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1.
Biotechnol Bioeng ; 120(1): 125-138, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36226467

RESUMO

The development of biopharmaceutical downstream processes relies on exhaustive experimental studies. The root cause is the poorly understood relationship between the protein structure of monoclonal antibodies (mAbs) and their macroscopic process behavior. Especially the development of preparative chromatography processes is challenged by the increasing structural complexity of novel antibody formats and accelerated development timelines. This study introduces a multiscale in silico model consisting of homology modeling, quantitative structure-property relationships (QSPR), and mechanistic chromatography modeling leading from the amino acid sequence of a mAb to the digital representation of its cation exchange chromatography (CEX) process. The model leverages the mAbs' structural characteristics and experimental data of a diverse set of 21 therapeutic antibodies to predict elution profiles of two mAbs that were removed from the training data set. QSPR modeling identified mAb-specific protein descriptors relevant for the prediction of the thermodynamic equilibrium and the stoichiometric coefficient of the adsorption reaction. The consideration of two discrete conformational states of IgG4 mAbs enabled prediction of split-peak elution profiles. Starting from the sequence, the presented multiscale model allows in silico development of chromatography processes before protein material is available for experimental studies.


Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Cromatografia por Troca Iônica/métodos , Termodinâmica , Imunoglobulina G/química , Anticorpos Monoclonais/química , Adsorção
2.
Biotechnol Bioeng ; 118(8): 2923-2933, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33871060

RESUMO

A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early-phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate. While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time-to-market pressure and an abundance of monoclonal antibodies (mAbs) in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform. This study investigates the influence of amino acid substitutions in the complementarity-determining region (CDR) of a full-length IgG1 mAb on the elution behavior in preparative cation exchange chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared with the wild-type antibody. Furthermore, the substitution of tryptophan with lysine in the H-CDR3 increased charge heterogeneity of the product. A multiscale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing.


Assuntos
Substituição de Aminoácidos , Anticorpos Monoclonais , Imunoglobulina G , Modelos Moleculares , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Cromatografia por Troca Iônica , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética
3.
Nutrients ; 14(19)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36235551

RESUMO

In 2020, charitable food organizations began adopting Healthy Eating Research (HER) nutrition guidelines, which rank individual foods in tiers (e.g., green, yellow, or red) based on each food's nutrient profile. This study aimed to validate this HER tier-ranked system against the Healthy Eating Index-2015 (HEI) and develop a formula to summarize the percentages of tier-ranked foods in an overall nutritional quality index that correlated with HEI. Using secondary data of foods selected by clients in 16 Minnesota food pantries (n = 503 "client carts"), we generated an HEI score and percentages of green, yellow, and red foods for each cart. As validation, we tested the association between HEI scores and the percentages of tier-ranked foods and compared the means of the tier-ranked variables using quintiles of the HEI scores. HEI scores were positively associated with percentages of green foods and negatively associated with percentages of red foods. Next, we used statistical learning to generate weights to maximize the correlation of the tier-ranked variables and the HEI scores and used these weights to create an index. The index demonstrated a moderate-to-strong correlation with HEI and can be used as a single measure to summarize the overall nutritional quality for sets of tier-ranked foods.


Assuntos
Dieta Saudável , Alimentos , Humanos , Política Nutricional , Estado Nutricional , Valor Nutritivo
4.
Bioorg Med Chem ; 18(7): 2602-10, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20299229

RESUMO

Natural products serve as a great reservoir for chemical diversity and are the greatest source for antibacterial agents. Recent discoveries of platensimycin and platencin as inhibitors of bacterial fatty acid biosynthesis enzymes supplied new chemical scaffolds for potential antibacterial agents to overcome resistant pathogens. Discovery of natural congeners augment chemical modification in understanding of structure-activity relationship (SAR). Chemical and biological screening of the extracts led to isolation of three hydroxylated analogs of platencin. The C-12, C-14 and C-15 hydroxylated analogs showed attenuated activities which provided significant understanding of functional tolerance in the diterpenoid portion of the molecule. A truncated and oxidized C-13 natural congener was isolated which suggested direct intermediacy of ent-copalyl diphosphate for the biosynthesis of platensimycins and platencins.


