytic enzymes

Proteolytic Enzyme	Origin	Opinion of Islam ^a
Trypsin	Porcine ^b	Haram to be consumed
Trypsin	Bovine ^c	Halal to be consumed if extracted from bovine slaughtered with the name of Allah. Otherwise, considered as part of carcasses, thus, haram to be consumed.
TrypLE	Recombinant fungi ^d	Halal to be consumed if genetic materials used are originated from halal sources. Natural microorganisms, including fungi itself, are halal according to sharia laws unless prohibited by the competent authority.
Accutase	Marine life (crustacean) ^d	Halal to be consumed unless extracted from amphibians, disgusting, poisoning, and intoxicating organisms.

^aBased on sharia laws as stated in Standards Malaysia.⁷

^bInformation extracted from Khoo et al.⁴

^cInformation extracted from Mótyán, Tóth, and Tőzsér.⁶⁵

^dInformation extracted from Rourou et al.⁶⁷

To meet the demand for animal-free products, a recombinant fungal protease, namely trypLE, has been generated through the fermentation process and is currently commercialized as a trypsin substitute with similar functions to trypsin.^67,68 TrypLE has superior effectiveness as a cell detachment agent for various cell types with greater stability and lower toxicity effects compared with traditional animal-derived trypsin.^67–70 The origin of trypLE, however, cannot be traced meticulously due to proprietary issues. Impermissible genetic materials sourced from forbidden animals might be used during production of the recombinant protease, thus it is prone to be excluded from sharia-compliant processing unless additional information proves otherwise.

In order to eliminate any doubts, accutase, a trypsin replacement prominently claimed by suppliers as a non-bacterial and mammalian-free protease and collagenase mixture^71,72 extracted from marine life⁷³ or specifically from an invertebrate species⁷⁴ such as a crustacean⁶⁷ seems the best option available. The selection was made based on sharia laws allowing the consumption of all kinds of marine life merely living in water except disgusting, poisoning, and intoxicating organisms.^48,55 In terms of performance, the capacity of accutase to detach and dissociate viable cells is lower than that of trypsin, but a few days after trypsinization, accutase-treated cells successfully recovered more cells and rapidly grow better than trypsin-treated cells.⁷⁵ The cellular behavior of accutase-treated cells also remained unchanged, and they could proliferate longer than cells dissociated with trypsin.⁷⁶ Addition of accutase prior to influenza virus infection also slightly improved virus propagation in the host cells (MDCK), although its effectiveness was incomparable to that of trypsin.⁷⁷ With those abilities, accutase could be a potential alternative to be used in sharia-compliant influenza vaccine production.

Gelatin

In vaccine production, maintaining the properties and structures of the active ingredients, including protein antigen(s), in vaccines is very important to avoid degradation and denaturation of components, which can lead to decreased effectiveness and potency of the vaccines. For the preparation of freeze-dried vaccine, a stabilizer is added prior to the lyophilization process to provide a matrix that can contain and protect vaccine components from the harsh conditions of the process.¹⁶ Gelatin and human serum albumin (HSA) are frequently used stabilizers for live attenuated and inactivated vaccines (including influenza vaccine), which contain high complexity, intrinsic instability, and environmental stress sensitivity of active components.^16,21,78 In terms of safety, both materials could transmit adventitious agents, but the application of HSA involves stricter requirements from authorities, and the source can only be certain certified manufacturers.¹⁶ In contrast, gelatin is a more feasible option with a more readily available supply.

Gelatin is a biopolymer protein partially hydrolyzed from collagen abundantly found in animal skins, bones, cartilages, and connective tissues.^79–81 Out of 108 vaccines analyzed and listed by Cardoso, Petrovajová, and Horňáková,²¹ 24 contain stabilizers made from gelatin, 15 from HSA, and 5 from both HSA and gelatin. The origins of the gelatins were not specified, and only six of them were specified as porcine-derived gelatin. The unknown gelatins were most likely of porcine origin, as nearly half of the global production of gelatin is sourced from pig skin (46%), followed by bovine hides (29.4%), pork and cattle bones (23.1%), and other sources (1.5%) such as fish and poultry skins and bones.^80,81 Economical production, a shorter pre-treatment process, and less generation of wastewater have become the main reasons why porcine gelatin is produced on a massive scale compared with bovine gelatin.⁸²

