**3.1 Mammalian production systems**

*Monoclonal Antibodies*

complementary interface [46].

[50, 52] and the problem persists.

linear antibody residue sequences [58].

and the increasing availability of the generated data in public domains provided a

analyses of antibody structures modeled from query residue sequences. These models are typically obtained by homology with precompiled antibody scaffold templates [44], which is possible because, despite the unique spatial identity of mAbs, the geometry of their variable regions is well conserved, with most CDR loops having a limited number of conformations, known as canonical classes [11]. In general, the established modeling tools coupled with refined protein–protein docking [45] and machine learning methods have been found useful for predicting the VH and VL domain arrangements and the potential antibody electrostatic

Computer-assisted design of new mAbs consists of high-throughput algorithm

Examples of platforms available for antibody modeling are the "Prediction of ImmunoGlobulin Structure" (PIGS) [47], the Rosetta Antibody Modeling [48], and the "Web Antibody Modeling" (WAM) [49]. These servers comprise fully automated homology-based modules that predict with high accuracy the tridimensional antibody structure, including most of the hypervariable regions of the antigenbinding site [50]. An exception is the H3 loop. Unlike the other CDRs, the H3 structure has unique conformations that do not follow a canonical form and are also not found in any described protein, with ~75% of its fragments not having structural neighbors in the known non-immunoglobulin protein world [51]. Therefore, the H3 loop cannot be predicted by selecting templates from a database and this is an important obstacle for the *in silico* antibody design. Some alternative algorithms, based on candidate conformations obtained computationally and energy functions, have been developed, but they often fail to produce sub-angstrom structure models

Other concerns also affect antibody modeling. The limited number of highquality X-ray crystal structures of mAbs in public protein databases may not be sufficient to allow a proper antibody shape prediction [50]. Regarding the docking protocols, it should be noted that, despite the great advances in the bioinformatic field, most of the antibody algorithms still need to be optimized to consider the molecular backbone flexibility and the transient conformational changes following protein–protein interactions [53, 54]. Another relevant point is the time needed for antibody modeling. High-throughput computational design of mAbs can still be as time-consuming as experimental cellular approaches, even when well-consolidated prediction systems are used. As an example, the Rosetta Antibody server was previously found to take 570,000 CPU hours to generate ~2,000 antibody models [55]. With many challenges ahead, currently there are few reports of functional antibodies completely designed by in-silico approaches. A successful attempt in this field is the mAb described by Nimrod and co-workers, which was based on robust predictions of specific residue-residue interactions rather than modeling the entire antigen–antibody complex [56]. On the other hand, computational protocols have been used with increasing frequency to improve the physicochemical properties of previously generated mAbs, as well as to engineer humanized versions of murine full-length immunoglobulins, making them like those found in humans [57]. Molecular structure-based iterative algorithms have been shown to optimize the generation of humanized antibody scaffolds without a significant drop in affinity and specificity toward the antigen, compared to the original murine one, and with reduced occurrence of structure failures, important drawbacks commonly found following conventional humanization techniques, which are mostly guided by

The overall computational antibody discovery scenario is promising and, although the design of new biologically active mAbs is still deeply dependent on

fundamental basis to the in-silico generation of mAbs.

**28**

As previously mentioned, one of the major utilities of the mammalian expression systems is to produce complex biomolecules such as antibodies that require posttranslational modifications like glycosylation [38]. Though other eukaryotic systems do provide this modification, their capability of doing so is limited and might result in the addition of glycans that are not common in human proteins [59]. This event might result in misfolding and biologically inactive immunoglobulins, undesirable features in human therapeutic and diagnostic monoclonal antibodies [60]. Also, expression in prokaryotic systems might lead to contamination with endotoxins, which increases downstream processes to clear these endotoxins from the final product. Thereby, the mammalian expression systems are valuable tools to produce monoclonal antibodies as well as other proteins with proper structure and activity. Indeed, there are numerous FDA-approved mAbs produced in mammalian expression systems in contrast to prokaryotic systems and other eukaryotic cells [61].