Assuntos
Adamantano/análogos & derivados , Aminobenzoatos/química , Aminofenóis/química , Antibacterianos/química , Compostos Policíclicos/química , Streptomyces/química , Adamantano/química , Adamantano/farmacologia , Aminobenzoatos/farmacologia , Aminofenóis/farmacologia , Antibacterianos/farmacologia , Cromatografia Líquida de Alta Pressão , Fermentação , Haemophilus influenzae/efeitos dos fármacos , Hidroxilação , Indicadores e Reagentes , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Compostos Policíclicos/farmacologia , Espectrofotometria Ultravioleta , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
5.
Bioprocess Biosyst Eng ; 32(4): 443-58, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18853195

RESUMO

A pilot-scale fermentation was developed for an antifungal compound produced by a filamentous fungus. Replacement of galactose with lactose (20-fold cost savings) and a threefold phosphate reduction (15 to 5 g/L) improved productivity 2.5-fold. Addition of supplements--glycine, cobalt chloride, and trace elements--resulted in a further twofold productivity increase, greater process robustness, and less foaming which reduced antifoam addition tenfold (30 to <3 mL/L). Mid-cycle lactose limitations were addressed by raising initial lactose levels (40 to 120 g/L) resulting in another twofold productivity increase. Overall, peak titers increased tenfold from 45 +/- 9 to 448 +/- 39 mg/L, and productivities improved from 3 to 25 mg/L day. Despite its high productivity, process scale up was challenged by high broth viscosity (5,000-6,000 cP at 16.8 s(-1)). Gassed power requirements at the 600 L scale (4.7 kW/1,000 L) exceeded available power at the 15,000 L scale (3.0 kW/1,000 L), and broth transfer to the downstream isolation facility was hindered. Mid-cycle broth dilution with up to five 10 vol% additions of 12 wt% lactose solution or whole medium-reduced viscosity three- to fivefold (1,000-1,500 cP at 16.8 s(-1)), gassed power within scale-up limits (2.5 kW/1,000 L), and peak titer by up to 45%. The process was scaled up to the 15,000 L working volume based on constant aeration rate (vvm) and peak impeller tip speed, raising superficial velocities at similar shear. This strategy maximized mass transfer rates at target gassed power per unit volume levels, and along with controlled broth viscosity, precluded multiple dilution additions. A final titer of 333 mg/L with one dilution addition was achieved, somewhat lower than expected, likely owing to inhibition from some unmeasured volatile compound (not believed to be carbon dioxide) during an extended period of high back-pressure in the early production phase.


Assuntos
Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Engenharia Biomédica , Reatores Biológicos , Meios de Cultura , Fermentação , Galactose/metabolismo , Glicerol/metabolismo , Lactose/metabolismo , Fosfatos/metabolismo , Projetos Piloto , Viscosidade
6.
J Antibiot (Tokyo) ; 60(9): 554-64, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17917238

RESUMO

Thiazolyl peptides are a class of rigid macrocyclic compounds richly populated with thiazole rings. They are highly potent antibiotics but none have been advanced to clinic due to poor aqueous solubility. Recent progress in this field prompted a reinvestigation leading to the isolation of a new thiazolyl peptide, thiazomycin, a congener of nocathiacins. Thiazomycin possesses an oxazolidine ring as part of the amino-sugar moiety in contrast to the dimethyl amino group present in nocathiacin I. The presence of the oxazolidine ring provides additional opportunities for chemical modifications that are not possible with other nocathiacins. Thiazomycin is extremely potent against Gram-positive bacteria both in vitro and in vivo. The titer of thiazomycin in the fermentation broth was very low compared to the nocathiacins I and III. The lower titer together with its sandwiched order of elution presented significant challenges in large scale purification of thiazomycin. This problem was resolved by the development of an innovative preferential protonation based one- and/or two-step chromatographic method, which was used for pilot plant scale purifications of thiazomycin. The isolation and structure elucidation of thiazomycin is herein described.


Assuntos
Actinomycetales/química , Antibacterianos/isolamento & purificação , Peptídeos Cíclicos/isolamento & purificação , Tiazóis/isolamento & purificação , Actinomycetales/classificação , Antibacterianos/química , Cromatografia Líquida/métodos , Fermentação , Bactérias Gram-Positivas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Mutação , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos Cíclicos/química , Solubilidade , Tiazóis/química
7.
J Biosci Bioeng ; 102(4): 251-68, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17116571

RESUMO

Facility experience primarily in drug-oriented fermentation equipment (producing small molecules such as secondary metabolites, bioconversions, and enzymes) and, to a lesser extent, in biologics-oriented fermentation equipment (producing large molecules such as recombinant proteins and microbial vaccines) in an industrial fermentation pilot plant over the past 15 years is described. Potential approaches for equipment design and maintenance, operational procedures, validation/verification testing, medium selection, culture purity/sterility analysis, and contamination investigation are presented, and those approaches implemented are identified. Failure data collected for pilot plant operation for nearly 15 years are presented and best practices for documentation and tracking are outlined. This analysis does not exhaustively discuss available design, operational and procedural options; rather it selectively presents what has been determined to be beneficial in an industrial pilot plant setting. Literature references have been incorporated to provide background and context where appropriate.


