Application of the Protein Maker as a platform purification system for therapeutic antibody research and development.

Within the research and development environment, higher throughput, parallelized protein purification is required for numerous activities, from small scale purification of monoclonal antibodies (mAbs) and antibody fragments for in vitro and in vivo assays to process development and optimization for manufacturing. Here, we describe specific applications and associated workflows of the Protein Maker liquid handling system utilized in both of these contexts. To meet the requirements for various in vitro assays, for the identification and validation of new therapeutic targets, small quantities of large numbers of purified antibodies or antibody fragments are often required. Reducing host cell proteins (HCP) levels following capture with Protein A by evaluating various wash buffers is an example of how parallelized protein purification can be leveraged to improve a process development outcome. Stability testing under various conditions of in-process intermediates, as an example, the mAb product from a clarified harvest, requires parallelized protein purification to generate concurrent samples for downstream assays. We have found that the Protein Maker can be successfully utilized for small-to-mid scale platform purification or for process development applications to generate the necessary purified protein samples. The ability to purify and buffer exchange up to 24 samples in parallel offers a significant reduction in time and cost per sample compared to serial purification using a traditional FPLC system. By combining the Protein Maker purification system with a TECAN Freedom EVO liquid handler for automated buffer exchange we have created a new, integrated platform for a variety of protein purification and process development applications.

Confinement of caspase-12 proteolytic activity to autoprocessing.

Caspase-12 is a dominant-negative regulator of caspase-1 (IL-1beta-converting enzyme) and an attenuator of cytokine responsiveness to septic infections. This molecular role for caspase-12 appears to be akin to the role of cFLIP in regulating caspase-8 in the extrinsic cell death pathway; however, unlike cFLIP/Usurpin, we demonstrate here that caspase-12 is catalytically competent. To examine these catalytic properties, rat caspase-12 was cloned, and the recombinant enzyme was used to examine the cleavage of macromolecular and synthetic fluorogenic substrates. Although caspase-12 could mediate autoproteolytic maturation of its own proenzyme, in both cis and trans, it was not able to cleave any other polypeptide substrate, including other caspase proenzymes, apoptotic substrates, cytokine precursors, or proteins in the endoplasmic reticulum that normally undergo caspase-mediated proteolysis. The dearth of potential substrates for caspase-12 also was confirmed by whole-cell diagonal-gel analysis. Autolytic cleavage within the caspase-12 proenzyme was mapped to a single site at the large-small subunit junction, ATAD(319), and this motif was recognized by caspase-12 when incorporated into synthetic fluorogenic substrates. The specific activity of caspase-12 with these substates was several orders of magnitude lower than caspases-1 and -3, highlighting its relative catalytic paucity. In intact cells, caspase-12 autoproteolysis occurred in the inhibitory complex containing caspase-1. We propose that the proteolytic activity of caspase-12 is confined to its own proenzyme and that autocleavage within the caspase-1 complex may be a means for temporal limitation of the inhibitory effects of caspase-12 on proinflammatory cytokine maturation.

Enzyme replacement therapy for murine hypophosphatasia.

INTRODUCTION: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment. MATERIALS AND METHODS: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2-/-), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, microCT, and histomorphometry. RESULTS: Akp2-/- mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. CONCLUSIONS: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2-/- mice.

Human recombinant endopeptidase PHEX has a strict S1' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein.

The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o -aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor-acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S(1)' subsite, with a strong preference for aspartate. Subsites S(2)', S(1) and S(2) exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P(2)', or valine, isoleucine or histidine in P(1) precluded hydrolysis of the substrate by the enzyme. The peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P(2) to P(2)' positions, was hydrolysed by PHEX at the N-terminus of aspartate with a k(cat)/ K(m) of 167 mM(-1) x s(-1). In addition, using quenched fluorescence peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein sequences flanked by Abz and N -(2,4-dinitrophenyl)ethylenediamine, we showed that these physiologically relevant proteins are potential PHEX substrates. Finally, our results clearly indicate that PHEX does not have neprilysin-like substrate specificity.