Design of salt‐tolerant membrane adsorbers for viral clearance

Strong anion exchange chromatography has frequently been employed as a viral clearance step during downstream processing of biological therapeutics. When challenged with viruses having only slightly acidic isoelectric points, the performance of the anion exchange operation becomes highly dependent on the buffer salt concentration, with the virus log reduction value (LRV) dropping dramatically in buffers with 50–150 mM salt. In this work, a series of anion exchange membrane adsorbers utilizing alternative ligand chemistries instead of the traditional quaternary amine (Q) ligand have been developed that overcome this limitation. Four different ligands (agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide, and polyethyleneimine) achieved >5 LRV of bacteriophage ΦX174 (pI ～ 6.7) at pH 7.5 and up to 150 mM salt, compared to 0 LRV for the Q ligand. By evaluating structural derivatives of the successful ligands, three factors were identified that contributed to ligand salt tolerance: ligand net charge, ligand immobilization density on the membrane, and molecular structure of the ligand‐binding group. Based on the results of this study, membrane adsorbers that incorporate alternative ligands provide a more robust and salt tolerant viral clearance‐processing step compared to traditional strong anion exchange membrane adsorbers. Biotechnol. Bioeng. 2009;103: 920–929. © 2009 Wiley Periodicals, Inc.     

A case study in converting disposable process scouting devices into disposable bioreactors as a future bioprocessing tool

In this study, we perform mass transfer characterization (k_La) on a novel mechanically driven/stirred Process Scouting Device, PSD, (SuperSpinner D 1000®, SSD) and demonstrate that this novel device can be viewed as disposable bioreactor. Using patch‐based optical sensors, we were able to monitor critical cell culture environmental conditions such as dissolved oxygen (DO) and pH in SSD for comparison to a 1 L standard spinner (SS) flask. We also coupled these mass transfer studies with mixing time studies where we observed relative high mixing times (5.2 min) that are typically observed in production scale bioreactors. Decreasing the mixing time 3.5‐fold resulted in 30% increase in k_La (from 2.3 to 3.0 h^?1) and minimum DO level increased from 0% to 20% for our model hybridoma cell line. Finally, maximum viable cell density and protein titer stayed within ±20% of historical data, from our standard 5 L stirred bioreactor (Biostat®) operated under active DO control. Biotechnol. Bioeng. 2012; 109: 2790–2797. © 2012 Wiley Periodicals, Inc.     

Monoclonal antibody capture and viral clearance by cation exchange chromatography

Traditionally, post-production culture harvest capture of therapeutic monoclonal antibodies (mAbs) is performed using Protein A chromatography. We investigated the efficiency and robustness of cation exchange chromatography (CEX) in an effort to evaluate alternative capture methodologies. Up to five commercially available CEX resins were systematically evaluated using an experimentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capture a model mAb with a neutral isoelectric point, (b) clear three model viruses (porcine parvovirus, CHO type-C particles, and a bacteriophage). This approach identified a narrow operating space where yield, purity, and viral clearance were optimal under a CEX capture platform, and revealed trends between viral clearance of PPV and product purity (but not yield). Our results suggest that after unit operation optimization, CEX can serve as a suitable capture step.     

Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures—Systems biology‐based interpretation using genome‐scale metabolic flux balance model and multivariate data analysis

Genome‐scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745–753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome‐scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163–1173, 2016     

Development of a pressure-measuring device to optimize compression treatment of lymphedema and evaluation of change in garment pressure with simulated wear and tear

The use of compression garments in treating lymphedema following treatment of genital (penis, testes, uterus, cervical) and breast cancer treatment is a well-established practice. Although compression garments are classified in compression classes, little is known about the actual subgarment pressure exerted along the extremity. The aims of this study were to establish an in vitro method for measuring subgarment pressure along the extremity and to analyze initial and over time subgarment pressure of compression garments from three manufacturers. The measurements were performed with I-scan(?) (Tekscan Inc.) pressure measuring equipment once a week during a period of 4 weeks. Wear and tear was simulated by washing and putting on the garments on plastic legs every day. There was a statistically significant difference between the garments of some of manufacturers. There was no difference between garments from the same manufacturer. No significant decrease of subgarment pressure was observed during the trial period. The study demonstrated that Tekscan pressure-measuring equipment could measure subgarment pressure in vitro. The results may indicate that there was a difference in subgarment pressure exerted by garments from different manufacturers and that there was no clear decrease in subgarment pressure during the first four weeks of usage.     

