Mammalian Cells: 

About 60-70% of all recombinant protein pharmaceuticals and 95% of approved antibodies are still produced in mammalian cell lines despite relatively high produciton costs and diffiulty in handling. However, the advanced mamalian folding, secretion and post-translational apparatus is capable of producing antibodies indistinguishable from those in the human body with least concerns for immunogenic modifications. Moreover, it is also highly efficient for secretion of large and complex IgGs and in combination with the folding and post-translational control it results in high product quality which reduces efforts and costs in the subsequent and more expressive downstream processing steps. The generaiton of stable master cell lines is a prerequesite for GMP complaint IgG production. Strong promoters like the immediate early cytomegalovirus (CMV) or the cellular elongation factor (EF) 1-alpha promter and polyadenylation sites form the simian virus (SV) 40 or the bovine growth hormone (BGH) for improved mRaNA stability and translation efficiency are usually implemented into the expression vector. Furthermore splicing of mRNA is known to promote mRNA packaging and transfer into the cytosol in order to stabilize and enhance gene expression as well as to reduce silencing of heterologous transgenes. For IgG expression, two different genes must be stably transfected into one cell clone, either by cotransfection or by using bicistronic expression vectors. Bicistronic vectors employing internal ribosomal entry sites (IRES) allow the translation of two or more cistrons form teh same transcript. Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013) Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Mammalian systems are the only platform applicable to routeinly produce biopharmaceutical proteins harboring human like complex glycosylation at an industrial scale. Consequently most FDA approved drugs of this category are currently either purified direclty from mammalian plasma or milk, or derived from mammalian cell lines such as Chine Hamster Ovary (CHO) cells, baby hamster kidney (BHK-21) cells, murine and hybridoma cells lines (NSO and Sp2/0) and to a lesser extent, human cells. However, mammalian cell lines can express terminal alpha-Gal strutures and Neu5Gc, two non-human epitopes against which humans have an antibody repertorie. Even human cell lines used to produce biotherpeutic glycoproteins may express Neu5Gc containing glycoproteins if animal sera and/or animal derived media additive are used in the produciton process. IN addition to cell lines, this problem extends to transgenic animals which can have large amounts of Neu5Gc. 

Antibodies require glycosylation and therefore generally are expressed in eukaryotic expression systems employing eukaryotic cells. such as CHO, PER.C6, NSO, BHK or Sp2/0 cells. (Spitali, WO2012/013682) 

Chinse hamster ovary (CHO) cells are one of the most univeral hosts in biopharmaceutical productions. They have several advantages such as high growth rate and productivity. A number of approved therpaeutic antibodies are produced in CHO cells, including Rituxan and herception. (Yamane-Ohuki (Biotechnology & bioengineering, 87(5), 2004).

Goletz (US2010/0028947) disloses a method for producing a protein having a defined glycosylation pattern by introducing in a host cell which is an immortalized human blood cells at least one nucleic acid encoding at least a part of said protein, culturing the host cells and isolating the protein. 

Hegen (US2012/0014975) teaches expression of a SDAB bivalent humanized polypeptide in a CHO mammalian expression system and then purification by protein A affinity cpature.

Insect cells: represent an alterntaive system for efficient expression of recombinant proteins. However, recombinant proteins derived from insect cells also carry glycan structures that differ significanlty from those present in mature huamn glycoproteins. Gharderi, Biotechnolgy and Genetic Engineering Reviews 28, 147-176 (2012).

Production in separate vectors:

Generating a mAb producing cell line starts with transfecting CHO cells with plasmid vectors carrying the LC, HC and selection marker genes. Stably trasnfected clones with plasmid vectors integrated into the genome are then screened for high producitvity, table production and gene product quality. LC, HC and the marker are often co-trasnfected on separate vectors or placed on a single vector with multiple promoters which each gene is under the control of its own promoter. Poor coupling of the mAb and seleciton marker genes for such designs resutls in a significant proportion of non-exressing clones surviving drug slection. Anotehr diasdvantage of having separate expression units is the lack of accurate control of the relative LC and HC expression levels. (Yang “Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells”. MAbs7:2, 403-412, 2015). 

