Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors

Abstract

The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens . Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH.

Stable Protein Sialylation in Physcomitrella

“…For MS analysis gel bands corresponding to high molecular weight range proteins were excised. Gel band preparations, MS measurements on a QExative Plus instrument and data analysis were performed as described previously (Top et al, 2019). Raw data were processed using Mascot Distiller V2.5.1.0 (Matrix Science, USA) and the peak lists obtained were searched with Mascot V2.6.0 against an in-house database containing all P. patens V3.3 protein models (Lang et al, 2018) and the sequence of the reporter protein.…”

A synthetic protein as efficient multitarget regulator against complement over-activation

“…This vector was obtained via site-directed mutagenesis using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The vector pAct5-MFHR1 38 was used as template with the primers MFHR1_I62_fwd and MFHR1_I62_rev…”

Stable Protein Sialylation in Physcomitrella

“…For MS analysis gel bands corresponding to high molecular weight range proteins were excised. Gel band preparations, MS measurements on a QExative Plus instrument and data analysis were performed as described previously (Top et al, 2019). Raw data were processed using Mascot Distiller V2.5.1.0 (Matrix Science, United States) and the peak lists obtained were searched with Mascot V2.6.0 against an in-house database containing all Physcomitrella V3.3 protein models (Lang et al, 2018) (Halim et al, 2014).…”