Although some Muslim scholars allow the use of gelatins sourced from any type of animal, including pigs, which they believe have been through an istihalah (transformation) process,^6,83,84 gelatins sourced from halal animals must be prioritized to avoid any argument. Nowadays, halal bovine gelatin is already manufactured and commercialized for Muslim consumption, but the supplies are limited, and the product niche is not as large as that of other types of gelatin.^8,48,81 To satisfy the requirement of sharia-compliant processing, other than using halal bovine gelatin, fish gelatin,^79,81 plant-based alternative of gelatin,⁴⁸ and other types of sugar alcohols, such as sorbitol, lactose, inulin, and trehalose,²¹ have great potential to be used as vaccine stabilizers instead of HSA, porcine, and bovine gelatins with unknown status. The invention of bacterial cellulose as gelatin substitute in future also might be another great option available to be used.⁸⁵

The application of gelatin is not only limited as a vaccine stabilizer but is also useful as a coating material or ligand for microcarriers.^86–88 Microcarriers, bead-like materials used to grow adherent cells in suspension form, are regularly encapsulated with gelatin to improve their biocompatibility through imitation of innate conditions favorable for cell adhesion and growth. In nature, cells tend to adhere to each other or to the extracellular matrix made up of collagens, proteoglycans, fibronectin, laminin, elastin, or chondronectin to nurture efficient cell functions, particularly for cell metabolism and differentiation.⁸⁷ Most of the commercial microcarriers, such as Cytodex 3, Cultispher S, Cultispher G, FACT III, Gelibeads, and Ventregel, have surface coatings made of porcine gelatin;^86–88 therefore, those microcarriers are barred from sharia-compliant processing. As alternatives, ionic microcarriers made up of cellulose, dextran, or synthetic polymers (e.g., polyacrylamide, polystyrene, and polyurethane) treated and substituted with ionic materials (e.g., diethylaminoethanol (DEAE)) can be used instead.^87,88 The charge density overlaid on the surface of the ionic microcarriers will promote cell attachment and spreading. Cytodex 1, Cytoline 2, and Cytopore 2 are exemplars of commercial ionic microcarriers that have been well studied and used for scale-up production of viral vaccine⁸⁶ and suitably implemented in sharia-compliant processing.

Recommendations for sharia-compliant cell-based influenza vaccine manufacturing

Based on the findings collected throughout this study, a few alternative suggestions, as summarized in Table 4, could be implemented in the manufacturing process of sharia-compliant cell-based influenza vaccines. The alternative suggestions for host cells and growth media may not obey sharia laws fully, but they are the best and closest options available today. Better options that obey sharia laws might be reserved in future if more studies related to this development can be carried out. The effectiveness of each alternative proteolytic enzyme, microcarrier, and gelatine stabilizer need to be tested to verify the suitability of those alternatives for producing safe, effective, and profitable influenza vaccines.

Table 4. Impermissible materials used in current production of cell-based influenza vaccines and their corresponding sharia-compliant alternatives

Types of materials	Impermissible materials	Alternative materials
Host cell	MDCK cell line	Duck (EB66, AGE1.CR, DuckCelt-T17) or chick cell lines (PBS-1)
Growth media	Serum-containing or animal-component media	Serum-free and animal-component- free media
Proteolytic enzyme	Porcine trypsin	Accutase, trypLE, or religiously slaughtered bovine trypsin
Microcarriers	Gelatin-coated microcarriers (FACT III, Cytodex 3, Cultispher S, Cultispher G)	Ionic microcarriers (Cytopore 2, Cytoline 2, Cytodex 1)
Stabilizer	Porcine or non-slaughtered bovine gelatin	Sugar alcohol (sorbitol, trehalose, inulin, etc.), religiously slaughtered bovine or fish gelatin.

In selecting those suggestions, chemical-based, plant-based, and animal-component-free materials are prioritized over animal-based materials to avoid intricate verification and possible fraudulent claims of raw material status. All human-based materials also must be avoided at all costs in sharia-compliant vaccine manufacturing.

Discussion

Based on the results, the current production of cell-based influenza vaccine does involve the use of impermissible or haram ingredients in the form of host cells, growth media, proteolytic enzymes, and gelatin. Other ingredients classified as mashbooh are not discussed in details in this study as the alternatives of those ingredients are available and optionally used by manufacturers. The authors would like to emphasize that the determination of the halal status of ingredients used in the cell-based influenza vaccine production and the selection of corresponding sharia-compliant alternatives as discussed in this paper were mostly made based on guidelines published in the world first halal pharmaceutical standard, MS 2424:2019.⁷ The halal definition and requirements as stated in the standard and applied in this study may differ from other countries or Muslim communities as they follow different schools of thought like Hanafi, Hanbali, Maliki, and Shafi’i.^89–91 In future studies, other standards certified by other countries or certifying bodies should be considered in defining the exact definition and requirements of halal and sharia-compliant production. The creation of a global halal standard is essential for the establishment of halal products including vaccines which are well accepted by all Muslim communities.

