Development of a novel affinity chromatography resin for platform purification of lambda fabs.

Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established.

Development of a fermentation process based on a defined medium for the production of pregallidermin, a nontoxic precursor of the lantibiotic gallidermin.

In this work, a defined medium was developed and optimized for the mutant strain Staphylococcus gallinarum DeltaP, which produces pregallidermin (PGDM), a nontoxic precursor of the lantibiotic gallidermin (GDM). The availability of a defined medium is a prerequisite for a rational process development and the investigation of medium effects on final product concentration, yield, and volumetric productivity. We identified four vitamins and three metal ions as essential for growth and PGDM production with S. gallinarum DeltaP. The strain was capable of growing without any added amino acids, but the addition of proline had a strong growth-stimulatory effect. The concentrations of all essential compounds were balanced in a continuous culture using a medium-shift technique. Based on this balanced medium, a fed-batch process was developed in which S. gallinarum DeltaP was grown up to a biomass concentration of 67 g l(-1) and produced 1.95 g l(-1) PGDM, equivalent to 0.57 mM. In the fermentation broth, we identified other GDM precursors in addition to those with a 12 or 14-amino-acid-long leader peptide that had been observed previously. Including those precursors with shorter leader sequences, the final concentration would correspond to 0.69 mM. In molar terms, this represents a roughly fourfold or fivefold increase, respectively, over established, complex medium-based gallidermin production processes (Kempf et al. 2000). With the same medium and feed protocol, the maximum concentration of mature GDM produced by wild-type S. gallinarum Tü 3928 was only 0.08 mM.

Development of a high cell-density protocol for the production of pregallidermin, a non-toxic precursor of the lantibiotic gallidermin.

Gallidermin, produced by Staphylococcus gallinarum Tü 3928, is a type-A lantibiotic with potential for the treatment of multidrug-resistant infections from Gram-positive pathogens such as methicillin-resistant S. aureus. In order to eliminate product inhibition as a reason for so far very modest product titers in S. gallinarum cultivations, we recently developed a novel two-stage production strategy based on the production of a non-toxic gallidermin precursor - pregallidermin - by an engineered strain and the subsequent conversion of the precursor to gallidermin in a separate step. This directed our efforts to the identification and alleviation of cultivation constraints for the engineered strain in fed-batch cultivations based on complex media supplemented with carbon sources, reasoning that extending the biomass production phase would lead to an extended pregallidermin production and higher titers. Substantial accumulation of acetate occurred in fed-batch cultivations with either maltose or glycerol - but not succinate - as an additional carbon source. Reductions in feeding rate to limit acetate accumulation led in turn to increased product degradation. Based on these observations, we developed an optimized exponential feeding strategy that allowed the process to reach a biomass concentration of 120gL(-1) and a product concentration of 1.23gL(-1) pregallidermin, corresponding to 0.780gL(-1) mature gallidermin, a 2.5-fold increase over previous processes.