Improving Downstream Process Related Manufacturability Based on Protein Engineering-A Feasibility Study.

While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process-related manufacturability, that is, how easily can a protein be purified. Process-related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety. Therefore, during molecule design, the surface charge of the target molecule should preferably differ sufficiently from the surface charge of the impurities to enable an efficient purification strategy. In this feasibility study, we evaluated the possibility of improving manufacturability by adapting the surface charge of the target protein. We generated several variants of a GLP1-receptor-agonist-Fc-domain-FGF21-fusion protein and demonstrated proof of concept exemplarily for an anion exchange chromatography step which then can be operated at high pH values with maximal product recovery allowing removal of HCP and viruses. Altering the surface charge distribution of biotherapeutic proteins can thus be useful allowing for an efficient manufacturing process for removing HCP and viruses, thereby reducing manufacturing costs.

A multivalent antibody assembled from different building blocks using tag/catcher systems: a case study.

The field of therapeutic antibodies and, especially bi- or multispecific antibodies, is growing rapidly. Especially for treating cancers, multispecific antibodies are very promising, as there are multiple pathways involved and multispecific antibodies offer the possibility to interfere at two or more sites. Besides being used as therapeutic, multispecific antibodies can be helpful tools in basic research. However, the design and choice of the most appropriate multispecific antibody format are far from trivial. The generation of multispecific antibodies starts with the generation of antibodies directed against the desired targets and then combining the different antigen-binding sites in one molecule. This is a time-consuming and laborious approach since the most suitable geometry cannot be predicted. The SpyTag technology is based on a split-protein system, where a small peptide of said protein, the SpyTag, can bind to the remaining protein, the SpyCatcher. An irreversible isopeptide bond between the SpyTag and the SpyCatcher is formed. A related Tag-Catcher system is the SnoopTag-SnoopCatcher. These systems offer the opportunity to separately produce proteins fused to the tag-peptides and to the catcher-domains and assemble them in vitro. Our goal was to design and produce different antibody fragments, Fab domains and Fc-containing domains, with different tags and/or catchers as building blocks for the assembly of different multivalent antibodies. We have shown that large multivalent antibodies consisting of up to seven building blocks can be prepared. Binding experiments demonstrated that all binding sites in such a large molecule retained their accessibility to their corresponding antigens.

Impact of lipopolysaccharides on cultivation and recombinant protein expression in human embryonal kidney (HEK-293) cells.

The human embryonal kidney 293 cell (HEK-293) is a widely used expression host for transient gene expression. The genes or plasmids used for the transient transfections are usually propagated and extracted from the gram-negative bacterium Escherichia coli, the workhorse for molecular biologists. As a gram-negative bacterium E. coli has an outer membrane (OM) containing lipopolysaccharides (LPS) or endotoxins. LPS are very potent inducers of inflammatory cytokines in the body. In early research phases DNA intended for transient transfections is not routinely checked for LPS-levels. In this study we addressed the question whether LPS has an impact on the cultivation and production of a recombinant antibody. At high concentrations the presence of LPS has a detrimental impact on cell viability and recombinant protein expression. But low LPS concentrations are tolerated and might even enhance protein expression levels.

An end-to-end automated platform process for high-throughput engineering of next-generation multi-specific antibody therapeutics.