The primary technique for mAb obtention was already originally dependent on a mammalian cell: the hybridoma cell [7]. As the high specificity of the monoclonal antibodies was making these molecules increasingly useful for various applications, a hybridoma large-scale cultivation became a great demand in the industry. Therefore, the ascites method production was no longer enough to supply bulk production, nor feasible due to ethical matters. That way, most research and diagnostic proposed mAbs are now produced *in vitro,* through the harvest and following purification of mAb-enriched media obtained in dynamic or non-dynamic cell culture systems [62].

In a therapeutic context, although the hybridoma cell lines are still responsible for the generation of more than 50% of the FDA-approved mAbs [63], these antibodies are bulk produced in other mammalian host systems [64]. This is due mainly to the highly immunogenic nature of murine mAbs for humans, demanding the antibodies to be genetically modified (humanization or generation of fragments) for human therapeutic use [65]. Besides, many of these mammalian cells had their expression machinery highly optimized for recombinant protein production [66].

In the mammalian expression system, cells are readily transfected or transduced to introduce foreign DNA that codes for the target protein and then, they are cultivated preferably in suspension in a chemically defined serum-free media [59].

The preferred mammalian cell lines for protein expression in research and industrial fields are CHO and HEK-293 cells [67]. CHO cells are dominant in heterologous protein production in industry, mainly because of advantages like the property to provide complex post-translational modifications similar to those of humans, their ease to scale-up, and for being easily adapted to grow in serum-free suspension cultures [65]. CHO cells are more suited for stable expression, for its transfection renders low yields of recombinant protein secretion in this lineage. Since establishing a stable cell line is time and labor-consuming, transient transfection is a suitable option to gather high amounts of proteins in a shorter period. In this case, HEK cells might represent an interesting option, since they are wellknown for being rather suitable for transient transfection. This cell line also has rapid doubling time and grows in high-density concentrations, just like CHO cells,

and presents productivity of grams of protein per liter of culture [67, 68], though they have somewhat more tendency to clump [69, 70].

The PER.C6 cells are human embryonic retinal cells, and like HEK cells are pointed out to promote human glycosylation profiles. They were projected to be grown in high-density conditions [71], with stable expression and also offer production yields similar to CHO cells, indicating that human cell lines will be more economically viable and more easily scalable options for antibody productions [72]. There is even description of a production with titers of 27 g/L of antibody, astounding yields when compared to a medium CHO cell production of around 12 g/L of antibodies [73]. Although there is still no FDA-approved mAb produced in this system, there are already some ongoing clinical and preclinical studies carried out with mAbs and other biological products purified from this system, like vaccines for influenza, HIV, and Ebola [74–77].

There are other suitable host cell lineages such as murine lymphoid cell lines like NS0 and Sp2/0-Ag14, derived from BALB/c mice plasmocytomas, corresponding for almost 25% production systems of FDA-approved monoclonal antibodies. One of their major advantages is being originated from naturally high immunoglobulin producing parental cells. Though, their murine origin is not to be underestimated, for there are reports that they do generate immunogenic glycoforms of the expressed antibodies [78].

Concerning the expression vectors for mAb production, usually the plasmids carrying the heavy and light chain genes are constructed based mainly into two kinds of systems: the dihydrofolate reductase (DHFR) system or glutamyl synthetase (GS) based system, both acting as selection markers [65]. In DHFR, selection occurs through glycine, hypoxanthine, and thymidine depletion from the cell culture medium. Selected clones are subjected to the addition of methotrexate, a folate analog that poisons the cells deficient in DHFR, obliging the cells to further synthesize the enzyme with consequent co-amplification of the IgG genes. In the GS system, the selection is done in the absence of glutamine, in a way that only cells with GS can survive by synthetizing glutamine from glutamate and ammonium. Here, the selective pressure is made through increasing doses of the GS inhibitor methionine sulfoximine, pushing the cells to amplify GS and IgG genes [79]. Promoter characteristics, inclusion of antibiotic resistance genes, transcription termination sequences [poly(A)], and translation control sequences should also be taken into account when designing these vectors [80].

