Technology

QVQ’s imaging agents are suitable for correlative imaging from nanometer to meter scale. (animation by ScienceTransmitter)

QVQ is a company performing Contract Research for industrial partners and works together with Universities and medical centres in Collaboration Agreements.

QVQ provides expertise in each step of Llama Antibody Technology [VHH Technology], from immunizations, selections and screening of functionalities to production.

QVQ has a considerable number of high quality VHH for Sales for Research in areas like Cancer, Age related Diseases and Infectious Diseases

Basic Monoclonal VHH development

Immunization and library construction
QVQ collaborates with Kaneka Eurogentec on the immunization of llamas. The immunizations are conducted according to the applicable animal welfare act at place. All immunizations are reviewed by the internal animal experiment commission of the company before starting the immunization.

QVQ has experience of using several antigens forms ranging from purified natural and recombinant protein up to complex cells and tissue for immunization. The optimized protocols allow for immunization with low amounts of recombinant protein with high success rates.

2 animals are scheduled for each immunization to ensure diversity of the selected VHH and to avert possible non-responder animals. Four injections with antigen are included in the standard 43 day protocol, which can be extended with additional boosts for generation of very high specific or affinity VHH.

Antibody titers directed against the antigen of interest are measured using “in-house made” polyclonal antibody directed against isolated VHH. In the case of a proper immune response the RNA isolated from the blood of the animals is used for construction of a phage display library. QVQ considers only libraries with size exceeding 107 different clones and insert frequency of more than 90% qualified to be used for selection of VHH binding to desired antigen.

Phage diplay selection and screening for binding

QVQ always proposes a strategy to achieve selection of VHH binders by phage display based on own experience and available tools. Such strategy is carefully designed to include customer’s requests and wishes and after agreement on the selection strategy, QVQ commits to select at least 6 VHH belonging to at least 2 different families based on the V-D-J gene combinations.

Importantly, already in the phage display selection the requirements for using the VHH in their final application are taken into consideration. Agonistic or antagonistic effects, preferred epitopes, high stability or high specificity can be selected for using the expertise of the QVQ team.

Production and delivery

VHH are characterized using the tools available for binding, competition and functionality. Moreover, sequence of the different VHH is determined. The binding data, sequence data and if possible functional data obtained by the customer are combined to select a lead panel of 6 VHH. These are cloned in an expression plasmid and purified using IMAC from the periplasm of Escherichia coli.

Data describing all the above mentioned steps are collected in the form of a report, which is send to the customer along with purified VHH protein (0,5 mg of each of the 6 lead clones)

Optional Customization of your product

Custom Labelling

Aim of QVQ is to deliver VHH that are exclusively labeled directionally. This is achieved by extending the VHH protein with a tag containing a cysteine residue at the C terminus. This unpaired cysteine is than used to couple to Maleimide containing molecules at a site that was proven to not affect the paratope of the VHH, while random labeling causes usually inactivation of the paratope.

QVQ delivers VHH that are labeled directionally at the C terminus with different fluorescent dyes (FITC, Hilyte, IRDye), biotin, DOTA, NOTA and HRP. Moreover, QVQ also labels the VHH on request with the preferred maleimide label or dye.

Optimization

QVQ disposes of a large database of VHH sequences, which are used for analysis of VHH production, stability and protease resistance. Moreover, due to wealth of information from mutagenesis of VHH, QVQ is able to manipulate the sequence of the selected VHH to generate more stable VHH and VHH produced at large yield.

Since VHH are not of human origin, and although llama origin of VHH was never an impediment for its application, QVQ can modify the VHH sequence to render it more human-like sequence, based on the information disclosed in the paper of Vincke et all (2012) and other proprietary information.

QVQ has ample experience with the production of recombinant VHH from bacterial and yeast hosts for samples up to 500 mg. Moreover, QVQ disposes of plasmids for the expression of VHH with a myriad tags at the C terminus. VHH purification is either by affinity chromatography or IEX-chromatography.

QVQ has gained experience on translating the production knowledge to perform a GMP approved process for production of VHH that will be applied for diagnostic imaging in human.

Fermentation

QVQ has invested in fermentation equipment and can now capitalize on the longstanding knowledge to obtain very high quantities of VHH in a fermentative process.

Two standard protocols are available which are suitable for very well producing VHH (> 1 g/L) and one for more difficult to produce VHH (50-800 mg/L)

Fermentative production of VHH using a special strain of S. cerevisiae and a proprietary process.