Impact of photo-oxidation on long term storage of affinity chromatography media used in multi-specific antibody manufacturing processes.

Large scale manufacture of complex biotherapeutic formats such as multi-specific antibodies can require development of custom biomanufacturing platforms, particularly for purification processes. Substantial advances in affinity chromatography media have allowed monoclonal antibody-like processes for these formats, and simplified process development to enable fast speed to the clinic. Thorough assessment of chromatography media performance and stability is critical to ensure robust operation and consistent product quality over repeated cycles throughout its lifetime. However, evaluation of repeated cycling and extended storage for chromatography media is resource consuming, which typically delays rigorous study to later development stages and often is acquired through increased operational experience. These areas can present quality risks if not properly understood. In this work, a class of affinity chromatography media employing camelid antibody-fragment ligands were evaluated for extended storage in benzyl alcohol solution as an alternative to ethanol storage. Initial laboratory studies revealed resin discoloration after 12 months of exposure to ambient light at room temperature. Resin photo-stress studies confirmed light exposure as the root cause, with benzyl alcohol storage conditions producing a substantially greater degree of discoloration compared to ethanol. Extreme photo-stress over the course of 7 days was also found to negatively impact resin dynamic binding capacities, with more severe declines observed with benzyl alcohol storage conditions. Binding capacity loss of 54 % was observed for photo-stressed CaptureSelect Kappa XP compared to control conditions. Addition of antioxidants reduced or eliminated resin discoloration during photo-stress, indicating that benzyl alcohol storage accelerates photo-oxidation of the affinity chromatography ligands. The addition of l-methionine to benzyl alcohol solution prevented resin discoloration and maintained a dynamic binding capacity of 41 g/L for CaptureSelect Kappa XP even after extreme photo-stress. Of practical importance, a study of LambdaFabSelect resin used and stored in benzyl alcohol solution, under recommended conditions (2-8 °C storage, protected from light) in an internal GMP facility over a period of three years, showed no impact to resin color, performance, or product quality.

Leveraging light chain binding avidity for control of mispaired byproducts during production of asymmetric bispecific antibodies.

Many biotherapeutic formats leverage antibody light chain affinity chromatography to enable robust manufacturing processes and to streamline process development. These include multi-specific antibody and antibody fragment platforms which are often designed for specific capture purification methods that can provide efficient removal of commonly expressed product-related impurities. Recently, several accounts of product-related impurity separation by leveraging binding avidity during affinity chromatography have been described in the literature. However, a more comprehensive evaluation of avidity-based separations, particularly for light chain affinity media with specificity for constant regions of antibody light chains, is valuable for development of emerging multi-specific and fragment antibody formats. Results in this work demonstrate the capability of camelid antibody-based light chain affinity media to separate asymmetric bispecific antibody heterodimers from impurities possessing more than one light chain of the same class that the media binds to, including mispaired variants, aggregates, and fragment impurities. Largest resolution for respective mispaired species were provided by CaptureSelect KappaXP and LambdaXP chromatography media. The addition of elution modifiers provided increased impurity separation, with CaptureSelect KappaXP requiring up to 500 mM concentrations of elution modifiers to produce substantial improvements to resolution, and LambdaXP showing much higher sensitivity. Isocratic elution methods developed for lambda light chain affinity chromatography media provided near complete removal of mispaired variants, and substantial removal of aggregates and fragment impurities. Addition of just 20 mM of elution modifiers such as NaCl are shown to drive increased binding strength and separation of heterodimer species from impurities on CaptureSelect LambdaXP. These results provide scalable and transferable methods for product-related impurity control for various biotherapeutic modalities by lambda light chain affinity chromatography.

Identification of compendial nonionic detergents for the replacement of Triton X-100 in bioprocessing.

We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×-100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real-world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno-associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X-100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.

Development and scale-up of a commercial fed batch refolding process for an anti-CD22 two chain immunotoxin.

We describe the development and scale-up of a novel two chain immunotoxin refolding process. This work provides a case study comparing a clinical manufacturing process and the commercial process developed to replace it. While the clinical process produced high quality material, it suffered from low yield and high yield variability. A systematic approach to process development and understanding led to a number of improvements that were implemented in the commercial process. These include a shorter inclusion body recovery process, limiting the formation of an undesired deamidated species and the implementation of fed batch dilution refolding for increased refold titers. The use of a combination of urea, arginine and DTT for capture column cleaning restored the binding capacity of the capture step column and resulted in consistent capture step yields compared to the clinical process. Scalability is shown with data from 250 L and 950 L scale refolding processes. Compared to the clinical process it replaces, the commercial process demonstrated a greater than fivefold improvement in volumetric productivity at the 950 L refolding scale.

Recovering corn germ enriched in recombinant protein by wet-fractionation.

Corn wet-fractionation processes (quick-germ fractionation and traditional wet milling) were evaluated as means of recovering fractions rich in recombinant collagen-related proteins that were targeted for expression in the germ (embryo) of transgenic corn. Transgenic corn lines accumulating a recombinant full-length human collagen type-I-alpha-1 (full-length rCIalpha1) or a 44-kDa rCIalpha1 fragment targeted for seed expression with an embryo-specific promoter were used. Factors to consider in efficient recovery processes are the distribution of the peptides among botanical parts and process recovery efficiency. Both recombinant proteins were distributed 62-64% in germ comprising about 8.6% of the dry grain mass; 34-38% in the endosperm comprising 84% of the dry grain mass; 1.7% in the pericarp comprising about 5% of the dry mass; and 1% in the tip-cap comprising 1.5-2% of the dry mass. The quick-germ method employed a short steeping period either in water or SO(2)-lactic acid solution followed by wet-milling degermination to recover a germ-rich fraction. Of the total recombinant protein expressed in germ, the quick-germ process recovered 40-43% of the total recombinant protein within 6-8% of the corn mass. The traditional corn wet-milling process produced higher purity germ but with lower recovery (24-26%) of the recombinant protein. The two quick-germ methods, using water alone or SO(2)-lactic acid steeping, did not substantially differ in rCIalpha1 recovery, and the quick-germ processes recovered germ with less leaching and proteolytic losses of the recombinant proteins than did traditional wet milling. Thus, grain fractionation enriched the recombinant proteins 6-fold higher than that of unfractionated kernels. Such enrichment may improve downstream processing efficiency and enable utilizing the protein-lean co-products to produce biofuels and biorenewable chemicals by fermenting the remaining starch-rich fractions.