In comparison to other production systems, mammalian cells are more fastidious to culture than bacteria and fungi, for they are larger and do not possess tough cell walls like other microorganisms, making them more sensitive to impurities naturally occurring from the production system itself. Having them to thrive and reproduce in culture after modifications to turn them stable and in conditions to secrete the aimed molecule with high yields is a challenge in itself [61, 67]. If one is not choosing for the transient transfection, having the stable lineages may also be costly and time-consuming.

Independent of the expression system, the correct choice of the production scale should be made accordingly to the given necessity. The simplest culture system is the static culture, consisted of T bottles with screw caps kept horizontally in an incubator. Because of its low maintenance profile and low costs, it is the most widely used culture method in the academic research context. It is possible to use this system for clone screening and determining experimental conditions, but its small-scale nature might not render enough mAb quantity for some other types of assays. An option to circumvent this matter might be the use of the rolling systems that offer a medium-scale mAb yield. In this condition, roller bottles are positioned in a rotation system that causes all cells to be in constant movement, and therefore,

**31**

*Alternative Methods to Animal Use for Monoclonal Antibody Generation and Production*

for the cultivation of mammalian cells, both on a pilot and industrial scales.

all of the components of the culture (cells, nutrients, dissolved gases, and metabolites) are uniformly distributed throughout the volume of the medium. This system requires gradual adaptation to cell growth in suspension, starting from very low

Currently, the bulk production of mAbs in agitated bioreactors is the predominant cell culture system in the industry because it allows constant control and monitoring of the process. The area of research for innovation in these bioreactors has advanced dramatically. In general, bioreactors are used to achieve high cell densities and thus increase the production of monoclonal antibodies, biopharmaceuticals, and vaccines [84]. Different types of agitated bioreactors have been used

The use of microorganisms such as bacteria and yeasts is widely used in science for several purposes, generally related to antibiotics and probiotics [73]. However, both bacteria and yeasts have been getting space in the production of mAbs for immunological therapy due to the biopharmaceutical demand and technological advances about their ability to produce antibodies by reducing the use of animals in the manufacturing process [73]. The motivation behind investments that seek to optimize the means of production of mAbs in alternative models stems from the manufacturing disadvantages presented in the traditional method with mammalian cells, which have been predominantly employed in the expression of these antibodies due to their ability to introduce post-translational modifications similar to those human cells [85]. The mammalian expression system is expensive and time-consuming, and efforts have been made to express them in different systems. Microbial cells of yeasts and bacteria have many advantages, such as typical rapid growth, low cultivation costs, and genetics well known in the literature [86]. Microorganisms can produce high molecular weight compounds like proteins, perform highly selective reactions by their native enzymatic machinery, and also allow the repeated introduction of enzymes or immobilized cells [87]. In addition, finally, processes that use microorganisms do not generate organic and inorganic pollutants, such as mercury and toluene [88]. Still, it was complicated to produce complete antibodies in prokaryotes to the detriment of the insecurity of microbial products for human use [73]. Fortunately, the FDA published a special set of rules called "Generally Recognized as Safe" (GRAS), which guarantees the human safety of microbial products and the production of monoclonal antibodies [89]. Thus, several microorganisms were explored. In the case of gram-negative bacteria, *Escherichia coli* stands out, once it has two compartments for protein expression - the cytoplasm and the periplasmic space [86]. Gram-negative bacteria also have an oxidizing environment

*DOI: http://dx.doi.org/10.5772/intechopen.95485*

**3.2 Bacterial and yeast production systems**

which allows the correct formation of disulfide bonds [90].

bacteria, do not produce endotoxins [93].

