Process development and characterization for integrated continuous bioprocesses-Highlights from N-mAb.

The N-mAb case study was produced by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) to support teaching and learning for both industry and to accelerate adoption of advanced manufacturing process technologies such as integrated continuous bioprocesses (ICB) for mAbs. Similar to the A-mAb case study, N-mAb presents the evolution of an integrated control strategy, from early clinical through process validation and commercial manufacturing with a focus on elements that are unique to integrated continuous bioprocesses. This publication presents a summary of the process design and characterization chapters to allow a greater focus on the unique elements relevant to that phase of development.

Managing integrated continuous bioprocesses in real time: Deviations in product quality.

The N-mAb case study was produced by the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) to support teaching and learning for both industry and regulators around adoption of advanced manufacturing process technologies such as integrated continuous bioprocesses (ICB) for monoclonal antibodies (mAbs). N-mAb presents the evolution of an integrated control strategy, from early clinical through process validation and commercial manufacturing with a focus on elements that are unique to ICB. The entire N-mAb case study is quite comprehensive, therefore this publication presents a summary of the chapter on managing deviations from a state of control in real time. This topic is of critical importance to ICB and is also applicable to batch processes operated at a rapid cadence.

Improved process intermediate stability through the identification and elimination of reactive glycation residues - a monoclonal antibody case study.

The manufacturing of therapeutic biologics can result in a heterogeneous population of charge variants, encompassing many quality attributes which could impact activity and pharmacokinetics. Monitoring the relative abundance of these charge variants to demonstrate process consistency is an expectation of regulatory agencies. Control of the relative abundance of charge variants is also necessary to ensure product comparability across the product lifecycle. We have observed a significant shift in the relative abundance of charged species, as measured by capillary isoelectric focusing, during clarified cell culture fluid holds for several monoclonal antibodies. This lack of stability requires that the hold time for this process intermediate be significantly curtailed, eliminating manufacturing flexibility. We have identified the cause of this shift in relative abundance of charged species as changes in glycation levels, focused predominantly on three conserved, solvent accessible, lysine residues. Mutants of a model protein were generated that show increased charge state stability can be gained by eliminating these reactive lysines. Further, characterization studies were conducted on these mutants to determine the impact to biological activity and stability of the molecule, with no detrimental effects observed. Incorporating this knowledge into the assessments of candidate drugs could allow for the selection of molecules less susceptible to this product degradation pathway, allowing for greater manufacturing flexibility. This process of identifying and removing reactive lysine residues could be useful in the design of drug candidates with improved charge state stability, across a range of modalities.

Kinetic modeling as a tool to understand the influence of cell culture process parameters on the glycation of monoclonal antibody biotherapeutics.

Glycation, the nonenzymatic reaction between the reducing sugar glucose and the primary amine residues on amino acid side chains, commonly occurs in the cell culture supernatant during production of therapeutic monoclonal antibodies (mAbs). While glycation has the potential to impact efficacy and pharmacokinetic properties for mAbs, the most common undesirable impact of glycation is on the distribution of charged species, often a release specification for commercial processes. Existing empirical approaches are usually insufficient to rationalize the effects of cell line and process changes on glycation. To address this gap, we developed a kinetic model for estimating mAb glycation levels during the cell culture process. The rate constant for glycation, including temperature and pH dependence, was estimated by fitting the kinetic model to time-course glycation data from bioreactors operated at different process settings that yielded a wide range of glycation values. The parameter values were further validated by independently estimating glycation rate constants using cell-free incubation studies at various temperatures. The model was applied to another mAb, by re-estimating the activation energy to account for effect of a glycation "hotspot". The model was further utilized to study the role of temperature shift as an approach to reduce glycation levels in the manufacturing process for mAb2. While a downshift in temperature resulted in lowering of glycation levels for mAb2, the model helped elucidate that this effect was caused due to contribution from changes in glucose consumption, mAb secretion and temperature, instead of a direct impact of temperature alone on the kinetic rate of glycation.

CLPTM1L promotes growth and enhances aneuploidy in pancreatic cancer cells.

Genome-wide association studies (GWAS) of 10 different cancers have identified pleiotropic cancer predisposition loci across a region of chromosome 5p15.33 that includes the TERT and CLPTM1L genes. Of these, susceptibility alleles for pancreatic cancer have mapped to the CLPTM1L gene, thus prompting an investigation of the function of CLPTM1L in the pancreas. Immunofluorescence analysis indicated that CLPTM1L localized to the endoplasmic reticulum where it is likely embedded in the membrane, in accord with multiple predicted transmembrane domains. Overexpression of CLPTM1L enhanced growth of pancreatic cancer cells in vitro (1.3-1.5-fold; PDAY7 < 0.003) and in vivo (3.46-fold; PDAY68 = 0.039), suggesting a role in tumor growth; this effect was abrogated by deletion of two hydrophilic domains. Affinity purification followed by mass spectrometry identified an interaction between CLPTM1L and non-muscle myosin II (NMM-II), a protein involved in maintaining cell shape, migration, and cytokinesis. The two proteins colocalized in the cytoplasm and, after treatment with a DNA-damaging agent, at the centrosomes. Overexpression of CLPTM1L and depletion of NMM-II induced aneuploidy, indicating that CLPTM1L may interfere with normal NMM-II function in regulating cytokinesis. Immunohistochemical analysis revealed enhanced staining of CLPTM1L in human pancreatic ductal adenocarcinoma (n = 378) as compared with normal pancreatic tissue samples (n = 17; P = 1.7 × 10(-4)). Our results suggest that CLPTM1L functions as a growth-promoting gene in the pancreas and that overexpression may lead to an abrogation of normal cytokinesis, indicating that it should be considered as a plausible candidate gene that could explain the effect of pancreatic cancer susceptibility alleles on chr5p15.33.

SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient- and exercise-induced stress.

AIMS: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. RESULTS: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP(K139) directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. INNOVATION: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. CONCLUSION: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins.

BRD4 short isoform interacts with RRP1B, SIPA1 and components of the LINC complex at the inner face of the nuclear membrane.

Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.

The UBIAD1 prenyltransferase links menaquinone-4 [corrected] synthesis to cholesterol metabolic enzymes.

Schnyder corneal dystrophy (SCD) is an autosomal dominant disease characterized by germline variants in UBIAD1 introducing missense alterations leading to deposition of cholesterol in the cornea, progressive opacification, and loss of visual acuity. UBIAD1 was recently shown to synthesize menaquinone-4 (MK-4, vitamin K(2) ), but causal mechanisms of SCD are unknown. We report a novel c.864G>A UBIAD1 mutation altering glycine 177 to glutamic acid (p.G177E) in six SCD families, including four families from Finland who share a likely founder mutation. We observed reduced MK-4 synthesis by UBIAD1 altered by SCD mutations p.N102S, p.G177R/E, and p.D112N, and molecular models showed p.G177-mutant UBIAD1 disrupted transmembrane helices and active site residues. We show UBIAD1 interacts with HMGCR and SOAT1, enzymes catalyzing cholesterol synthesis and storage, respectively, using yeast two-hybrid screening and immunoprecipitation. Docking simulations indicate cholesterol binds to UBIAD1 in the substrate-binding cleft and substrate-binding overlaps with GGPP binding, an MK-4 substrate, suggesting potential competition between these metabolites. Impaired MK-4 synthesis is a biochemical defect identified in SCD suggesting UBIAD1 links vitamin K and cholesterol metabolism through physical contact between enzymes and metabolites. Our data suggest a role for endogenous MK-4 in maintaining cornea health and visual acuity.

Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes.

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.

A method of HIV-1 inactivation compatible with antibody-based depletion of abundant proteins from plasma.

We have examined several methods, including heat treatment and treatment with detergents, to inactivate HIV-1 present in plasma to be depleted of abundant proteins utilizing an antibody-based technology. Treatment with Triton X-100 was not compatible with abundant protein depletion with an antibody column and heat treatment alters the composition of the plasma proteome. However, treatment with 1.2% N-octylglucoside for 5 min completely inhibited HIV-1 infectivity. The detergent was easily removed through buffer exchange, and this treatment had no discernable effect on protein depletion.

A bacterial one-hybrid selection system for interrogating zinc finger-DNA interactions.

We have developed two bacterial one-hybrid systems for interrogating and selecting zinc finger-DNA interactions. Our systems utilize two plasmids: a zinc finger-plasmid containing the gene for the zinc finger fused to a fragment of the alpha subunit of RNA polymerase and a reporter plasmid where the zinc finger-binding site is located upstream of a reporter gene-either the gene encoding the green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT). Binding of the zinc finger domain to the target binding site results in a 10-fold increase in chloramphenicol resistance with the CAT reporter and an 8- to 22-fold increase in total cell fluorescence with the GFP reporter. The CAT reporter allows for sequence specific zinc fingers to be isolated in a single selection step whereas the GFP reporter enables quantitative evaluation of libraries using flow cytometry and theoretically allows for both negative and positive selection. Both systems have been used to select for zinc fingers that have affinity for the motif 5'-GGGGCAGAA-3' from a library of approximately 2 x 10(5) variants. The systems have been engineered to report on zinc finger-DNA binding with dissociation constants less than about 1 microM in order to be most applicable for evaluating binding specificity in an in vivo setting.

Mathematical expressions useful in the construction, description and evaluation of protein libraries.

The creation of protein libraries by random mutagenesis and cassette mutagenesis has proven to be a successful method of protein engineering. Appropriate statistical analysis is important for the proper construction of these libraries and even more important for the interpretation of data from these libraries. We present simple mathematical expressions useful in the creation and evaluation of such libraries. These equations are useful in estimating the distribution of mutations, the degeneracy of the library and the frequency of a particular clone in the library. In addition, general equations addressing the probability that a particular clone is in a library, the probability that a library is complete, and as the consequences of retransformation of the library on these probabilities are presented.