Application of platform process development approaches to the manufacturing of Mabcalin™ bispecifics.

Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3 L bioreactors and production was scaled-up to up to 1000 L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic.

Retrocyte Display® technology: generation and screening of a high diversity cellular antibody library.

Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).

Redirecting catalysis from proteolysis to perhydrolysis in subtilisin Carlsberg.

Enzyme promiscuity describes the ability of biocatalysts to catalyze conversions beyond their natural reactions. Enzyme engineering to promote side reactions is attractive for synthetic and industrial applications. For instance, a subtilisin Carlsberg protease variant (T58A/L216W) catalyzes in addition to its proteolytic activity the generation of peroxycarboxylic acids from corresponding esters in the presence of hydrogen peroxide. In the current study we used a semi-rational design approach to shift the specificity of subtilisin Carlsberg towards production of peroxycarboxylic acid. Among other identified amino acid substitutions, position Gly165 in the S1 binding pocket provided insights in subtilisin Carlsberg's promiscuity by promoting ester perhydrolysis. Catalytic constants of subtilisin Carlsberg for perhydrolysis of methyl-propionate, methyl-butyrate and methyl-pentanoate were increased up to 3.5-, 5.4- and 5.5-fold, respectively, while proteolysis was decreased up to 100-fold for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate (suc-AAPF-pNA).

Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions.

A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15% of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12% of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.

Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method.

Many ligase-independent cloning methods have been developed to overcome problems of standard restriction cloning such as low transformation efficiency and high background of vector with no insert. Most of these methods are still enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. Thus, with the aim to establish a robust enzyme/ligase-free method, we developed the phosphorothioate-based ligase-independent gene cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution. After optimization of polymerase chain reaction (PCR) and DNA cleavage conditions, PLICing performs competitively with all commercialized methods in terms of handling and transformation efficiency. In addition, PLICing is absolutely sequence independent and surpasses other concepts regarding cloning efficiency given that none of the 240 analyzed clones showed any religation event for three different model genes. A developed fast PLICing protocol does not require any purification step and can be completed within 10 min. Due to its robustness, reliability, and simplicity, PLICing should prove to be a true alternative to other well-established cloning techniques.

Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases.

BACKGROUND: The major peanut allergens are Ara h 1, Ara h 2 and Ara h 6. Proteolytic processing has been shown to be required for the maturation process of Ara h 6. The aim of this study was to examine whether Ara h 2 undergoes proteolytic processing and, if so, whether proteolytic processing influences its ability to bind human immunoglobulin E (IgE). RESULTS: Ara h 2 isolated from peanut extract under conditions of protease inhibition revealed a single additional peak for its two known isoforms (Ara h 2.01 and Ara h 2.02), corresponding to a C-terminally truncated form lacking a dipeptide (RY). Ara h 2 isolated in the absence of protease inhibition, however, yielded two additional peaks, identified as C-terminally truncated forms lacking either a dipeptide (RY) or a single tyrosine residue. The IgE-binding capacity of the Ara h 2 truncated forms was not altered. CONCLUSION: Ara h 2 undergoes proteolytic processing by peanut proteases that involves C-terminal removal of a dipeptide. Hence Ara h 2 isolated from peanut extract is a complex mixture of two isoforms expressed by different genes, Ara h 2.01 and Ara h 2.02, as well as truncated forms generated by the proteolytic processing of these isoforms.

Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant.

One of the main obstacles in employing P450 monooxygenases for preparative chemical syntheses in cell-free systems is their requirement for cofactors such as NAD(P)H. In order to engineer P450 BM3 from Bacillus megaterium for cost-effective process conditions in vitro, a validated medium throughput screening system based on cheap Zn dust as an electron source and Cobalt(III)sepulchrate (Co(III)sep) as a mediator was reported. In the current study, the alternative cofactor system Zn/Co(III)sep was used in a directed evolution experiment to improve the Co(III)sep-mediated electron transfer to P450 BM3. A variant, carrying five mutations (R47F F87A V281G M354S D363H, Table I), P450 BM3 M5 was identified and characterized with respect to its kinetic parameters. P450 BM3 M5 achieved for mediated electron transfer a 2-fold higher k(cat) value and a 3-fold higher catalytic efficiency compared with the starting point mutant P450 BM3 F87A (k(cat): 62 min(-1) compared with 28 min(-1); k(cat)/K(m): 62 microM(-1)min(-1) compared to 19 microM(-1)min(-1)). For obtaining first insights on electron transfer contributions, three reductase-deficient variants were generated. The reductase-deficient mutant of P450 BMP M5 exhibited a catalytic efficiency of 69% and a k(cat) value of 89% of the values obtained for P450 BM3 M5.

Quantification of Art v 1 and Act c 1 being major allergens of mugwort pollen and kiwi fruit extracts in mass-units by ion-exchange HPLC-UV method.

A simple ion-exchange HPLC-UV method was developed for determination of major allergens from mugwort pollen and kiwi fruit extracts in mass-units. The separation of Art v 1 and Act c 1 from other components in the extracts was achieved in one step. The extinction coefficients used in the study were theoretically determined and compared to the extinction coefficients determined by gravimetry. We also reported a close correlation of the major allergen contents with the overall allergenic potency of the extracts determined by inhibition ELISA. This method could be a useful tool for standardization of allergenic extracts for clinical use.