Next-generation multi-specific antibody therapeutics (MSATs) are engineered to combine several functional activities into one molecule to provide higher efficacy compared to conventional, mono-specific antibody therapeutics. However, highly engineered MSATs frequently display poor yields and less favorable drug-like properties (DLPs), which can adversely affect their development. Systematic screening of a large panel of MSAT variants in very high throughput (HT) is thus critical to identify potent molecule candidates with good yield and DLPs early in the discovery process. Here we report on the establishment of a novel, format-agnostic platform process for the fast generation and multiparametric screening of tens of thousands of MSAT variants. To this end, we have introduced full automation across the entire value chain for MSAT engineering. Specifically, we have automated the in-silico design of very large MSAT panels such that it reflects precisely the wet-lab processes for MSAT DNA library generation. This includes mass saturation mutagenesis or bulk modular cloning technologies while, concomitantly, enabling library deconvolution approaches using HT Sanger DNA sequencing. These DNA workflows are tightly linked to fully automated downstream processes for compartmentalized mammalian cell transfection expression, and screening of multiple parameters. All sub-processes are seamlessly integrated with tailored workflow supporting bioinformatics. As described here, we used this platform to perform multifactor optimization of a next-generation bispecific, cross-over dual variable domain-Ig (CODV-Ig). Screening of more than 25,000 individual protein variants in mono- and bispecific format led to the identification of CODV-Ig variants with over 1,000-fold increased potency and significantly optimized production titers, demonstrating the power and versatility of the platform.

Production of a novel heterodimeric two-chain insulin-Fc fusion protein.

Insulin is a peptide hormone produced by the pancreas. The physiological role of insulin is the regulation of glucose metabolism. Under certain pathological conditions the insulin levels can be reduced leading to the metabolic disorder diabetes mellitus (DM). For type 1 DM and, dependent on the disease progression for type 2 DM, insulin substitution becomes indispensable. To relieve insulin substitution therapy for patients, novel insulin analogs with pharmacokinetic and pharmacodynamic profiles aiming for long-lasting or fast-acting insulins have been developed. The next step in the evolution of novel insulins should be insulin analogs with a time action profile beyond 1-2 days, preferable up to 1 week. Nowadays, insulin is produced in a recombinant manner. This approach facilitates the design and production of further insulin-analogs or insulin-fusion proteins. The usage of the Fc-domain from immunoglobulin as a fusion partner for therapeutic proteins and peptides is widely used to extend their plasma half-life. Insulin consists of two chains, the A- and B-chain, which are connected by two disulfide-bridges. To produce a novel kind of Fc-fusion protein we have fused the A-chain as well as the B-chain to Fc-fragments containing either 'knob' or 'hole' mutations. The 'knob-into-hole' technique is frequently used to force heterodimerization of the Fc-domain. Using this approach, we were able to produce different variants of two-chain-insulin-Fc-protein (tcI-Fc-protein) variants. The tcI-Fc-fusion variants retained activity as shown in in vitro assays. Finally, prolonged blood glucose lowering activity was demonstrated in normoglycemic rats. Overall, we describe here the production of novel insulin-Fc-fusion proteins with prolonged times of action.

Expression and purification of the extracellular domains of human glycoprotein VI (GPVI) and the receptor for advanced glycation end products (RAGE) from Rattus norvegicus in Leishmania tarentolae.

Glycosylation is one of the most complex post-translational modifications and may have significant influence on the proper function of the corresponding proteins. Bacteria and yeast are, because of easy handling and cost reasons, the most frequently used systems for recombinant protein expression. Bacteria generally do not glycosylate proteins and yeast might tend to hyperglycosylate. Insect cell- and mammalian cell-based expression systems are able to produce complex N-glycosylation structures but are more complex to handle and more expensive. The nonpathogenic protozoa Leishmania tarentolae is an easy-to-handle alternative expression system for production of proteins requiring the eukaryotic protein folding machinery and post-translational modifications. We used and evaluated the system for the secretory expression of extracellular domains from human glycoprotein VI and the receptor for advanced glycation end products from rat. Both proteins were well expressed and homogeneously glycosylated. Analysis of the glycosylation pattern identified the structure as the conserved core pentasaccharide Man3GlcNac2.

Inhibition of diacylglycerol-sensitive TRPC channels by synthetic and natural steroids.

TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca(2+) signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC(50)s of 3-5 µM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 µM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC(50)s ranging from 6 to 18 µM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy.

Identification of a novel AS160 splice variant that regulates GLUT4 translocation and glucose-uptake in rat muscle cells.

