Comparing nitric acid treatment and microwave digestion for efficiency of metal extraction from bioprocess samples.

Metal ions may act as enzyme cofactors and influence the kinetics of biochemical reactions that may also influence the biological production of therapeutic proteins and quality attributes such as glycosylation. Because sample preparation is a significant step in the reliable analysis of metals, we compared two sample preparation procedures for metal analysis of bioreactor culture media samples by ICP-MS: (i) samples were diluted in 2 % nitric acid (treatment with nitric acid, TNA); and (ii) samples were mixed with equal volume of 5 % nitric acid and closed vessel digestion was performed in a microwave (closed vessel digestion, CVD). In the comparison of extraction efficiencies between TNA and CVD procedures, CVD showed better extraction for Ca and Cu among bulk metals (∼30 %) and for Ni among the trace metals (∼65 %) for the bioreactor broth supernatant samples.

Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture.

The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA).

Single in-line biomass probe detects CHO cell growth by capacitance and bacterial contamination by conductivity in bioreactor.

Background: Real time process data facilitates timely decisions, enables better process control, and can increase quality assurance. Biological drugs (mol. Wt.

Zinc supplementation improves the harvest purity of β-glucuronidase from CHO cell culture by suppressing apoptosis.

The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-μM concentrations to determine the impact on the production and quality of β-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system.

Differential effects of bioreactor process variables and purification on the human recombinant lysosomal enzyme β-glucuronidase produced from Chinese hamster ovary cells.

β-Glucuronidase is a lysosomal enzyme and a molecular model of a class of therapeutics approved as enzyme replacement therapies for lysosomal storage diseases. Understanding the effect of bioreactor process variables on the production and quality of the biologics is critical for maintaining quality and efficacy of the biotherapeutics. Here, we have investigated the effect of three process variables, in a head-to-head comparison using a parallel bioreactor system (n = 8), namely 0.

An ICP-MS platform for metal content assessment of cell culture media and evaluation of spikes in metal concentration on the quality of an IgG3:κ monoclonal antibody during production.

Metal ions can be enzyme cofactors and can directly influence the kinetics of biochemical reactions that also influence the biological production and quality attributes of therapeutic proteins, such as glycan formation and distribution. However, the concentrations of metals in commercially available chemically defined media can range from 1 to 25,000 ppb. Because such concentration changes can impact cell growth, manufacturing yield and product quality the alteration/fluctuation in media composition should be well controlled to maintain product quality.

Direct quantification of protein glycan phosphorylation.

Phosphorylation is an important post-translational modification (PTM) of proteins and a critical quality attribute for protein therapeutics, especially if it is required for protein function or sub-cellular targeting. Most current methods to quantify phosphorylation are time-consuming, indirect, or require specific instrumentation and technical skills. Here, we report the adaptation of a phosphate-specific binding dye and common laboratory instruments for quantification of relative amounts of phosphorylated glycans as well as phosphorylation of amino acid residues on the backbones of proteins.

An improved purification method for the lysosomal storage disease protein β-glucuronidase produced in CHO cells.

Human β-glucuronidase (GUS; EC 3.2.1.

Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant β-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs.

Nuclear-cytoplasmic localization of acetyl coenzyme a synthetase-1 in the rat brain.

Acetyl coenzyme A synthetase-1 (AceCS1) catalyzes the synthesis of acetyl coenzyme A from acetate and coenzyme A and is thought to play diverse roles ranging from fatty acid synthesis to gene regulation. By using an affinity-purified antibody generated against an 18-mer peptide sequence of AceCS1 and a polyclonal antibody directed against recombinant AceCS1 protein, we examined the expression of AceCS1 in the rat brain. AceCS1 immunoreactivity in the adult rat brain was present predominantly in cell nuclei, with only light to moderate cytoplasmic staining in some neurons, axons, and oligodendrocytes.

Metabolic acetate therapy improves phenotype in the tremor rat model of Canavan disease.

Genetic mutations that severely diminish the activity of aspartoacylase (ASPA) result in the fatal brain dysmyelinating disorder, Canavan disease. There is no effective treatment. ASPA produces free acetate from the concentrated brain metabolite, N-acetylaspartate (NAA).

Methamphetamine-induced neuronal protein NAT8L is the NAA biosynthetic enzyme: implications for specialized acetyl coenzyme A metabolism in the CNS.

