Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs
© de Marco. 2015
Received: 19 July 2015
Accepted: 20 August 2015
Published: 2 September 2015
Antibodies have been a pillar of basic research, while their relevance in clinical diagnostics and therapy is constantly growing. Consequently, the production of both conventional and fragment antibodies constantly faces more demanding challenges for the improvement of their quantity and quality. The answer to such an increasing need has been the development of a wide array of formats and alternative production platforms. This review offers a critical comparison and evaluation of the different options to help the researchers interested in expressing recombinant antibodies in their choice.
Rather than the compilation of an exhaustive list of the recent publications in the field, this review intendeds to analyze the development of the most innovative or fast-growing strategies. These have been illustrated with some significant examples and, when possible, compared with the existing alternatives. Space has also been given to those solutions that might represent interesting opportunities or that investigate critical aspects of the production optimization but for which the available data as yet do not allow for a definitive judgment.
The take-home message is that there is a clear process of progressive diversification concerning the antibody expression platforms and an effort to yield directly application-adapted immune-reagents rather than generic naked antibodies that need further in vitro modification steps before becoming usable.
KeywordsAntibody fragments Bacterial antibody display Antibody-based fusion proteins Neutralizing antibodies scFv VHH
The worldwide therapeutic antibody market is estimated as over $50b/year, diagnostic market over $10b/year, and research market accounts for $3b/year, whereas the average annual growth rate for all antibody applications in the last 15 years is over 5 % and the recombinant antibody sector is one of the fastest growing. These numbers explain clearly the interest in all the biotechnological innovations aimed at improving the antibody production process in terms of absolute yields, structural stability and functional reliability of the final product. Specifically, the increasing attention dedicated to recombinant antibodies is due to the possibility of: (1) performing straightforward engineering by means of simple molecular biology techniques; (2) developing binders of variable formats and fused to different effectors and tags; (3) producing the final constructs inexpensively in microbial factories; (4) maintaining stable material clonality, the mandatory pre-requisite for result reproducibility . This trend will probably accelerate in the foreseeable future because of the demanding structural features of the emerging antibody-based reagents such as Immunotoxins, Antibody Drug Conjugates (ADCs), Bispecific Antibodies, and Bispecific T cell Engager (BiTE) [2, 3]. Clearly, the great structural variability of the designed immune-reagents has its reason in the necessity to obtain effective reagents that are possibly also simple to use. At the same time, it implies the necessity of developing customized expression and purification procedures adapted to the construct specificities in order to assure sufficient yields. Such a complex context requires the capacity of comparing advantages and shortcomings of alternative platforms. This review aims at summarizing the recent trends, tries to evaluate the level of feasibility and reliability reached by the different methodologies, and will describe some recent innovative proposals.
Production of antibody fragments in prokaryotic systems
The conventional approach: expression in Escherichia coli (E. coli)
Expression rate control is a key parameter in recombinant production  and alternatives to the classical method based on lac promoter and its derivatives can contribute to higher antibody fragment yields. In the case of the pair formed by the Pm promoter and the benzoic acid-inducible XylS transcription activator , the direct evolution of XylS resulted in mutants that enabled ninefold higher yields of the tested scFv-phOx construct. Unfortunately, the paper does not report data describing the structural and functional features of the (over)-produced immune-reagent.
The strategies used for increasing the solubility of recombinant proteins expressed in E. coli have prevalently focused on conditions known for improving folding efficiency. Proteins such as isomerases and chaperones have been overexpressed, the bacterial growth has been performed at low temperatures and in the presence of osmolytes and alcohols known for stimulating heat-shock response, and even specific strains have been developed [7–9, 20]. By contrast, the optimization of medium composition and fermentation conditions is often neglected in academia with the exception of labs performing more industrial-oriented R&D. However, significantly higher amounts of antibody fragments can be recovered when the physical–chemical culture conditions are improved, and this is possible even in the absence of sophisticated fermentation equipment. Cell density can be increased tenfold in standard Erlenmeyer flasks when bacteria are grown using rich medium instead of LB  and the yields can be further improved by exploiting LEX™ bioreactors  or baffled flasks in combination with optimized shaking velocity .
