Antibodies are glycoproteins that specifically recognize antigen.
They’re produced by cells of the immune system - called matured B cells or plasmocytes - in order to block and neutralize foreign agents while infection occurs. In case of auto-immunity diseases (e.g. diabetes, lupus…), antibodies are secreted against host molecules, and are so named auto-antibodies.
During the immune response, an organism synthesizes antibodies directed against several epitopes of an antigen: this in-vivo reaction is called polyclonal immune response (1891, Emil von Behring and Shibasaburo Kitasato).
The polyclonal antibodies (pAbs) are a collection of antibodies recognizing different epitopes on a given antigen, each idiotype being secreted by a different plasmocyte. Each individual of this mixture, is a monoclonal antibody (mAb), recognizing only one epitope of a given antigen and coming from only one single B clone. When this last clone is fused to an immortal myeloma cell line (or fusion partner), it gives birth to a hybridoma cell that can produce large quantities of identical antibody (1975, César Milstein and Georges Köhler – 1983, Hervé Bazin).
But since the polyclonal approach generates a great deal of variability from one batch to another - because they are produced from different animals at different times - the market naturally turned to the monoclonal approach.
But monoclonals also have their own limitations.
1. Disadvantages of Monoclonal Antibody approach
In fact, compared to polyclonals, monoclonal antibodies (mAbs) are :
- More costly to produce since they require more time, equipment and cell culture capabilities,
Myeloma-dependent, so potentially
- containing additional transcripts and leading to extra light-chain and heavy-chain generation,
- confering genetic instability to hybridoma clone and a loss of protein expression
- not available for certain species
- Too specific for certain application (e.g. in food industry diagnostic to detect modified allergens after heat treatment, to detect different mutants of a virus strain…)
- More sensitive to environmental stress conditions (pH, buffer…) and so potentially useless if the binding site is modified (contrary to polyclonals which can afford the loss of a single binding site).
And to circumvent this issues, several alternative strategies can be considered.
2. Alternatives to Monoclonal Antibody generation
a. Custom Polyclonal antibodies generation & immuno-affinity purification
Polyclonals have the great advantage to be generated in a vast range of different animal hosts, allowing researchers to have access to a large amount of cost-effective material issued from various immune repertoires.
At SYnAbs, we offer polyclonal from rabbits, rats, mice and guinea pigs. In order to approach the specificity that monoclonals provide, SYnabs offers to its partners the purification of anti-serum on immuno-affinity column against your antigen. Antigen affinity purification results in the elimination of the bulk of the non-specific IgG fraction (95 to 99,5% of the all antiserum), and enriches the fraction of immunoglobulin that specifically reacts with the target antigen. These polyclonals can quickly capture the required antigen and offer a great sensitivity for your immuno-assay.
Nevertheless, in case of polyclonal antibody generation, the constant use of animals and the limited supply of material for passive immunotherapy application remain major hurdles.
Soon, another strategy had been figured in Denmark thanks to the rise of genetic engineering.
b. Manufacturing of recombinant polyclonal antibodies
Back in 2000, Symphogen (Servier, since June 2020) decided to launch symphobody Sym001 (Rozrolimupab), a recombinant polyclonal antibody consisting of 25 different anti-Rhesus D (RhD) antibodies to replace existing anti-RhD hyperimmune immunoglobulins for the treatment of Idiopathic Thrombocytopenic Purpura (ITP) and the prevention of Hemolytic Disease of Newborns (HDN) (1).
To control manufacturing costs, and thanks to its unique SympressTM technology (site specific integration), Symphogen was able to tackle the issue of large-scale industrial production of recombinant human polyclonal antibodies creating the first polyclonal Master Cell Bank. With this same economic view, in 2010, Merus & Crucell announced the establishment of a single PER.C6 clonal production cell line to manufacture mixtures of recombinant polyclonal human antibodies (2).
In the same way that Symphogen generated its library, Diaclone offers to its clients to isolate antigen-specific antibodies coming from polyclonal immune response and converting them into a phage display library. From immunised animals, with strong antibody responses to your specific antigen, Diaclone isolates total RNA, generates cDNA by reverse transcription, and then amplifies the VH and VL region gene repertoires. The VH and VL region genes are separately cloned into a phagemid vector to generate original Fab phage display library.
But how do you manage to select each component of your mixture based on affinity, epitope specificity and particular features ?
In this case, a new scenario can then be considered.
c. Oligoclonal antibody cocktails or multiclonals
A more costly approach, compared to previous methodologies, is to construct a pool of several monoclonal antibodies or an oligoclonal cocktail of qualified monoclonals based on their specificity and affinity for the target. These multiple antibodies or multiclonals are expressed and purified in parallel before combining.
At SYnAbs, this is typically the case for our following products:
Despite the fact that polyclonals can offer many advantages in the case of particular immuno-assays or as a first and cost-effective tool for primary research, polyclonal generation strategy shows its limits in several situations.
For example, in the case of small epitopes modifications. In fact, if you need to target epigenetic modifications (acetylation, methylation, mutation, phosphorylation), neo-epitopes after enzyme cleavage, or small molecules, these antigens will not trigger a sufficient polyclonal response and the risk of non-specific antibody development is pretty high.
(1) Wiberg, F.C., Rasmussen, S.K., Frandsen, T.P., Rasmussen, L.K., Tengbjerg, K., Coljee, V.W., Sharon, J., Yang, C.‐Y., Bregenholt, S., Nielsen, L.S., Haurum, J.S. and Tolstrup, A.B. (2006), Production of target-specific recombinant human polyclonal antibodies in mammalian cells. Biotechnol. Bioeng., 94: 396-405. doi:10.1002/bit.20865