Production as one transcript:

Tricistronic vecotrs using internal ribosomes entry sites (IRES) or 2A peptides to express the LC, HC and a selection marker gene in one trasncript provides accurate control of the relative expression of LC over HC. Expressing the LC, HC and marker genes on one transcript also minimizes non-expressing clones becasue none of the 3 genes will be expressed should the vector be fragmented. IRES and 2A peptides use different mechanisms for co-epxression of multiple genes in one transcript. When using IRES to express multiple genes in one mRNA, the gene directly downstream of the promoter is translated by the canonical cap-dependent mechanisms, and thsoe downstream of IRES are translated by a cap-independent mechanisms. In contrast, 2A linked genes are translated in one open reading fram and “self-cleavage” occurs co-translationally to give equal amounts of the co-expressed prtoeins. Many 2A peptides have been idnetified from viruses. 2A peptides have about 20 amino acids and “self-clevage” occurs between the last 2 amino acids, glycine and protein. Adding a furin recognition sequence between the first gene and 2A aids in removing 2A residues from the upstream gene. (Yang “Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells”. MAbs7:2, 403-412, 2015). 

Plant Systems: 

Plant based systems have been established as an alternative produciton platform for bioactive therpauetic proteins and have several advantags such as the ease of scale up for industrial produciton at relatively low cost. However, the N-glycosylation machinery of plants and humans is distinctive and results in significant structual differences of the N-glycan repertoire. Gharderi, Biotechnolgy and Genetic Engineering Reviews 28, 147-176 (2012).

Genetically modifed plants are created using techniques that are different from those employed to create genetically modifed animals. Transgenic plants can be created by introducing foreing DNA into plant cells by electroporation, physical bombardment, chemical treatment and bacterial transfer. Transgenes can be transferred into the gehome of certain plant species form the plant pathogen Agrobacterium tumefacien. This bacterium harbors a plamide called the Ti (tumor-inducing) plasmid. 

Standardized plant antibody production methodolgies are typically dependent on generation of transgenic plant lines that require months to derive and up to 3 years to generate sufficient seed for full sscale production. The utilizaiton of virus vectors shortens such production time lines but each production process followed in such instances is typically conductcted much like mammalina cell production activities, which thus requires high specificity and increased chances of off quality batches. Morton (US 15/110200)

Morton (US 15/110200) discloses mAb production in plants which includes sublconing the HC and LC antibody chains into tobamovirus and potexvirus expression vectors in cDNA form. Unitque T-DNA constructs are transformed in the Agrobacterium strains and plants are inoculated with the two distinct strains containing either tobamovirus or potexvisu vectors. Different signal peptides are fused to eahc test HC and LC genes and compared for pexpression levels and mAb assemble. Victors showing similar epxression are choosin for production. Once the plant source is harvested, extraction of the desired biomass from the plant is accomplished through utilizaiton of a suitable buffer and target antibodies are extracts with 100 mM Tris-Base + 40 mM Ascorbic Acid + 1 mM EDTA, pH 8.5. Residue cellulosic plant fiber is then removed and the resulting exptract is adjusted to a pH of about 8 with NaOH. Clairificaiton of samples is then udnertaken with diatomaceous earth and filter pressed (depth filtration step). At this point, the filtrate sample provides the ending point of the upstream processing steps leading to downstream processing steps for purificaiton of anitobdies. (see Antibody purificaiton). 

Some crops, including potato can be maintained and multipliedk through clonal propagation of tubers or roots, allowing for preservation of both existing and ectopically introduced trains, for example, geenrated by precision gene editing technologies. Cultivated potato is autotetraploid (2n=4x=48) and highly heterozygous with a genome of 12 chromosomes and its mintenance is essential to avoid inbreeding depression, cinluding reduct fetility and productivity. Sexual reproduction by seed is associated with unpredictable segreation and high recombination rates, potentially resulting in loss of existing traits, for example, laborioously acquired from earlier breeding efforsts. (Petersen “Strategies and Protocols for optimized genome editing in potato” CRISPR Journal, volume 00, number 00, 2024). 