In a recent contribution , the yields of two constructs formed of the same scFv fused to the sequences corresponding to a toxin of either bacterial (Pseudomonas exotoxin A) or plant (saporin) origin were compared in E. coli and P. pastoris. Exotoxin A-scFv was apparently expressed better in bacteria, whereas saporin-scFv production was more successful in yeast. The authors’ conclusions were that the toxin origin strongly influences the production of functional immune-reagents in evolutionary related or distant organisms. This would be an interesting hypothesis to assess thoroughly. However, the experimental data are difficult to interpret since the immune-toxins accumulated as inclusion bodies when expressed in bacteria and the reported yields correspond to the amounts of soluble protein after refolding. Consequently, the variable yields could be due to the differential refolding efficiency rather than to specificities of the microbial factories. Furthermore, crucial experimental characterizations and descriptions necessary to evaluate the structural features of the immune-toxins are missing thus rendering final judgment impossible.
A very interesting development is represented by the attempt of obtaining homogeneous N-glycosylated recombinant antibodies in E. coli with the long-term aim of recovering the effector functions and tissue targeting specificity proper of IgG molecules . The authors demonstrated that it is possible to achieve a homogenously glycosylated and degradation-resistant scFv by designing a glycosylation site in the linker region and using an E. coli strain in which the N-glycosylation machinery of the ε-proteobacterium Campylobacter jejuni has been transferred . The post-transcriptional modification significantly improved the scFv stability and solubility, while did not affect its binding capacity because glycosylation has a distal location with respect to the antibody paratope.
Escherichia coli extract has been successfully used also for the cell-free production of “knobs-into-holes” bispecific BiTE IgG antibodies . The knobs-into-holes technology enables the pairing of complimentary antibody arms but the efficiency of the IgG reconstitution is low when the two complimentary molecules are co-expressed in cells due to the usual variable accumulation rate of the two constructs. The cell-free approach overcomes this major drawback and results in high yields (several hundred mg/L) of functional antibodies that can be further improved by optimizing the reagent composition .
Alternative bacteria suitable for specific applications
Exotic bacteria such as the psychrophilic Pseudoalteromonas haloplanktis TAC125 have been proposed as an alternative to E. coli for VHH and scFv production [37, 38]. Although the choice of a microbial cell factory working at low temperature is interesting because it might favor the proper folding of the recombinant proteins, the practical advantages of this approach remain elusive, whereas some drawbacks should be considered, e.g. higher energy costs for bacterial culture, limited availability of expression vectors with application-friendly tags. Much more convincing are the data published recently by Mizukami et al.  that illustrate the enormous potential of Brevibacillus choshinensis for expressing recombinant antibody fragments. Two VHHs were expressed using a secretion vector and accumulated in the culture medium at concentrations of hundreds of mg/L when grown in flasks and topped at 3 g/L when produced in fermenter. The extremely accurate biophysical characterization of the resulting nanobodies confirms that they were functional and unquestionably monodispersed. It will be very interesting to follow the development of this production platform based on the “Brevibacillus in vivo cloning (BIC)” technology [40, 41] to understand if the excellent quantitative and qualitative features obtained with these model VHHs will be confirmed by using nanobodies with variable propensity to aggregation. It seems that larger/more complex proteins such as scFvs cannot benefit as much as VHHs [40, 41] from this expression methodology. At the moment it is difficult to evaluate whether the observed limitation is intrinsic of the organism folding machinery or of still-to-be-optimized expression vectors and fermentation conditions. The available information is not sufficient to evaluate the possible advantages of expressing recombinant antibody fragments in Bacillus megaterium and Corynebacterium glutamicum [42, 43]. Also Pseudomonas putida KT2440 does not seem to be a superior alternative to E. coli in terms of productivity, but at least this strain already obtained the biosafety certificate  and this may constitute a critical advantage for the preparation of therapeutic antibody fragments.
