By: Dr Leah Quinn, Product Development Scientist & Dr Hannah Byrne,Head of Biologics
Antibody therapeutics are revolutionising the treatment of diseases such as cancer and autoimmune disease. All the while, significant advances are being made to modify antibody formats, making them more effective and potent therapies. ValitaCell’s Quantum platform technology, based on fluorescence polarisation technology, enables rapid and accurate measurement of protein: protein or protein: peptide interactions.Utilising this technology platform, ValitaCell have developed smart analytics to quantify whole and fragment antibodies. This provides a complete antibody screening tool to support efficient and high quality manufacture of cell-based biologics.
Introduction to antibodies
Immunoglobulins (IgG), also known as antibodies, are multifunctional components of the immune system. Antibodies which are produced as part of the normal immune response are polyclonal, which means that they are produced from different B-lymphocytes and have different specificities for the target antigen. This is vital to allow for defence against a wide range of pathogens. The main functions of antibodies are elimination of infection, phagocytosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated lysis of pathogens and infected cells. 
Given their powerful cytotoxic and immunogenic properties, research has evolved over the last five decades to exploit antibodies as therapeutic targets in the clinical management of numerous diseases such as cancer, autoimmune disorders and infectious diseases. In 1975, Kohler and Milstein developed the hybridoma method, which allowed the production of large quantities of antibodies derived from a single B-lymphocyte with the same antigen specificity. These are known as monoclonal antibodies (mAb’s) and have revolutionised the treatment of cancer and autoimmune disorders. In the past five years, therapeutic antibodies have become the best-selling drugs in the pharmaceutical market, with an expected revenue of $300 billion by the year 2025. In 2018, six mAb products had individual sales of over $6 billion each, with many more reaching sales above $3 billion.3
Novel antibody formats
This success has prompted further research to improve antibody therapeutics. Given the inherent ‘modular’ nature of antibodies, both structurally and functionally, the generation of novel antibody formats have been explored, leading to the generation of smaller ‘antigen binding fragments’.
There are three main formats of antibody fragments: Fragment Variable (Fv)-based formats, Fab (fragment antigen binding)- based formats and single-domain antibodies (see Figure 1 for overview). These antibody fragments can be used on their own or linked to other molecules or fragments to create bi-specific, multi-specific, multimeric or multifunctional molecules. 4 These formats allow a single antibody to bind to multiple targets, and therefore possess novel and vast biological functions. Antibody fragments have numerous advantages compared to traditional antibodies, in terms of both their production and biological effects. Rather than mammalian expression platforms, antibody fragments can be produced in microbial expression systems, which leads to faster production, higher yields and lower costs. Given their small size, they are easily accessible to challenging epitopes and can penetrate tumours, making them more effective therapeutics. They also have reduced immunogenicity and reduced ‘bystander activation’ of the immune system, which occurs when immune cells are activated in an antigen-independent manner.
Figure 1. Diagrammatic representations of whole IgG antibody structure. (A) Classical Y-shaped IgG composed of two heavy (blue–red) and two light (grey–green) chains that are further divided into variable (VH in red and VL in green) and constant domains (CH in blue and CL in grey). The fragment variable (Fv) domain is the smallest fragment of an antibody required for binding and is composed of the VH and VL domains which house the complementarity determining regions (CDRs). (B) The introduction of a flexible linker to the VL–VH (or VH–VL) gives rise to the single chain fragment variable (scFv). (C) The fragment antigen binding (Fab) can be generated by both recombinant and enzymatic approaches as can the F(ab)2 fragment (D), which is composed of two Fab fragments.
ValitaCell’s Quantum Technology
At ValitaCell, our aim is to develop novel analytical technologies to enhance the quality and manufacture of cell-based biologics. Valitacell have established their Quantum platform, which encompasses a range of high-throughput, simple assays for protein screening throughout drug discovery and development. The Quantum platform is underpinned by fluorescence polarisation technology, which is a simple, rapid and efficient method to measure protein-protein or protein-peptide interactions. Fluorescence Polarisation effectively analyzes changes in the size of molecules. “Fixed” fluorophores that are excited by polarized light preferentially emit light in the same plane of polarization. However, rotation of the molecules between absorption and emission of the photon has the effect of “twisting” the polarization of the light. Small molecules tumble faster in solution than larger molecules. Hence, the change of size of molecules, with an associated fluorophore, can be measured using the degree of light de-polarization. Consequently, when a fluorescently labelled target-specific binding peptide is unbound, it tumbles rapidly and depolarizes the light more than when it is bound to its target (which is typically ~5 times larger). FP is measured by exciting the solution with plane polarized light and measuring the intensity of light emitted in the plane parallel to the exciting light (polarized proportion) and perpendicular to the exciting light (depolarized portion). The FP is expressed as a normalized difference of these two intensities, which is typically in millipolarization units (mP). This technology is the basis of our Quantum platform of products, specifically our antibody quantification tools.
Figure 2: Assay Principle: The assay applies fluorescence polarization to quantify IgG. Small, unbound molecules rotate rapidly in solution (top), while large, bound molecules rotate slowly (bottom).
ValitaCell’s Smart Analytical Tools for Relative Antibody Quantification
Valita®Titer and Valita®Titer Plus provide a cost-effective and robust method to accurately measure whole IgG titre over a wide functional range (2.5 mg/L – 2000 mg/L). Our fluorescently-labelled Fc-specific probe interacts with the Fc domain of IgG’s, providing a high-throughput and rapid quantification tool. Both assays, have a two-step workflow (overviewed in Figure 2) and a 5-minute associated incubation time allowing the preparation and quantification of 96 test IgG samples in <20 minutes (based on manual off-line preparation of plates).
Figure 3: Assay Schematic of Valita®Titer Plus assay for IgG quantification using fluorescence polarization. (A) Each well of the plate is pre-coated with a fluorescently labelled Fc-specific probe. An IgG sample binds to the probe (B). Binding is measured via fluorescence polarization and rotational diffusion (C).
In line with our ethos of developing novel analytics to enhance biomanufacturing, ValitaCell are expanding our quantification product portfolio to include antibody fragments, allowing the quantification of all CH1-containing antibodies. We are developing a novel assay, namely Valita®CH1, which relies on the detection of CH1-domain interactions with a fluorescently labelled anti-CH1 aptamer using Fluorescence Polarisation. This allows for the quantification of fragment antibodies, including monovalent F(ab) and divalent F(ab’)2 fragments (Figure 1).
The use of antibodies as therapeutic targets has been hugely successful, with antibody-based products dominating the pharmaceutical market. Novel antibody formats are actively being developed, offering more effective and less immunogenic alternatives to traditional whole IgG products. At ValitaCell, our existing Valita®Titer platform along with our expanding portfolio of novel antibody quantification assays means that we can offer a full antibody screening tool for a variety of antibody formats, which are cost-effective, robust, and high-throughput.
 Manis, JP. (2019). Overview of therapeutic monoclonal antibodies. UpToDate, www.uptodate.com/contents/overview-of-therapeutic-antibodies (Accessed 25/03/2020).
 Forthal, D (2014). Functions of Antibodies. Microbiology Spectrum 2(4): 1-17.
 Lu, R-M et al (2020). Development of therapeutic antibodies for the treatment of diseases. Journal of Biomedical Science 27(1): 1-30.
 Bates, A and Power, C.A (2019). David vs Goliath: The Structure, Function and Clinical Prospects of Antibody Fragments. Antibodies 8(28): 1-31.