Whole antibodies can be produced in bacteria and this process is dependent on periplasm, which is an essential region for folding the proteins and chains that make up the structure of antibodies. Unfortunately, studies reveal very low levels of periplasm, which limits the yield for mAbs production [91]. Efforts to produce antibodies in the cytoplasm have not been successful until recently [92]. Gram-positive bacteria are more advantageous than gram-negative bacteria because they do not produce endotoxins - a highly immunogenic lipopolysaccharide (LPS) produced by gram-negative bacteria. Fewer complex eukaryotes such as yeasts have also been exploited for the production of mAbs. They have the advantage over prokaryotes in similarity with the mammalian protein expression system, allowing the expression and folding of complex proteins more easily, and yet, as well as gram-positive

rotation speeds [81–83].

*Alternative Methods to Animal Use for Monoclonal Antibody Generation and Production DOI: http://dx.doi.org/10.5772/intechopen.95485*

all of the components of the culture (cells, nutrients, dissolved gases, and metabolites) are uniformly distributed throughout the volume of the medium. This system requires gradual adaptation to cell growth in suspension, starting from very low rotation speeds [81–83].

Currently, the bulk production of mAbs in agitated bioreactors is the predominant cell culture system in the industry because it allows constant control and monitoring of the process. The area of research for innovation in these bioreactors has advanced dramatically. In general, bioreactors are used to achieve high cell densities and thus increase the production of monoclonal antibodies, biopharmaceuticals, and vaccines [84]. Different types of agitated bioreactors have been used for the cultivation of mammalian cells, both on a pilot and industrial scales.

#### **3.2 Bacterial and yeast production systems**

*Monoclonal Antibodies*

and presents productivity of grams of protein per liter of culture [67, 68], though

The PER.C6 cells are human embryonic retinal cells, and like HEK cells are pointed out to promote human glycosylation profiles. They were projected to be grown in high-density conditions [71], with stable expression and also offer production yields similar to CHO cells, indicating that human cell lines will be more economically viable and more easily scalable options for antibody productions [72]. There is even description of a production with titers of 27 g/L of antibody, astounding yields when compared to a medium CHO cell production of around 12 g/L of antibodies [73]. Although there is still no FDA-approved mAb produced in this system, there are already some ongoing clinical and preclinical studies carried out with mAbs and other biological products purified from this system, like vaccines

There are other suitable host cell lineages such as murine lymphoid cell lines like NS0 and Sp2/0-Ag14, derived from BALB/c mice plasmocytomas, corresponding for almost 25% production systems of FDA-approved monoclonal antibodies. One of their major advantages is being originated from naturally high immunoglobulin producing parental cells. Though, their murine origin is not to be underestimated, for there are reports that they do generate immunogenic glycoforms of the

Concerning the expression vectors for mAb production, usually the plasmids carrying the heavy and light chain genes are constructed based mainly into two kinds of systems: the dihydrofolate reductase (DHFR) system or glutamyl synthetase (GS) based system, both acting as selection markers [65]. In DHFR, selection occurs through glycine, hypoxanthine, and thymidine depletion from the cell culture medium. Selected clones are subjected to the addition of methotrexate, a folate analog that poisons the cells deficient in DHFR, obliging the cells to further synthesize the enzyme with consequent co-amplification of the IgG genes. In the GS system, the selection is done in the absence of glutamine, in a way that only cells with GS can survive by synthetizing glutamine from glutamate and ammonium. Here, the selective pressure is made through increasing doses of the GS inhibitor methionine sulfoximine, pushing the cells to amplify GS and IgG genes [79]. Promoter characteristics, inclusion of antibiotic resistance genes, transcription termination sequences [poly(A)], and translation control sequences should also be

In comparison to other production systems, mammalian cells are more fastidious to culture than bacteria and fungi, for they are larger and do not possess tough cell walls like other microorganisms, making them more sensitive to impurities naturally occurring from the production system itself. Having them to thrive and reproduce in culture after modifications to turn them stable and in conditions to secrete the aimed molecule with high yields is a challenge in itself [61, 67]. If one is not choosing for the transient transfection, having the stable lineages may also be