N-acetylaspartate (NAA) is a concentrated, neuron-specific brain metabolite routinely used as a magnetic resonance spectroscopy marker for brain injury and disease. Despite decades of research, the functional roles of NAA remain unclear. Biochemical investigations over several decades have associated NAA with myelin lipid synthesis and energy metabolism.

Antipsychotic drugs increase N-acetylaspartate and N-acetylaspartylglutamate in SH-SY5Y human neuroblastoma cells.

N-Acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) are related neuronal metabolites associated with the diagnosis and treatment of schizophrenia. NAA is a valuable marker of neuronal viability in magnetic resonance spectroscopy, a technique which has consistently shown NAA levels to be modestly decreased in the brains of schizophrenia patients. However, there are conflicting reports on the changes in brain NAA levels after treatment with antipsychotic drugs, which exert their therapeutic effects in part by blocking dopamine D(2) receptors.

N-acetylaspartate synthesis in the brain: mitochondria vs. microsomes.

Several reports during the last three decades have indicated that biosynthesis of N-acetylaspartate (NAA) occurs primarily in the mitochondria. But a recent report by Lu et al. in this journal [2004; 122: 71-78] and subsequent two reports that cited those data suggested a predominant microsomal localization of the NAA biosynthetic enzyme, which is surprising in view of what is known about the biological functions of NAA.

Mutational analysis of aspartoacylase: implications for Canavan disease.

Mutations that result in near undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-l-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A.

N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology.

The brain is unique among organs in many respects, including its mechanisms of lipid synthesis and energy production. The nervous system-specific metabolite N-acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A in neurons, appears to be a key link in these distinct biochemical features of CNS metabolism. During early postnatal central nervous system (CNS) development, the expression of lipogenic enzymes in oligodendrocytes, including the NAA-degrading enzyme aspartoacylase (ASPA), is increased along with increased NAA production in neurons.

Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators.

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both.

Aspartoacylase is a regulated nuclear-cytoplasmic enzyme.

Mutations in the gene for aspartoacylase (ASPA), which catalyzes deacetylation of N-acetyl-L-aspartate in the central nervous system (CNS), result in Canavan Disease, a fatal dysmyelinating disease. Consistent with its role in supplying acetate for myelin lipid synthesis, ASPA is thought to be cytoplasmic. Here we describe the occurrence of ASPA within nuclei of rat brain and kidney, and in cultured rodent oligodendrocytes.

Canavan disease and the role of N-acetylaspartate in myelin synthesis.

Canavan disease (CD) is an autosomal-recessive neurodegenerative disorder caused by inactivation of the enzyme aspartoacylase (ASPA, EC 3.5.1.

Progress toward acetate supplementation therapy for Canavan disease: glyceryl triacetate administration increases acetate, but not N-acetylaspartate, levels in brain.

Canavan disease (CD) is a fatal genetic neurodegenerative disorder caused by mutations in the gene for aspartoacylase, an enzyme that hydrolyzes N-acetylaspartate (NAA) into L-aspartate and acetate. Because aspartoacylase is localized in oligodendrocytes, and NAA-derived acetate is incorporated into myelin lipids, we hypothesize that an acetate deficiency in oligodendrocytes is responsible for the pathology in CD, and we propose acetate supplementation as a possible therapy. In our preclinical efforts toward this goal, we studied the effectiveness of orally administered glyceryl triacetate (GTA) and calcium acetate for increasing acetate levels in the murine brain.

Defective N-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan's disease.

Canavan's disease (CD) is a fatal, hereditary disorder of CNS development that has been linked to mutations in the gene for the enzyme aspartoacylase (ASPA) (EC 3.5.1.

SH-SY5Y neuroblastoma cells: a model system for studying biosynthesis of NAAG.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide that is thought to modulate neurotransmitter release through pre-synaptic mGluR3 receptors. Despite years of research into NAAG biochemistry, almost nothing is known about NAAG biosynthesis. To date, NAAG biosynthesis has only been demonstrated conclusively in explanted animal neural tissues, including frog retina, rat dorsal root ganglia and crayfish nerve cord, but not in human cells or tissues.

Immunohistochemical localization of aspartoacylase in the rat central nervous system.

Aspartoacylase (ASPA; EC 3.5.1.