Besides the absence of the global halal standard^55,89,90 and halal-listing pharmacopoeia,⁹² long duration of R&D stage, high cost production and operation, as well as stringent requirements for safety and halal aspects of the whole manufacturing process become the major constraints for the establishment of the halal vaccine industry.⁹³ In particular, the current production of influenza vaccine has a relatively low-profit margin due to its low product selling price. Toward the establishment of the sharia-compliant influenza vaccine manufacturing, changes of the production process and raw materials as recommended in the Results section will increase the overall production costs and create time-consuming regulatory challenges such as the need for clinical trials to confirm the effectiveness and safety of the produced vaccine and the lengthy procedures of halal certification. Inaccessible and shortage of halal raw materials supplies such as gelatin⁸ and lack of human capital who are competent in both manufacturing fields and sharia laws⁵⁵ also contribute to other challenges for manufacturers. Due to those constraints, manufacturers will be reluctant to implement the new sharia-compliant process. Although the establishment of the sharia-compliant or halal-only facilities for vaccine production seem impractical and not the best action plan for the meantime, the production of halal vaccine is expected to bring additional profits to manufacturers in future, as there are huge potential market and increasing demands for halal pharmaceutical products.⁸

Granted that there are a lot of hurdles toward the establishment of the sharia-compliant vaccine manufacturing, Muslims must keep working on this establishment for the sake of Muslims well-being. With the creation of a halal vaccine, the demands of halalopathy⁹⁴ and ethnocentric medicines⁹⁵ among Muslims and non-Muslims can be attained, as some similar vaccine components, are also forbidden in other religions, as described by Grabenstein.⁸⁴ The successful establishment of sharia-compliant vaccine manufacturing in the future will contribute immensely to the expansion of the halal pharmaceutical industry, which is estimated to be worth USD 131 USD billion by the end of 2023.⁸

This study has several limitations. Owing to the proprietary nature of vaccine development and production details, this review might not capture all related issues accurately. As some information for this study was collected through industrial visits, there might be some bias in the analysis as those issues that could be ascertained during the observation and interview process are less proprietary hence discoverable, whereas the most proprietary information was not revealed during the observation and interview process and thus could not become part of this study and analysis.

Conclusions

Increasing vaccine hesitancy cases due to religious and safety grounds cannot be ignored. When provided with better vaccine alternatives, consumers, especially Muslims, might change their minds and perceptions of vaccines. Unfortunately, the establishment of sharia-compliant vaccine production cannot become a reality in just a few years. The complexity of vaccine processing itself and inaccessible supplies of sharia-compliant materials has hindered, for the most part, this endeavor. High infectivity and rapid mutation of the influenza virus complicate this endeavor even more as Muslims desperately need sharia-compliant influenza vaccine supplies each year to protect themselves.

As presented above, at this moment, the establishment of sharia-compliant influenza vaccine production still cannot be realized fully. Host cells essentially needed for influenza virus propagation are mostly derived from human, dog, monkey, insect, or avian carcasses, which are prohibited from use in sharia-compliant processing. Blood, human-, porcine-, and non-halal bovine-derived proteins, as well as mashbooh fermentation and recombinant products used in the preparation of growth media applicable to host cell propagation are currently irreplaceable and represent the greatest challenge to be resolved. Impermissible proteolytic enzymes and gelatin application in influenza vaccine production, fortunately, can be replaced with permissible ingredients, although the ingredients are not equally effective. All alternative ingredients suggested in this review were generated solely on the basis of combined information gathered from previous studies, and those ingredients might be impractical for use in real production of influenza vaccine unless they are tested on the laboratory and industrial scales. To verify and uphold those suggestions, more intensive and scientific studies should be done in the near future. New inventions of sharia-compliant cell lines and applicable growth media may also resolve existing problems with sharia-compliant processing.

Although most parts of this review focused on the establishment of sharia-compliant vaccine processing, the outputs might be useful for befitting the creation of safer animal-free vaccines that meet strict regulation policies. In sharia-compliant or halal processing, all ingredients employed for food and medicine production must be sourced from safe, clean, and nutritious supplies, notably involving animal-derived products that compulsorily originated from religiously slaughtered animals and passed high safety screening as exercised under the concept of halalan toyyiban. This review may also be used as a reference for amending more specific and standard guidelines for halal vaccine certification. The authors hope that this review could be a precursor and great help to other researchers who have the same interests in developing sharia-compliant vaccine processing.

Declaration of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgments

Acknowledgements are extended to Department of Chemical and Process Engineering, UKM for their support, and also to Biomedical Research Centre, Northwest Minzu University and Jilin Guan Jie Biotechnology Co. Ltd. for their kind assists and shared knowledge during our visits.

Additional information

Funding

This work is supported by Universiti Kebangsaan Malaysia, Research University Grant DCP-2017-010/1 for Sharia-compliant Vaccine Program (DCP-2017-010).