Independent of the expression system, the correct choice of the production scale

should be made accordingly to the given necessity. The simplest culture system is the static culture, consisted of T bottles with screw caps kept horizontally in an incubator. Because of its low maintenance profile and low costs, it is the most widely used culture method in the academic research context. It is possible to use this system for clone screening and determining experimental conditions, but its small-scale nature might not render enough mAb quantity for some other types of assays. An option to circumvent this matter might be the use of the rolling systems that offer a medium-scale mAb yield. In this condition, roller bottles are positioned in a rotation system that causes all cells to be in constant movement, and therefore,

they have somewhat more tendency to clump [69, 70].

taken into account when designing these vectors [80].

for influenza, HIV, and Ebola [74–77].

expressed antibodies [78].

costly and time-consuming.

**30**

The use of microorganisms such as bacteria and yeasts is widely used in science for several purposes, generally related to antibiotics and probiotics [73]. However, both bacteria and yeasts have been getting space in the production of mAbs for immunological therapy due to the biopharmaceutical demand and technological advances about their ability to produce antibodies by reducing the use of animals in the manufacturing process [73]. The motivation behind investments that seek to optimize the means of production of mAbs in alternative models stems from the manufacturing disadvantages presented in the traditional method with mammalian cells, which have been predominantly employed in the expression of these antibodies due to their ability to introduce post-translational modifications similar to those human cells [85]. The mammalian expression system is expensive and time-consuming, and efforts have been made to express them in different systems. Microbial cells of yeasts and bacteria have many advantages, such as typical rapid growth, low cultivation costs, and genetics well known in the literature [86]. Microorganisms can produce high molecular weight compounds like proteins, perform highly selective reactions by their native enzymatic machinery, and also allow the repeated introduction of enzymes or immobilized cells [87]. In addition, finally, processes that use microorganisms do not generate organic and inorganic pollutants, such as mercury and toluene [88]. Still, it was complicated to produce complete antibodies in prokaryotes to the detriment of the insecurity of microbial products for human use [73]. Fortunately, the FDA published a special set of rules called "Generally Recognized as Safe" (GRAS), which guarantees the human safety of microbial products and the production of monoclonal antibodies [89]. Thus, several microorganisms were explored. In the case of gram-negative bacteria, *Escherichia coli* stands out, once it has two compartments for protein expression - the cytoplasm and the periplasmic space [86]. Gram-negative bacteria also have an oxidizing environment which allows the correct formation of disulfide bonds [90].

Whole antibodies can be produced in bacteria and this process is dependent on periplasm, which is an essential region for folding the proteins and chains that make up the structure of antibodies. Unfortunately, studies reveal very low levels of periplasm, which limits the yield for mAbs production [91]. Efforts to produce antibodies in the cytoplasm have not been successful until recently [92]. Gram-positive bacteria are more advantageous than gram-negative bacteria because they do not produce endotoxins - a highly immunogenic lipopolysaccharide (LPS) produced by gram-negative bacteria. Fewer complex eukaryotes such as yeasts have also been exploited for the production of mAbs. They have the advantage over prokaryotes in similarity with the mammalian protein expression system, allowing the expression and folding of complex proteins more easily, and yet, as well as gram-positive bacteria, do not produce endotoxins [93].