Simplified approaches: the opportunities offered by cell display
Eukaryotic expression systems for antibody fragments
Yeasts as microbial cell factories
The idea of displaying antibodies on whole cells has been long used in yeast for panning antibody libraries but only recently it has been proposed to exploit antibody-displaying whole cells as ready-to-use reagents. Binders were first selected from a scFv pre-immune library in yeast display format and the recovered cells were directly lyophilized for storage . They retained their initial activity and specificity and could be used for few weeks representing a cheap and fast alternative to purified antibodies for at least some immune-capture applications such as the identification of soluble and membrane-bound pathogen antigens.
Pichia pastoris is the workhorse yeast for conventional production of scFv-derived antibody fragments. The protocols for obtaining secreted constructs are standardized and efficient [49–51] and most of the lately proposed improvements concern incremental features such as the length of the pre-induction glycerol feeding during fermentation, the codon usage, the optimization of the starting culture temperature, of the methanol concentration, and of pH obtained by means of Design of Experiment approach [52–54].
Despite the fact that P. pastoris is one of the most established organisms for the expression of recombinant scFv and scFv-fusion proteins, only few reports describe the production of nanobodies in this yeast species. Since for many research labs yeast production might be technically more demanding than the golden standard E. coli, the choice would become meaningful only when specific advantages can be obtained. This evidence was missing in the first paper describing the production of a naked single-domain antibody in P. pastoris. Yields were comparable (10 mg/L) with those obtained with most of the VHHs expressed in bacteria . The approach was more clearly justified in the work of Ji et al.  who used Pichia to express the IgG-like construct composed by an anti-TNF VHH fused with a human Fc. The authors found that it was more functionally active than the same construct expressed in the periplasm of E. coli. Nevertheless, the exact reasons of the functional advantage remain unknown since the authors did not characterize the recombinant antibodies for their structural features. In terms of yields, these were lower (5 mg/L) in comparison to what was obtained by Djender et al.  who expressed comparable constructs in bacterial cytoplasm. S. cerevisiae offers an interesting alternative to Pichia, but despite some promising initial results , it has never become a popular expression systems for VHHs. Recently, an important contribution assessed the role of specific residues in the VHH sequences the optimization of which might contribute substantially to improve their final yields in S. cerevisiae . Specifically, the authors identified that some amino acids close to and inside the J segment are critical for VHH folding and secretion in yeast because they are involved in the chaperone recruitment in the ER. Their presence compensates for the recognition differences that exist between the mammalian and S. cerevisiae chaperone machineries and favor the VHH folding kinetics.
Any good reason for expressing recombinant antibodies in mammalian cells?
Mammalian cells represent the gold standard for producing therapeutic antibodies but at the research lab level other systems have been preferred. However, the mammalian origin of VHH and scFv fragments suggests that it could be meaningful not only to produce them in yeast co-expressing mammalian chaperones , but directly in evolutionarily related organisms such as mammalian cells that should possess conserved secretion/folding machineries. The resulting antibodies are expected to gain the specific post-transcriptional modifications that enable the immune-activation and target recognition mechanisms in mammalians. This would represent a clear advantage for evaluating the in vivo biological activity of antibodies conceived for therapeutic applications. From this perspective, it would be very interesting to have access to comparative studies evaluating the binding characteristics, the biodistribution and pharmacokinetic, the immunogenic and curative effects, and the production costs of recombinant antibodies expressed in different organisms. However, sufficient information is not available so far with only few reports of antibody fragments produced in mammalians, such as a VHH against GFP expressed in CHO cells and scFv-Fc fragments against Epstein Barr virus in HEK293 [63, 64]. It is difficult to infer from this lack of data if the scarce interest finds its reasons in technical, biological or economic considerations.
