Behind the scenes: Developing vWF Antigen

 

The thought of developing a vWF Antigen reagent has been with us since early in Nordic Biomarker’s history. Back then, R&D was far from the populous department it is today and resources were sparse. Now, we have finally released a product that meets our high set standards. Follow along on the journey!

The seed had been planted and was germinating. Customers kept asking us about a vWF antigen reagent, and it was a challenge that we really wanted to take on. A vWF reagent is complex, not in the least because the vWF molecule itself is very mutagenic, but it was a very intriguing project. 

 ”This is what we are experts in. We know latex immunoassays, we know coagulation, and we want to be a complete supplier of reagents for coagulation diagnostics”, says Malin Walfridsson, R&D director at Nordic Biomarker.  

 

Von Willebrand Factor 101

Von Willebrand Factor (vWF) is a large protein. Two or multiple vWF molecules bind together and form even larger molecules (dimers and multimers). vWF plays an important part in the coagulation process – if it does not function properly, your blood will not be able to clot as it is supposed to, which may lead to excessive bleeding. Treatments include tranexamic acid, desmopressin and vWF concentrate.  

vWF assays are one of the most often used specialty assays, as up to 1% of the population is affected by von Willebrand disease (vWD). vWD is the most common hereditary bleeding disorder. 

Further reading:  Whitepaper: Von Willebrand Factor and its Functions

 

In 2017, Jenny Pasto was recruited for the task. Jenny had previous research and lab experience from academia, and was tempted by the opportunity to work independently on a larger project. The practical focus was especially tempting: 

 “You start from nothing, and it becomes a product that will help patients”, Jenny says. 

The first step from ”nothing” to “patient benefits” was to find vWF specific antibodies.  

In Search of a Specific Antibody 

Nordic Biomarker’s approach has always been to use monoclonal antibodies for our latex immunoassays, as opposed to polyclonal antibodies. The production process starts the same way for both types of antibodies: you immunise mice or rabbits with vWF concentrate, which makes them produce antibodies against vWF. After that, the processes differ.  

For monoclonal antibodies, the spleen is extracted, and the spleen cells (antibody-producing lymphocytes) are isolated. The spleen cells are then fused with cancer cells to form so called hybridomas. Cancer cells’ ability to grow endlessly is catastrophic in the body, but an asset in the lab. The hybridomas will live in cell culturing flasks or machines and continue to divide and produce the same antibodies almost indefinitely.

Polyclonal antibodies, on the other hand, are extracted directly from the blood of vWF immunised rodents, and it is less certain that you get the exact same antibodies over time. 

 “Monoclonal antibodies give a lower risk of lot-to-lot variation in the long term”, Malin says. “Polyclonal antibodies may drift over time, but monoclonal stay the same.” 

We have chosen to use a mix of monoclonal antibodies in our vWF Antigen reagent to ensure optimal function.  

 “One antibody is technically enough, but vWF is naturally mutagenic, so in case there is a mutation on one of the binding sites, we have a mix of different antibodies”, Malin says.  

It is important to find hybridomas that produce antibodies specific to vWF, i.e. antibodies that can attach themselves to the vWF molecule, but to no other. Jenny went through a long and careful screening process before she found antibodies that met our demands on specificity and affinity, and were interesting to move forward with. 

Putting the Antibody in the Right Company 

To have specific antibodies is all well and good, but there is a plethora of other variables that need to fall into place before you have a viable product. Jenny exemplifies: 

 “Most important, there is the type of latex particle and the combination of latex particle and antibody. Then there is the coupling buffer, the storage buffer and the reaction buffer, which can be of different concentrations and have different additives, and then you can use different amounts of stabiliser…”  

The list is long, and the potential for failure is correspondingly huge. 

 “It is a lot of trial and error. It may be a little exaggerated, but you can say that there is a 98% fail rate”, says Jenny. 

Often the failure is instantly obvious. 

 “I could come back from lunch and see that a sample had self-aggregated, and that was that.” 

Sometimes the failure is a bit more hidden.  

 “It turned out that one promising combination of antibody and latex particle was way too sensitive for variation in antibody concentration. If the concentration was just slightly higher or lower, the assay didn’t work like it was supposed to.” 

That degree of sensitivity is not suitable for large-scale production, so Jenny needed to find a new combination.  

And finally, she succeeded.  

The Development Phase 

In December 2020 the research phase had come to an end, and the development phase could begin. Jenny chose to stay on the project and be the project manager for the development phase as well.  

 “I had lived with this project for four years, so I wanted to see it through. I also thought it would be an exciting opportunity to learn something new and grow – leading a development project is very different from leading a research project. In the research phase, much of the work is done alone or with someone from R&D, but in the development phase you get to work closely together with all departments.” 

The production department produces pilot lots, which the quality control department can test and perform stability studies on; the product specialists assist with testing out settings on different instruments; quality assurance keep track of regulatory requirements; sales and marketing are educated on the product and inform customers; the order department sends out reagent kits for evaluation – almost every department has been involved in a product even before it is released.  

MRX Green vWF Antigen was finally CE marked and released to the market in April 2022. 

 “I am so happy and proud that we have followed through with this project”, says Malin. “It has been our most complex one so far, but we have persisted and ended up with a great product.” 

What is Next? 

Jenny’s next project will be another development project, as she will join the team that is already at work with CE-marking products under IVDR. 

 “Sometimes I miss that deep dive that a research project is, but I need to recharge a bit first”, she says. 

As a company, we have many projects in the pipeline – Anti FXa, PT Quick Liquid and PT DOAC being some of them – and of course a vWF activity assay. But that is for another time…