Among yeasts, *Pichia pastoris* and *Saccharomyces cerevisiae* dominate the field in the production of antibodies [86]. *S. cerevisiae* is promising due to the advantage of being well characterized, but the correct folding of chains and proteins and low yields are problems to be faced. On the other hand, *P. pastoris* does not secrete many endogenous proteins that need to be removed in the mAbs production process [94]. Yeasts have cellular glycosylation machinery, however, their proteins exhibit types of glycosylation completely different from human proteins, and this results in a significant reduction in therapeutic effector functions [95]. Whether from yeasts or bacteria, native fulllength mAbs need to be glycosylated during their synthesis, but this is an obstacle that has yet to be overcome for better production efficiency in microbial hosts. The glycosylation status of the Fc region is critical for the recruitment of serum proteins from the complement system and the destruction of target cells by complement-dependent cytotoxicity (CDC) cascades [95]. This is the main reason why the method of producing mAbs in mammalian cells is still the most applied [91]. Until 2020, there are 151 recombinant therapeutic proteins approved by the FDA, one-third of them are mAbs but there are many other mAbs under development. Among these mAbs, only two are antigen-binding fragments (Fabs) that are produced in the periplasm of the bacteria *E. coli*: ranibizumab and certolizumab pegol. The first, ranibizumab, approved in 2006, is an IgG1 Fab fragment used to treat neovascular age-related macular generation and macular edema after retinal vein occlusion. Certolizumab pegol is also a humanized Fab fragment, approved in 2008 for the treatment of Crohn's disease and rheumatoid arthritis. Therefore, since the advent of mAb therapy, the biopharmaceutical industry has been investing considerable resources in new bioprocesses for the manufacture of glycosylated antibodies that attach human IgG-like glycans through alternative host expression [95].

#### **3.3 Plant-based antibody production systems**

To produce antibodies in plants, a transformation is mediated by a bacterium that infects plants, called *Agrobacterium*. The bacterium then loads the expression vector with the antibody gene, thus generating the transgenic plants that express the desired antibody. The transformed Agrobacterium is inoculated into the leaf slices of the plants. These slices regenerate in 3–4 weeks. Small shoots are then formed from the callus and transferred to a plant cultivation box *in vitro*. For the production of biomass, *in vitro* transgenic plants are transferred to a soil pot and grown in a greenhouse [96]. The most used plant systems are tobacco and alfalfa because they are the most accessible and common sources of leaf biomass. Tobacco has great advantages, such as high leaf biomass yield and rapid scaling up through easy seed production compared to other plant species. However, tobacco contains nicotine and other toxic alkaloids that need to be removed through an additional extraction step [97].

The plant system offers important advantages, such as high production capacity, low cost in the large-scale cultivation process, in addition to avoiding ethical problems associated with animals [98]. Another important advantage of using this system is found in post-translational protein modifications, which occur in plant cells in a similar way to animal cells, as well as in the correct assembly of complex molecules, such as antibodies, are aided by chaperones that mediate folding and the formation of disulfide bonds, while the addition of N-glycans is carried out by specific cellular glycosyltransferases. In fact, while core N-glycans are similar in plants and mammals, complex N-glycans show substantial differences with sialic acid [99–101].

In addition, there is a possibility to design a custom antibody glycosylation profile, and production can be enlarged simply by increasing the number of plants [102]. In comparison with the systems described earlier, the use of plants for the production of antibodies offers several irreplaceable benefits. Plants are

**33**

**Acknowledgements**

São Paulo (grant no. 16/14358-2; FAPESP, Brazil).

*Alternative Methods to Animal Use for Monoclonal Antibody Generation and Production*

widespread, abundant, and develop more quickly because they normally mature after a growing season. It is possible to put the product on the market quickly, which ends up decreasing the cost of production. Plants also reduce screening costs for bacterial toxins, viruses, and prions because they are less likely to introduce animal

The disadvantages of this system are found in the low yield of protein expression, the downstream processing problems related to the extraction of proteins

The first pioneering study on the production of full-size IgG in plants dates back almost 30 years ago [104]. Since then, different antibody formats have been expressed in plants, such as IgA, Fab fragments, minibodies, and scFvs [103]. The first drug from plant cells to receive FDA approval for human use was the enzyme β-glucocerebrosidase, commercially called ELELYSO, indicated for the treatment of patients with a confirmed diagnosis of Type 1 Gaucher disease [105]. Thereafter, Medicago Inc. developed a quadrivalent plant-derived seasonal influenza vaccine that recently completed Phase III clinical trials [106]. A study published in September 2020 positively demonstrated the expression of a scFv 13F6 antibody with binding activity against Ebola virus-like particles in a plant system [107, 108]. Of the antibodies produced by plants, there are already 6 against viruses, 5 against