Filamentous fungi, insect-based systems and further “niche” eukaryotic organisms
Trichoderma reesei and Aspergillus spp failed to achieve the leading role in the production of recombinant nanobodies that was envisaged almost 15 years ago . As suggested at that time, these expression systems should have been particularly effective for producing fusion proteins composed of antibody fragments linked to active enzymes such as oxidases and peroxidases that are structurally complex and consequently difficult to express correctly in bacteria. However, the recent report describing the expression in Aspergillus of a nanobody-glucoamylase fusion  does not provide sufficient information concerning yields and glycosylation patterns to evaluate the robustness of the method. Probably, the lack of a ready-to-use platform for cloning and expression discouraged many researchers to test filamentous fungi as a microbial cell factory for antibody fragment production. Another niche expression system that has been occasionally used to express recombinant antibody fragments and reconstituted IgG-like molecules is the Leishmania tarentolae eukaryotic parasite [66, 67]. The putative advantages of this method should reside in its codon usage but the reported yields are very low and probably do not justify the investment necessary to set this platform in a lab already equipped for recombinant production in another organism.
Insect cells and larvae have been proposed as nanobody expression living biofactories [68, 69]. Specifically, Trichoplusia ni larvae have been transformed by means of the efficient Improved Baculovirus Expression System to produce neutralizing nanobodies directed against diarrhea-inducing rotavirus A . The characterization of the resulting VHHs indicated that they were functional but the shown data are too preliminary to assess the real benefit of this production approach in terms of simplicity, cost-efficiency, and antibody quality.
Exploiting plants for antibody production
Recombinant antibody fragments have been successfully produced in different higher plants. For instance, an anti-murine TNF nanobody expressed in and purified from rice seeds was effective in neutralizing its antigen and suppressing the progression of collagen-induced arthritis in mice . The claimed advantages of this approach with respect to microbial protein factories are low production costs, scalability, reduced safety issues such as toxin and virus contaminations, controlled N-glycosylation, and the possibility to concentrate the product in specific organs, usually seeds or leaves. Nevertheless, the final antibody concentration in plant organs is generally low (not exceeding hundreds of mg/kg plant, with antibodies representing usually less than 5 % of the total seed proteins and less than 1 % of leaf proteins). The downstream processing is the major system drawback since the diluted target product is mixed with a high load of soluble and particulate contaminants that require demanding integrated technological procedures for their removal . Furthermore, recombinant protein production can induce physiological stress in the host plants resulting in the accumulation of heterogeneous products . The involved mechanisms are not yet completely understood but recently it was demonstrated that the expression of VHH-Fc and scFv-Fc fusions trigger unfolded protein response in Arabidopsis seeds . The most surprising result in this study was that neither the antibody expression levels nor the intrinsic structural stability of the constructs led to significant response differences. This observation suggests that the critical threshold for stress response is probably low, whereas other factors, such as the environmental conditions during seed development might contribute to final protein quality and yields. In general, the biophysical characterization of the antibodies expressed in plants have not been thoroughly addressed because the emphasis was rather placed on the (residual?) functionality of the purified material. Bivalent reconstituted Fc-VHH antibodies produced in Arabidopsis and Nicotiana benthamiana appeared partially degraded after the purification step but resulted effective in neutralizing avian influenza virus  and the α-cobratoxin venom . Also in other cases the structural heterogeneity of antibodies produced in plants does not appear to compromise significantly their functionality [72, 76–78], but it can probably complicate their acceptance as therapeutics. Likely, the methodology still requires the optimization of the expression conditions and of the binder formats. Forcing the antibody fragment accumulation in specific sub-cellular compartments and the use of plant codons, specific promoters, fusion partners, and tags can increase the final yields of stable recombinant antibodies [79–82], although the optimal stabilizing combination seems to be rather antibody-specific, as exemplified in the case of the fusion of VHHs with different Fc domains .
Immunogenicity can be induced by proteins expressed in higher-plant due to the presence of some plant-specific sugar residues. The problem has been addressed by using RNA interfering technology to down-regulate glycosylation. A more elegant and definitive alternative was the engineering of Nicotiana benthamiana and of the moss Physcomitrella patens to obtain fully human N-glycosylation [83, 84]. Furthermore, such moss is suitable for photobioreactor production of antibody fragments  as well as of therapeutic IgG antibodies with superior ADCC (antibody-dependent cell-mediated cytotoxicity) compared to the same constructs recovered from mammalian cultures .