Plant Tissue Culture: is a technique in which fragments of tissues from a plant (explants) are developed in vitro in an artifical medium under aseptic conditions. It involves culturing explaints (such as shoot tip, root tip, callus, seed, embryo, pollen grain, ovule or even a single cell) isoalted form motehr plant on asterile nutrient medium which elads to cell multiplicaiton and plant regeneration. The cmmonly sued medium in plant tissue culture is Murashigean Skoog (MS) basal medium supplemented with the required amounts of plant hormones which include auxins, cytokinins, abscisic acid, gibberellines, ethylene, and grwoth regulations with similar metabolic efffect. (Hasnain, “Plants in vitro propagation with its applicaitons in food, pharmaceuticals and cosmetic industries; current scenario and future approaches” Frontiers in Plant Science 2022)

Tissue culture technique offers several dvantages over plant propagation under natural conditions. It is a rapid procedure as thousands of seedling can be produced form small fragments of plants in a short period of time in contrast to conventionally propagated flora. This also ehlps to accelrate the production process of new crop varieties with superior traits as tissue culture experiments require less time and space compared to in-vivo plant growth. Tissue culture can be sued to propagate prennial plants, irrespective of weather or season. It also helps in the development of pahtgen-free micro-plants saved from various diseases adn the new plants produced by tissue culture udner aseptic conditions are also serile. (Hasnain, “Plants in vitro propagation with its applicaitons in food, pharmaceuticals and cosmetic industries; current scenario and future approaches” Frontiers in Plant Science 2022)

Plant micro propagation, also known as plant tissue culture, is a technique that isolates, sterilizes, and incubates cells, tissues or organs of chosen plants in a growth-promoting aseptic environment to create a large number of plantlets. The isolated cloning technique revleaed that, given the right conditions, somatic cells may develop into a complete plant. (Hasnain, “Plants in vitro propagation with its applicaitons in food, pharmaceuticals and cosmetic industries; current scenario and future approaches” Frontiers in Plant Science 2022)

Somatic embryogenesis is the process of producing embryos from somatic plant cells (any non-sexual cell) in order to produce a whole plant. (Hasnain, “Plants in vitro propagation with its applicaitons in food, pharmaceuticals and cosmetic industries; current scenario and future approaches” Frontiers in Plant Science 2022)

Protoplasts are plant cells with cell walls removed by enzymatic or mechanical methods. Protoplasts are obtained by immersing plant cells in a hypertonic solution which causes the plamsa mebrane to shrink off the cell waa due to water efflux. Cell wall may now be removed using either enzyamtic digestion with pectinase and cellulose or by mechanical tehniques. (Hasnain, “Plants in vitro propagation with its applicaitons in food, pharmaceuticals and cosmetic industries; current scenario and future approaches” Frontiers in Plant Science 2022)

Yeast Systems: 

Yeasts combine the properties of eukaryotic cells short generation time and ease of genetic manipulation with the robustness and simple medium requirements of unicellular microbial hosts. Moreover, yeasts have been used for fermentation in food production for sevral millennia; they do not produce bacterail endotoxins and have gained the GRAS status paving the way toward production of therpaeutic proteins. Pichia pastoris represetns the major yeast strain used for recombinant antibody production. (Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Allison (14/215370) dicloses the yeast Pichia pastoris as a cost-effective platform for producing functional antibodies. Other methylotrophic yeast strains include Pichia angusta. In an alternative embodiment, a filamentous fungal host cell is selected from Apergillus, Trichoderma, Penicillium, Rhizopus, Paecilomyces, Fusarium, Neurospora and Claviceps. 

Yeast have the ability to multiply rapidly to high densitites, have well characterized glycosylation machineries and the ability to scale up fermentation to industrial levels is well established. Although human and yeasts share the same N-glycan core structure, the hypermannosylated yeast N-glycans with up to 100 additional mannose residues is highly immunogenic for humans. However, great strides have been made to genetically modify yeasts to produce human like N-glycosylated therpaeutic glycoproteins Gharderi, Biotechnolgy and Genetic Engineering Reviews 28, 147-176 (2012).

Particular types of Yeast systems

P. pastoris: has been used for the expression of a ScFv fragment. (Joosten, “The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi” Microbial Cell Factories 2003, 2 pp. 1-15).

Production in Filamentous fungi

Filamentous fungi, in particular specie form the genera Trichoderma and Aspergillus have the capacity to secrete large amounts of proteins, metabolites and organic acis into thir culture medium. This property has been widely exploited by the food and beverage industries where compounds secreted by these filamentous fungal species have been used for decades. Filamentous fungi like A awamori, A. niger and A. oryzae are suitable organisms for the production of commerically interesting proteins. (Joosten, “The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi” Microbial Cell Factories 2003, 2 pp. 1-15).