Variation of the electron population by four units in the cluster series [(η-Cp′)3Mo3S4Co(L)] (L = I, CO, PPh3, NO; n = 0, 1)

The versatility of cuboidal Mo₃S₄Co clusters for the preparation of complexes with different numbers of valence shell electrons (VSE) in the cluster is described. The reaction of the geometrically incomplete cuboidal cluster salt [(η⁵-Cp′)₃Mo₃S₄][pts] (pts = p-toluenesulfonate) with one molar equivalent of [Co₂(CO)₈] afforded almost quantitatively the electroneutral 60 VSE cluster [(η⁵-Cp′)₃Mo₃S₄Co(CO)] (1), which previously has been prepared in low yield by Curtis et al. in autoclave syntheses [M.D. Curtis, U. Riaz, O.J. Curnow, J.W. Kampf, Organometallics 14 (1995) 5337]. Cluster 1 was also obtained in high yield by reaction of [(η⁵-Cp′)₃Mo₃S₄][pts] with [(η⁵-Cp^∗)Co(CO)₂]. Reaction of [(η⁵-Cp′)₃Mo₃S₄][pts] with two molar equivalents of [Co(I)(CO)₃(PPh₃)] led to a complex mixture of products, of which the electron deficient 58 VSE cluster salt [(η⁵-Cp′)₃Mo₃S₄Co(I)][Co(I)₃(thf)] ([2][Co(I)₃(thf)]) was isolated as single crystals. In the crystal structures of 1 and [2][Co(I)₃(thf)], the Co-Mo bond lengths are almost identical, indicating a delocalization of the electron deficiency in [2]⁺. The reduced form of [2]⁺, [(η⁵-Cp′)₃Mo₃S₄Co(I)] (2), was prepared by oxidative substitution of the carbonyl ligand in 1 by I₂. Further reactions of 1 with PPh₃ and NO leading to the 60 and 61 VSE cluster complexes [(η⁵-Cp′)₃Mo₃S₄Co(PPh₃)] (3) and [(η⁵-Cp′)₃Mo₃S₄Co(NO)] (4), respectively, enabled the preparation of Mo₃S₄Co clusters in altogether four different oxidation states.     

Chemometrics applications in biotechnology processes: Predicting column integrity and impurity clearance during reuse of chromatography resin

Separation media, in particular chromatography media, is typically one of the major contributors to the cost of goods for production of a biotechnology therapeutic. To be cost‐effective, it is industry practice that media be reused over several cycles before being discarded. The traditional approach for estimating the number of cycles a particular media can be reused for involves performing laboratory scale experiments that monitor column performance and carryover. This dataset is then used to predict the number of cycles the media can be used at manufacturing scale (concurrent validation). Although, well accepted and widely practiced, there are challenges associated with extrapolating the laboratory scale data to manufacturing scale due to differences that may exist across scales. Factors that may be different include: level of impurities in the feed material, lot to lot variability in feedstock impurities, design of the column housing unit with respect to cleanability, and homogeneity of the column packing. In view of these challenges, there is a need for approaches that may be able to predict column underperformance at the manufacturing scale over the product lifecycle. In case such an underperformance is predicted, the operators can unpack and repack the chromatography column beforehand and thus avoid batch loss. Chemometrics offers one such solution. In this article, we present an application of chemometrics toward the analysis of a set of chromatography profiles with the intention of predicting the various events of column underperformance including the backpressure buildup and inefficient deoxyribonucleic acid clearance. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012