Also micro-algae can represent a valid antibody factory. The unicellular organism Chlamydomonas reinhardtii was able to express functional immunotoxins in both scFv- and IgG-like formats . These large macromolecules have very complex structures that are difficult to recover correctly folded in most of the recombinant expression systems and this evidence can justify the interest in this protein factory.
A meaningful line of research is aimed at developing transgenic crops able to produce antifungal recombinant antibodies that neutralize pathogens in vivo. The approach is not absolutely new, but its efficacy strongly profited from the late technological progresses. By using such a strategy, it was possible to develop crops tolerant to different phytopathogen infections such as Brassica napus resistant to anti-Sclerotinia sclerotiorum scFvs , Ciitrus tolerant to Citrus tristeza virus , and soybean resistant to the Fusarium virguliforme toxin-1 that induces the sudden death syndrome .
Neutralizing recombinant antibody fragment expression for direct therapeutic applications
In one of the previous sections we have seen how Lactobacilli overexpressing anti-viral antibody fragments can be directly used as an effective “therapeutic food”. It is possible because a relatively small volume of their culture delivers enough functional antibodies to mitigate the disease symptoms [31, 33]. Vegetal organisms can be exploited as factories for antibody production and for expressing those antibody fragments that can protect them from phytopathogens [88–90] but would a plant-based diet be able to provide therapeutic antibody doses sufficient to induce a beneficial effect in fed mammalians? There are promising results indicating the feasibility of such a smart development by which plant-producing recombinant antibodies might provide “functional food” with direct therapeutic effect. Transgenic rice has been engineered to accumulate neutralizing anti-rotavirus nanobodies in its seeds (MucoRiseARP1) . The idea is that of protecting human populations against life-threatening diarrhea by feeding people with antibody-enriched seeds because rice is already part of their daily diet. Following the engineering of plants to suppress the upload of endogenous storage proteins, the nanobodies accumulated in the seeds at sufficiently high concentrations (g/kg of total weight and 11.9 % of total protein) to be effective in vivo as a prophylactic medicament. Furthermore, neutralizing nanobodies are water-soluble, resistant to both boiling and long-term storage (at least 1 year), and do not rely on cold-chain transport and storage. These features render the mutant rice not a simple basic research proof-of-principle but a product that could be potentially adopted by final users. In animal model the results seem very promising since even immunodeficient mice fed with MucoRiseArp1 resulted significantly protected against rotavirus infections. The strategy as well as the probable legal and cultural resistance is the same as described above for anti-virus neutralizing nanobodies expressed in Lactobacillus. It also reminds the logic that led to the development of the “Golden-rice” that was designed for providing a cheap source of beta-carotene to integrate the dietary Vitamin A shortage in rice-eating populations . Hopefully they will not share the same tormented story. Another meaningful example of therapeutic plant is given by the transgenic peas accumulating anti-Eimeria neutralizing scFvs in their seeds. Chickens fed with them showed significant mitigation against the parasite-induced coccidiosis .
A different therapeutic approach considers the adenovirus vector-dependent expression of a hetero-trimer composed of VHHs specific for independent epitopes of both Stx1 and Stx2 Shiga toxins. The expression vector is delivered by a single intramuscular injection and the resulting VHH-based neutralizing agent is released to the circulation. As the consequence of effective and systemic toxin targeting, the methodology resulted highly protective against hemolytic-uremic syndrome in piglet model  and, as a vaccination method, could represent a valuable curative approach for a pathology for which no suitable remedy is available. Another peculiar exploitation of VHH in vivo expression has been proposed by De Vooght et al.  for impairing the tsetse fly-dependent spreading of trypanosome parasites. The authors demonstrated that it was possible to stably infect the fly population with the genetically engineered bacterial symbiont Sodalis glossinidius expressing a strong anti-trypanosoma nanobody. The nanobodies were efficiently secreted by transformed bacteria in vitro  and in vivo  and such bacteria were transmitted—although not very efficiently—in the progeny and promoted the accumulation of the trypanolytic nanobodies in several tissues among which the midgut that is an obligatory environment for the trypanosome development. The paper does not clearly demonstrate that is possible to select long-term paratransgenic tsetse populations in which the parasite transmission is impaired by the nanobody activity. Nevertheless, it evidences the drawbacks that must be removed to achieve this goal, such as the preventive elimination of the wild type bacterial population to avoid competition with the recombinant bacteria and the selection of nanobodies structurally resistant to the acidic and proteolytic conditions typical of midgut.