Filamentous fungi of the genera Trichoderma and Aspergillus have the capacity to secrete large amounts of proteins and metabolites into the medium. They are widely used in the food and biotechnological industry, for example A. niger for citric acid production. Morevoer, A. niger and Aspergillus oryzae gained GRAS status. Two protmoters are typically  used for the expression of antibodies in fungi; the glucamylase promoter (glaA) and the endoxylanase A promoter (exIA). Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Production in Protozoa

Recently, the eukaryotic parasite Leishmania Tarentolae has been explored as an expression system for different recombinant proteins. One major advantage of this system is the mammalian like glycosylation pattern; this protozoa is able to perform O-glycosylation as well as N-glycosylation, which is highly conserved in mammalians. Concsequently, L. tarentolae has started to be used for the production of recombinant antibodies. Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Production in Insect Cells

To deliver a target gene into cells and achieve prtoein expression, insect cells require baculovirus as an intermediate. Baculoviruses represent a diverse group of DNA viruses that can infect more than 600 different types of insect cells. Baculoviruses serve as a shuttle to introduce a target gene into a host cell. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best characgerized baculovirus for that purpose,a nd is widely used for insect cell mediated protein expression. (BioProcess International, 2020 -Recombinant Protein Expression with a Baculovirus-Insect Cell System –Yuning Chen). 

Insect cells represent a very versatile eukaryotic expression system. They can be efficiently transfected with insect-specific viruses form teh family of Baculoviridae, particularly the Autographa californica nuclear polyhedrosis virus (AcNPV). Baculoviruses are hihgly species specific and are considered as safe for humans, mammalians and plants. Infeciton of human hepatocytes and mammalian cell lines including stable transduction has been demosntrated in cell culture without evience of viral replication or gene expression under the control of baculoviral promoters. Non-essential baculovirus genes invovled in the viral life cycle, like Polyhedrin, P10, or Basic can be replaced by heterologous genes. The flexible viral envelop allows packaging of large heterologous gene sequences of more than 20 kb. Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Transgenic Animals

Several researchers developed transgenic animals for the production and expression of human monoclonal and polyclonal antibodies, mostly in the milk of transgenic mice, goats and eggs fo transgenic chickens. Teh first step toward the generation of human antibodies in animals by immunization was the transfer of a human minilo-cus containing unrearranged immunoglobulin variable, diversity, and joining elements linked to a human u-chain into mice. In this study, about 4% of teh extracted B-lymphocytes expressed human antibodies. Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Prokayrotic expression systems:  See outline

Recombinant Expression of Antibody Genes (see outline)

Conditions of Cell Culture (Media, additives, etc): 

Addition of Metal Ions

Cu2+:

(Chaderjian, Biotechnol. Prog. 2005, 21 550-553) discloses addition of copper uslfate to a recombinant Chinease hamster ovary (CHO) cell culture production medium as an oxidizing agent to reduce free thiol due to unpaired cystein residues (Cu2+ facilitates disulfide bond formation). 

Kao (US8,574,869) teaches a method for preventing the reduction of disulfide bond during recombinant production of disulfide containing polypeptides such as antibodies by adding a thioredoxin inhibitor which is a metal ion such as Hg2+, Cu2+, Zn2+, Co2+ and Mn2+ between 15 and 50 uM. 

Schotte (US13/266503 and US2012/0157664) discloses that despite the high yield and functionality of domain antibodies produced in non-coli hosts, in particular yeast, there is a quantitatively significant fraction of product that represents a structural variant. In particular, a fraction of the product lacks at least one disulfide bond. It is consistently reported that conventional antibodies or fragments lacking at least one disulfide bond are characterized by a loss of function. Schotte provides a method of counteracting this. Schotte teaches a method of producing a domain antibody in yeast by applying conditions such as addition of oxidizing agents, preferably oxidizing metal ions such as Cu2+, Fe2+, Fe3+ and Zn2+ that promote the formation of disulfide bridges in the domain antibodies and/or removing domain antibodies lacking at least one disulfide bride. The method results in the production of domain antibodies wherein the quality of the domain antibodies is improved with a reduced level of free thiol or its absence.

Zn2+: 

Srivastava (WO2012/147053) disclsoes a method for reducing heterogeneity of antibodies during culturing by adding a divalent transitional metal ion such as zinc, Zn2+, to the cell culture medium.