Assuntos
Reatores Biológicos/microbiologia , Descontaminação/métodos , Contaminação de Equipamentos/prevenção & controle , Análise de Falha de Equipamento/métodos , Fermentação , Microbiologia Industrial/instrumentação , Manutenção/métodos , Descontaminação/instrumentação , Análise de Falha de Equipamento/instrumentação , Microbiologia Industrial/métodos , Projetos Piloto
8.
J Pediatr Endocrinol Metab ; 29(4): 451-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26669243

RESUMO

BACKGROUND: The metabolic syndrome (MS) in pediatrics is controversial. Rather than a dichotomous scale, a continuous metabolic syndrome score (cMets) has been proposed to evaluate MS. The aim of this study was to evaluate the utility of a cMets score among an ethnically diverse cohort of children with acanthosis nigricans (AN). METHODS: A retrospective chart review was used to extract clinical and laboratory information on a cohort of obese children. Criteria for MS components and the cMets score were established using published guidelines. Multiple linear regression evaluated the effect of AN status on MS and cMets. Fisher's exact test compared the race differential on the presence or absence of MS component disorders. RESULTS: MS diagnosis was non-significant when considering AN status (p=0.554) and ethnicity (p=0.431). Evaluation of the frequency of component disorders, revealed that Caucasians had significantly higher levels of abnormal triglycerides (TG) (35.1 vs. 10.3%; p<0.001), whereas African Americans had significantly higher homeostatic model assessment of insulin resistance (HOMA-IR) scores (51.3% vs. 22.3%; p<0.001). cMets was sensitive to identifying metabolic risk among Caucasians with AN, only (p=0.029). CONCLUSIONS: This study found differences in health risk among an obese, ethnically diverse sample of children. cMets is a more sensitive marker of metabolic change compared with MS, especially when AN status and race are considered. cMets may pose an opportunity for the clinician to evaluate the interaction of health risks on the health status of obese children.


Assuntos
Acantose Nigricans/fisiopatologia , Resistência à Insulina , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/etiologia , Obesidade/complicações , Biomarcadores/análise , Glicemia/análise , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Seguimentos , Hemoglobinas Glicadas/análise , Humanos , Masculino , Prognóstico , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Triglicerídeos/metabolismo
10.
Biotechnol Prog ; 25(1): 176-88, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19205044

RESUMO

An Amycolatopsis fastidiosa culture, which produces the nocathiacin class of antibacterial compounds, was scaled up to the 15,000 L working volume. Lower volume pilot fermentations (600, 900, and 1,500 L scale) were conducted to determine process feasibility at the 15,000 L scale. The effects of inoculum volume, impeller tip speed, volumetric gas flow rate, superficial gas velocity, backpressure, and sterilization heat stress were examined to determine optimal scale-up operating conditions. Inoculum volume (6 vs. 2 vol %) and medium sterilization (R(o) of 68 vs. 92 min(-1)) had no effect on productivity or titer, and higher impeller tip speeds (2.1 vs. 2.9 m/s) had a slight effect (20% decrease). In contrast, higher backpressure, incorporating increased head pressure at the 15,000 L scale (1.2 vs. 0.7 kg/cm(2)) and low gas flow rates (0.25 vs. 0.8 vvm), appeared to be problematic (40-50% decrease). High off-gas CO2 levels were likely reasons for observed lower productivity. Consequently, air flow rate for this 25-fold scale-up (600-15,000 L) was controlled to match off-gas CO2 profiles of acceptable smaller scale batches to maintain levels below 0.5%. The 15,000 L-scale fermentation achieved an expected nocathiacin I titer of 310 mg/L after 7 days. Other on-line data (i.e., pH, oxygen uptake rate, and CO2 evolution rate) and off-line data (i.e., analog production, glucose utilization, ammonium production, and dry cell weight) at the 15,000 L scale also tracked similarly to the smaller scale, demonstrating successful fermentation scale-up.


Assuntos
Actinobacteria/crescimento & desenvolvimento , Actinobacteria/metabolismo , Antibacterianos/metabolismo , Microbiologia Industrial/métodos , Peptídeos/metabolismo , Fermentação , Peptídeos e Proteínas de Sinalização Intercelular , Cinética
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