Another innovative development concerns the possibility to use plant-expressed antibodies for environmental remediation and by such a way preventing pollution conditions that are critical for human health. Specifically, Barbi et al.  developed transgenic tobacco expressing anti-microcystin-LR antibody fragments that are secreted and anchored to plasma membrane in leaf tissues. The membrane-retained antibodies were able to bind the microcystin initially present in hydroponic medium and formed stable complexes that could be eliminated removing the leaves.
Recombinant antibody fragments as effective intrabodies
VHHs are often stable in the absence of disulfide bonds [98–100]. This characteristic makes them valuable tools for cell biology studies  as well as candidates for therapeutic applications based on their expression and accumulation as intrabodies directly in the cytoplasm/nucleus of the host cells. In this subcellular compartment they can neutralize the corresponding antigen and block its activity, as already demonstrated in the case of anti-viral VHHs [98, 99]. In the specific case of the anti-Rev VHH Nb190 that is able to suppress HIV-1 replication, cells stably expressing the intrabody were protected against the virus-induced cytopathogenic effect .
Different strategies have been developed in order to select directly for functional recombinant intrabodies. A C-terminal GFP protein allowed for the fluorescence-based light-microscopy screening of the clones that expressed folded nanobodies binding selected cellular structures . However, the original method is demanding because the evaluation is made individually for each clone. Furthermore, the folding of several VHHs depended on DsbC isomerase and stabilizing tags that would not be available in clinical settings. Consequently, more convenient approaches have been considered and recently different variations of the two-hybrid technology were successfully applied for isolating both scFv and VHH intrabodies [100, 104–106]. Mukhtar and colleagues  successfully used the yeast two-hybrid technology to isolate human scFv intrabodies against nucleoproteins involved in influenza virus replication and transcription. Such intrabodies strongly bound their antigens and modified their cellular distribution and accumulation rate. When applied to porcine circovirus type 2-immunized VHH collections, yeast two-hybrid libraries enabled the isolation of intrabodies suitable for ELISA and immunocytochemistry on infected cells . More recently, some VHHs targeting HIV-1 proteins have been isolated by Sos Recruitment System, a variation of the classical two-hybrid methodology in which the bait-prey interaction happens in the cytoplasm instead of the nucleus . This alternative should prevent failures in all of the cases in which the folding efficiency of one of the two interacting polypeptides suffers from the conditions present in the nucleus milieu. However, only one of the selected candidates described in the paper was able to bind its viral antigen in the cytoplasm of eukaryotic cells leading to its delocalization and despite the strong binding the nanobody could not impair the cytostatic and apoptotic effects promoted by the antigen . The results confirm that the approach still needs to be improved but can be suitable for identifying functional binders useful for studying the molecular mechanisms regulating pathogen proteins. On the other hand, they question the hypothesis of using antibodies for buffering pathogenic factors because incomplete neutralization could determine the approach failure, whereas antibody excessive accumulation could result toxic for the host cell. The problem could also lie in the antibody quality obtained by yeast selection methods since the low transfection efficiency of this organism limits the overall original clone variability. Therefore, transposing the two-hybrid principle into a bacterial system makes sense due to higher transformation efficiency i.e. a larger clonal diversity would become available for selection. The suitability of bacterial two-hybrid for the selection of VHHs was demonstrated by Pellis et al.  who succeeded in recovering functional intrabodies directed against GFP, HIV-1 integrase, and T. vivax nucleoside hydrolase. The nanobodies had stability and binding characteristics similar to those of VHHs isolated by phage display libraries constructed using the same RNA pool and, in some cases, the same clone was recovered by both selection methods. The authors underline how important was having used material from immunized animals since somatic maturation would have provided clones of sufficient binding affinity for obtaining effective two-hybrid coupling. However, most of the selected clones had affinity in the nanomolar range, namely binding constants that can be normally recovered panning sufficiently large pre-immune libraries . Consequently, these should be considered also for this application. The results recently obtained using cytoplasmic Retained Display  confirm that pre-immune libraries are suitable for direct intrabody recovery, in this specific case scFvs with fixed human framework and CDR1/CDR2, and which can tolerate CDR3 diversification.