AS160 (AKT substrate of 160 kDa) is an important mediator of GLUT4 (glucose transporter 4) translocation and glucose-uptake in adipocytes and muscle cells. In our study we have identified a novel splice variant of AS160 (variant 2 of AS160, AS160_v2) that lacks exon 11 and 12. The protein is phosphorylated in response to insulin via the PI3K/AKT pathway. Expression of this splice variant in human tissues from different donors was examined with quantitative RT-PCR. Our data reveal a tissue specific distribution pattern of both isoforms with highest overall expression of AS160_v2. To investigate the function of the novel splice variant we established the doxycycline-inducible expression of the protein in a rat myoblast cell line co-expressing GLUT4-myc. In contrast to data reported for the full-length AS160 protein, over expression and activation of transcript variant 2 in this cell line increased GLUT4 translocation and glucose-uptake rates in response to insulin and IGF-1 but not in response to AICAR or metformin. Immunofluorescence based studies indicated a direct association of AS160_v2 with GLUT4 under basal but not under insulin-stimulated conditions. Additionally, over expression of AS160_v2 slightly improved glucose-uptake rates in a model of insulin resistance but was not able to fully prevent induction of insulin resistance. This was accompanied with decreased phosphorylation of AS160_v2 and AKT. Taken together, our data suggest a tissue specific distribution of full-length AS160 and the novel AS160 splice variant (AS160_v2) indicating different functions. In contrast to full-length AS160, transcript variant 2 of AS160 seems to be a novel regulator of glucose transport that positively influences glucose-uptake rates.

Properties of a time-dependent potassium current in pig atrium: evidence for a role of kv1.5 in repolarization.

Cardiac electrical activity is modulated by potassium currents. Pigs have been used for antiarrhythmic drug testing, but only sparse data exist regarding porcine atrial ionic electrophysiology. Here, we used electrophysiological, molecular, and pharmacological tools to characterize a prominent porcine outward K(+) current (I(K,PO)) in atrial cardiomyocytes isolated from adult pigs. I(K,PO) activated rapidly (time to peak at +60 mV; 2.1 +/- 0.2 ms), inactivated slowly (tau(f) = 45 +/- 10; tau(s) = 215 +/- 28 ms), and showed very slow recovery (tau(f) = 1.54 +/- 0.73 s; tau(s) = 7.91 +/- 1.78 s; n = 9; 36 degrees C). Activation and inactivation were voltage-dependent, and current properties were consistent with predominant K(+) conductance. Neurotoxins (heteropodatoxin, hongatoxin, and blood depressing substance) that block K(v)4.x, K(v)1.1, -1.2, -1.3, and -3.4 in a highly selective manner as well as H(2)O(2) and tetraethylammonium, did not affect the current. Drugs with K(v)1.5-blocking properties (flecainide, perhexiline, and the novel atrial-selective antiarrhythmic 2'-{2-(4-methoxyphenyl)-acetylamino-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide; AVE0118) inhibited I(K,PO) (IC(50) of 132 +/- 47, 17 +/- 10, and 1.25 +/- 0.62 microM, respectively). 4-Aminopyridine suppressed the current and accelerated its decay, reducing charge carriage with an IC(50) of 39 +/- 15 microM. Porcine-specific K(v) channel subunit sequences were cloned to permit real-time quantitative reverse transcription-polymerase chain reaction on RNA extracted from isolated cardiomyocytes, which showed much greater abundance of K(v)1.5 mRNA compared with K(v)1.4, K(v)4.2, and K(v)4.3. Action potential recordings showed that I(K,PO) inhibition with 0.1 mM 4-AP delayed repolarization (e.g., action potential duration at -50 mV increased from 45 +/- 9 to 69 +/- 5 ms at 3 Hz; P < 0.05). In conclusion, porcine atrium displays a current that is involved in repolarization, inactivates more slowly than classic transient outward current, is associated with strong K(v)1.5 expression, and shows a pharmacological profile typical of K(v)1.5-dependent currents.