Therefore, given the data presented and the clear advantages, we can say that the plant system is quite efficient and may, in the future, be widely used in the produc-

Bearing in mind that obtaining high specificity and affinity mAbs is not trivial, there is a great race to develop methodologies that can meet the most varied demands. An overview of the main technologies clearly shows that the total replacement of animals' use in the generation and production of mAbs is not possible for the moment. We believe that this will only be reached when the in silico technology is fully dominated. But as the implementation of alternative methods must be seen as a process, reducing and refining the use of animals are achievements. Thus, the different types of antibody display libraries represent a major breakthrough. As described, the source of genes for building the libraries may imply greater or lesser use of animals and only synthetic display libraries completely dispense the use of animals. In the same way, for the production of mAbs, several possibilities are currently available. The important thing in the production stage is that the use of ascites, a proceeding that brings pain and stress to animals, may already be eliminated in most cases. Invariably, the purpose and amount of the mAb to be produced will determine the choice of obtaining and production methodologies. Given the great utility and diversity of mAb uses, ranging from therapeutic application to essential research tools, and the wide range of technologies available today for obtaining and producing them, it seems a fact that

it is always possible to choose or design a path that meets the concept of 3Rs.

The authors acknowledge the support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), and Fundação de Amparo à Pesquisa do Estado de

tion of antibodies both in basic research and on an industrial scale.

*DOI: http://dx.doi.org/10.5772/intechopen.95485*

pathogens than mammalian cells or animals [98].

from leaves, and some regulatory obstacles [103].

tumors, and 3 against bacteria [97].

**4. Conclusions**

#### *Alternative Methods to Animal Use for Monoclonal Antibody Generation and Production DOI: http://dx.doi.org/10.5772/intechopen.95485*

widespread, abundant, and develop more quickly because they normally mature after a growing season. It is possible to put the product on the market quickly, which ends up decreasing the cost of production. Plants also reduce screening costs for bacterial toxins, viruses, and prions because they are less likely to introduce animal pathogens than mammalian cells or animals [98].

The disadvantages of this system are found in the low yield of protein expression, the downstream processing problems related to the extraction of proteins from leaves, and some regulatory obstacles [103].

The first pioneering study on the production of full-size IgG in plants dates back almost 30 years ago [104]. Since then, different antibody formats have been expressed in plants, such as IgA, Fab fragments, minibodies, and scFvs [103]. The first drug from plant cells to receive FDA approval for human use was the enzyme β-glucocerebrosidase, commercially called ELELYSO, indicated for the treatment of patients with a confirmed diagnosis of Type 1 Gaucher disease [105]. Thereafter, Medicago Inc. developed a quadrivalent plant-derived seasonal influenza vaccine that recently completed Phase III clinical trials [106]. A study published in September 2020 positively demonstrated the expression of a scFv 13F6 antibody with binding activity against Ebola virus-like particles in a plant system [107, 108]. Of the antibodies produced by plants, there are already 6 against viruses, 5 against tumors, and 3 against bacteria [97].

Therefore, given the data presented and the clear advantages, we can say that the plant system is quite efficient and may, in the future, be widely used in the production of antibodies both in basic research and on an industrial scale.

#### **4. Conclusions**

*Monoclonal Antibodies*

alternative host expression [95].