Any help from in silico resources?
Antibody fragment final yields are clearly influenced by their propensity to misfold and aggregate. A first step for the selective screening of stable binders issued from phage display libraries can be achieved before starting the panning procedure by introducing a heating treatment of the phage population . Since thermal stability and the capacity to refold into native structure inversely correlate with aggregation readiness, the heat-resistant clones are on average more stable than precipitated ones and can be purified by incubation at high temperatures . Bioinformatics and protein modeling could further help in identifying critical residues able to improve the stability of the recombinant antibodies selected after panning. This approach should be considered, although it is difficult to identify a consensus about the hot spots even in the case of simple single-domain molecules such as the VH(H)s [111–113].
Recombinant antibody production: hot topics and examples
Opportunity and challenges
Achievement reports (references)
Fusions of Ab fragments with active proteins (IL1, toxins, chromophores,…)
Bispecific T-cell engager
Possibility to interfere or to join two different pathways
Purification-independent methods for inexpensive Ab production
Development of self-assembling multivalent structures
In vivo expression and activity
Production of pathogen-neutralizing Abs directly in food and animals
Effective glycosylation for precise targeting and CDC/ADCC
Ab chemical functionalization for ADC, radiotherapy, and imaging
Immune-response and accumulation of active molecules
Necessity to fold into active form in cell reducing cytoplasm
Altogether, the picture looks optimistic because of the many published achievements. Nevertheless, the drawback of the present model for research production dissemination is its unbalance towards the exclusive publication of positive results. The consequence is that one has access to success stories but knows very little about conditions that resulted in failures and that would be useful to know in order to avoid similar mistakes. Systematic comparisons based on combinatorial approaches  and benchmarking initiatives [115, 116] are extremely useful to evaluate methods, experimental sets or reagents but are still rare in the field of recombinant antibody production [27, 60, 78, 86, 117]. Another observed limit is represented by the publication of papers in which controls and characterization data necessary to assess the reliability of the claimed conclusions are missing. For instance, data reporting yield improvement should be considered with some caution in the absence of control results showing that the antibody functionality is not affected by the quantitative increase since absolute and functional yields might be not coincident. Unluckily, the presentation of a complete set of control data is rare and it is a pity because potentially good ideas remain unfulfilled when reliable information is diluted in the presence of ambiguous results. Accordingly, methods presented without preliminary scientific reviewing, such as patent applications , are not discussed in this work. Probably, the community (authors, editors, funding agencies) should take more seriously the necessity to request minimal standards of biophysical  and functional characterization of immune-reagents and to reward the attempts to improve this praxis .
Summary of the evaluated expression systems for recombinant antibody production
Method robustness: specificities
References (for unusual organisms)
Prokaryotic gold standard, different methodologies
Effective diet therapy
Effective diet therapy
Effective cargo in vivo
Diet therapy to be demonstrated
Protein folding at low temperature: high energy needs?
Highly productive for VHHs
No shown advantage
No shown advantage
FDA biosafety certificate
Suitable for display and in vivo cargo applications, biosafe
Eukaryotic gold standard
Suitable for engineering
No shown advantage
Industrial gold standard: no shown advantage at research lab level
No shown advantage at research lab level
Not thoroughly characterized
Not thoroughly characterized
Favorable codon usage, low yields
Positive preliminary results
Optimized yields and quality
Suitable for photoreactor, promising for IgGs
Promising for large immune-reagents
The author wishes to thank Alicja Gruszka for her stimulating comments, the Creative Core AHA-MOMENT grant from Slovene Ministry of Economic Development and Technology as well as the European Fund for Regional Development—Cross-Border Cooperation Programme Italy-Slovenia 2007–2013, (Project PROTEO, Code N. CB166) for having supported this work with research funds.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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