A cross-industry forum on benchmarking critical quality attribute identification and linkage to process characterization studies.

Identification of Critical Quality Attributes (CQAs) and subsequent characterization in process development studies are the key elements of quality by design (QbD) for biopharmaceutical products. Since the inception of ICH Q8R2, several articles have been published on approaches to conducting CQA risk assessments as well as the application to process understanding. A survey was conducted by multiple companies participating in an International Consortium working group on the best practices for identifying CQAs with linkages to process characterization (PC) studies. The results indicate that the companies surveyed are using similar approaches/timing to identify CQAs during process development. Consensus was also observed among the companies surveyed with approaches to linkage of CQAs to process characterization studies leading to impact to control strategies and lifecycle management.

Evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation.

Inclusion of a detergent in protein biotherapeutic purification processes is a simple and very robust method for inactivating enveloped viruses. The detergent Triton X-100 has been used for many years and is part of the production process of several commercial therapeutic proteins. However, recent ecological studies have suggested that Triton X-100 and its break-down products can potentially behave as endocrine disrupters in aquatic organisms, raising concerns from an environmental impact perspective. As such, discharge of Triton X-100 into the waste water treatment plants is regulated in some jurisdictions, and alternative detergents for viral inactivation are required. In this work, we report on the identification and evaluation of more eco-friendly detergents as viable replacements for Triton X-100. Five detergent candidates with low to moderate environmental impact were initially identified and evaluated with respect to protein stability, followed by proof-of-concept virus inactivation studies using a model enveloped virus. From the set of candidates lauryldimethylamine N-oxide (LDAO) was identified as the most promising detergent due to its low ecotoxicity, robust anti-viral activity (LRV >4 at validation set-point conditions with X-MuLX), and absence of any negative impact on protein function. This detergent exhibited effective and robust virus inactivation in a broad range of protein concentrations, solution conductivities, pHs, and in several different cell culture fluid matrices. The only process parameter which correlated with reduced virus inactivation potency was LDAO concentration, and then only when the concentration was reduced to below the detergent's critical micelle concentration (CMC). Additionally, this work also demonstrated that LDAO was cleared to below detectable levels after Protein A affinity chromatography, making it suitable for use in a platform process that utilizes this chromatographic mode for protein capture. All these findings suggest that LDAO may be a practical alternative to Triton X-100 for use in protein therapeutic production processes for inactivating enveloped viruses. Biotechnol. Bioeng. 2017;114: 813-820. © 2016 Wiley Periodicals, Inc.

Evaluation of a novel methacrylate-based Protein A resin for the purification of immunoglobulins and Fc-fusion proteins.

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc-fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline-tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure-flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure-flow experiments in lab-scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up.

Production and purification of TAL6003: a novel version of the protease plasmin.

TAL6003 is an engineered form of human plasmin under development for thrombolytic therapy. TAL6003 (38.2kDa) contains nine disulfide bonds distributed within and between its two functional domains, a 25.2 kDa serine protease domain linked to a 13.0 kDa kringle I domain; kringles 2-5 present in native human plasmin have been deleted from this plasmin molecule. TAL6003 is expressed as its zymogen in Escherichia coli and is harvested in inclusion bodies. Following solubilization of inclusion bodies with urea as the chaotrope and glutathione as the reducing agent, this zymogen is refolded by dilution to a final concentration of 0.5mg/ml, with a yield of 48% (relative to total zymogen). Refolded TAL6003 zymogen is filtered, diafiltered, and filtered again prior to capture and purification on SP Sepharose, which is highly effective in removing host-cell protein. Subsequent affinity purification on ECH-Lysine Sepharose serves to capture polypeptide chains containing correctly refolded kringle 1 domain, which is the locus of the molecule's lysine-binding site, and to further eliminate host-cell protein. TAL6003 zymogen eluted from the ECH-Lysine Sepharose column is activated to TAL6003 with streptokinase, with an activity yield of approximately 80%. Proteolytically active TAL6003 is stripped of streptokinase by passage through an anion-exchange (Q) membrane and is then affinity purified on Benzamidine Sepharose, which serves to remove unreacted TAL6003 zymogen and proteolytically degraded TAL6003. An ultrafiltration/diafiltration step, to concentrate and to formulate TAL6003, completes the purification process. The final product exhibited a specific activity of close to unity and high purity by several relevant criteria.

Understanding the mechanism of beta-sheet folding from a chemical and biological perspective.

Perturbing the structure of the Pin1 WW domain, a 34-residue protein comprised of three beta-strands and two intervening loops has provided significant insight into the structural and energetic basis of beta-sheet folding. We will review our current perspective on how structure acquisition is influenced by the sequence, which determines local conformational propensities and mediates the hydrophobic effect, hydrogen bonding, and analogous intramolecular interactions. We have utilized both traditional site-directed mutagenesis and backbone mutagenesis approaches to alter the primary structure of this beta-sheet protein. Traditional site-directed mutagenesis experiments are excellent for altering side-chain structure, whereas amide-to-ester backbone mutagenesis experiments modify backbone-backbone hydrogen bonding capacity. The transition state structure associated with the folding of the Pin1 WW domain features a partially H-bonded, near-native reverse turn secondary structure in loop 1 that has little influence on thermodynamic stability. The thermodynamic stability of the Pin1 WW domain is largely determined by the formation of a small hydrophobic core and by the formation of desolvated backbone-backbone H-bonds enveloped by this hydrophobic core. Loop 1 engineering to the consensus five-residue beta-bulge-turn found in most WW domains or a four-residue beta-turn found in most beta-hairpins accelerates folding substantially relative to the six-residue turn found in the wild type Pin1 WW domain. Furthermore, the more efficient five- and four-residue reverse turns now contribute to the stability of the three-stranded beta-sheet. These insights have allowed the design of Pin1 WW domains that fold at rates that approach the theoretical speed limit of folding.

Synthesis, structure, and activity of diclofenac analogues as transthyretin amyloid fibril formation inhibitors.

Twelve analogues of diclofenac (1), a nonsteroidal antiinflammatory drug and known inhibitor of transthyretin (TTR) amyloid formation, were prepared and evaluated as TTR amyloid formation inhibitors. High activity was exhibited by five of the compounds. Structure-activity relationships reveal that a carboxylic acid is required for activity, but changes in its position as well as the positions of other substituents are tolerated. High-resolution X-ray crystal structures of four of the active compounds bound to TTR were obtained. These demonstrate the significant flexibility with which TTR can accommodate ligands within its two binding sites.