Services include llama immunization, construction of phage-display libraries, custom-tailored selection procedures, production of VHH, plain or equipped with different tags in bacteria and upscaling of production in yeast up to GMP production in collaboration with Orthros TR BV. The knowledge build up within QVQ can be used to optimize the VHH for stability, production levels and humanization.
Immunization and library construction
For the immunization of llamas QVQ collaborates with Kaneka Eurogentec.
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.
If desired, immunization can be prolonged.
Phage display selection and screening
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.
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).
VHH Custom labelling
Aim of QVQ is to deliver VHH that are exclusively labelled directionally.
Moreover, QVQ also labels the VHH on request with preferred maleimide label or dye.
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.
QVQ has invested in fermentation equipment and can now capitalize on the longstanding knowledge to obtain very high quantities of VHH in a fermatitive 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)