**3.3 Plant-based antibody production systems**

Among yeasts, *Pichia pastoris* and *Saccharomyces cerevisiae* dominate the field in the production of antibodies [86]. *S. cerevisiae* is promising due to the advantage of being well characterized, but the correct folding of chains and proteins and low yields are problems to be faced. On the other hand, *P. pastoris* does not secrete many endogenous proteins that need to be removed in the mAbs production process [94]. Yeasts have cellular glycosylation machinery, however, their proteins exhibit types of glycosylation completely different from human proteins, and this results in a significant reduction in therapeutic effector functions [95]. Whether from yeasts or bacteria, native fulllength mAbs need to be glycosylated during their synthesis, but this is an obstacle that has yet to be overcome for better production efficiency in microbial hosts. The glycosylation status of the Fc region is critical for the recruitment of serum proteins from the complement system and the destruction of target cells by complement-dependent cytotoxicity (CDC) cascades [95]. This is the main reason why the method of producing mAbs in mammalian cells is still the most applied [91]. Until 2020, there are 151 recombinant therapeutic proteins approved by the FDA, one-third of them are mAbs but there are many other mAbs under development. Among these mAbs, only two are antigen-binding fragments (Fabs) that are produced in the periplasm of the bacteria *E. coli*: ranibizumab and certolizumab pegol. The first, ranibizumab, approved in 2006, is an IgG1 Fab fragment used to treat neovascular age-related macular generation and macular edema after retinal vein occlusion. Certolizumab pegol is also a humanized Fab fragment, approved in 2008 for the treatment of Crohn's disease and rheumatoid arthritis. Therefore, since the advent of mAb therapy, the biopharmaceutical industry has been investing considerable resources in new bioprocesses for the manufacture of glycosylated antibodies that attach human IgG-like glycans through

To produce antibodies in plants, a transformation is mediated by a bacterium that infects plants, called *Agrobacterium*. The bacterium then loads the expression vector with the antibody gene, thus generating the transgenic plants that express the desired antibody. The transformed Agrobacterium is inoculated into the leaf slices of the plants. These slices regenerate in 3–4 weeks. Small shoots are then formed from the callus and transferred to a plant cultivation box *in vitro*. For the production of biomass, *in vitro* transgenic plants are transferred to a soil pot and grown in a greenhouse [96]. The most used plant systems are tobacco and alfalfa because they are the most accessible and common sources of leaf biomass. Tobacco has great advantages, such as high leaf biomass yield and rapid scaling up through easy seed production compared to other plant species. However, tobacco contains nicotine and other toxic

alkaloids that need to be removed through an additional extraction step [97].

complex N-glycans show substantial differences with sialic acid [99–101].

In addition, there is a possibility to design a custom antibody glycosylation profile, and production can be enlarged simply by increasing the number of plants [102]. In comparison with the systems described earlier, the use of plants for the production of antibodies offers several irreplaceable benefits. Plants are

The plant system offers important advantages, such as high production capacity, low cost in the large-scale cultivation process, in addition to avoiding ethical problems associated with animals [98]. Another important advantage of using this system is found in post-translational protein modifications, which occur in plant cells in a similar way to animal cells, as well as in the correct assembly of complex molecules, such as antibodies, are aided by chaperones that mediate folding and the formation of disulfide bonds, while the addition of N-glycans is carried out by specific cellular glycosyltransferases. In fact, while core N-glycans are similar in plants and mammals,

**32**

Bearing in mind that obtaining high specificity and affinity mAbs is not trivial, there is a great race to develop methodologies that can meet the most varied demands. An overview of the main technologies clearly shows that the total replacement of animals' use in the generation and production of mAbs is not possible for the moment. We believe that this will only be reached when the in silico technology is fully dominated. But as the implementation of alternative methods must be seen as a process, reducing and refining the use of animals are achievements. Thus, the different types of antibody display libraries represent a major breakthrough. As described, the source of genes for building the libraries may imply greater or lesser use of animals and only synthetic display libraries completely dispense the use of animals. In the same way, for the production of mAbs, several possibilities are currently available. The important thing in the production stage is that the use of ascites, a proceeding that brings pain and stress to animals, may already be eliminated in most cases. Invariably, the purpose and amount of the mAb to be produced will determine the choice of obtaining and production methodologies. Given the great utility and diversity of mAb uses, ranging from therapeutic application to essential research tools, and the wide range of technologies available today for obtaining and producing them, it seems a fact that it is always possible to choose or design a path that meets the concept of 3Rs.

#### **Acknowledgements**

The authors acknowledge the support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), and Fundação de Amparo à Pesquisa do Estado de São Paulo (grant no. 16/14358-2; FAPESP, Brazil).
