Streamlining the polishing step development process via physicochemical characterization of monoclonal antibody aggregates

Doss, Hannah R.; Raman, Mathura; Knihtila, Ryan; Chennamsetty, Naresh; Wang, David; Shupe, Alan; Mussa, Nesredin

doi:10.1016/j.chroma.2019.03.044

Cited by 13 publications

(8 citation statements)

References 45 publications

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“…Also, the potential heterogeneity of aggregate complexes studied in experiments may hamper the development of a unified aggregate clearance model. Indeed, recent studies show that aggregate surface properties are unique to each antibody (Doss et al, 2019), potentially requiring different conditions for clearance. In general, however, the majority of aggregates have more exposed hydrophobic surface areas, making nylon a potential aggregate clearance option (Telikepalli et al, 2014; Vázquez‐Rey & Lang, 2011).…”

Section: Discussionmentioning

confidence: 99%

Improving viral filtration capacity in biomanufacturing processes using aggregate binding properties of polyamide‐6,6

Stanevich

Pachalla

Nunez

et al. 2020

Biotech & Bioengineering

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Virus retention filtration is a common step in modern biopharmaceutical manufacturing as it enables efficient removal of potential adventitious and endogenous viruses via size exclusion. Modern parvovirus retention filters have significantly improved fluxes and parvovirus retention in comparison to earlier versions of these filters. However, these filters may be more susceptible to premature fouling and require more effort for process optimization. Here, we demonstrate that polyamide‐6,6 (nylon‐6,6) membranes when used as prefilters can increase the capacity of these Parvovirus retentive filters that are less susceptible to premature fouling. We found that the mechanism of polyamide‐mediated filtration improvement can be explained by the binding of monoclonal antibody (mAb) aggregates with a diameter of 20–100 nm, and we show that this mechanism is shared by other types of adsorptive prefilters. Finally, by the combination of mobile phase screening, additive spiking, and molecular dynamics simulations, we show that polyamide‐6,6 removes mAb aggregates through hydrophobic interactions making its design space potentially complementary to other available prefilters. Our studies support the aggregate‐mediated mechanism of flux decay during viral filtration and suggest that polyamide‐6,6 could be considered as an alternative cost‐effective option to extend the capacity of viral filters.

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Section: Discussionmentioning

confidence: 99%

Improving viral filtration capacity in biomanufacturing processes using aggregate binding properties of polyamide‐6,6

Stanevich

Pachalla

Nunez

et al. 2020

Biotech & Bioengineering

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“…The surface hydrophobicity of the model proteins was also evaluated experimentally by analytical Hydrophobic Interaction Chromatography (HIC) following the protocol presented by Doss et al 20 with a small modification of the mobile phase. Samples were analyzed using a TOSOH Bioscience® TSKgel Butyl‐NPR column (4.6 mm × 10 cm, Tokyo, Japan) run on an Agilent HPLC 1100 system (Agilent Technologies, Santa Clara, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Binding capacities evaluated by numerical integration of breakthrough curves out to C/C o = 0.9.F I G U R E 5Breakthrough curves for filtration of 0.2 g/L solutions of myoglobin, albumin, ribonuclease A, and α-chymotrypsin in 30 mM PBS through PDH4 depth filters at a constant filtrate flux of 0.6 LMM consideration of the detailed surface characteristics or heterogeneity of the protein (e.g., location, proximity or size of hydrophobic patches on the protein surface). In order to obtain a more direct measure of the protein hydrophobicity, we used analytical hydrophobic interaction chromatography (HIC) to measure protein retention time on a highly hydrophobic TSKgel Butyl-NPR column with 2.5 μm nonporous particles as the stationary phase following the procedures developed by Doss et al20 No attempt was made to optimize the buffer conditions to maximize protein recovery since the goal was to evaluate the hydrophobicity based on the measured retention time.The proteins are initially denatured in a 2.3 M ammonium sulfate buffer, allowing the hydrophobic patches to strongly interact with the stationary phase. The proteins are then eluted using a 0.1 M phosphate buffer with a linear gradient in the ammonium sulfate concen-…”

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confidence: 99%

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

Nejatishahidein

Borujeni

Roush

et al. 2020

Biotechnology Progress

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The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.

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“…Figure 3 shows an overlay of the MHIC elution profiles for suspensions of 200 nm PS NPs (PS), 200 nm silica NPs (Si), and an LAV with similar particle size. 23 The PS were examined in the presence of 0.01% Tween 20 in the binding and elution buffers; the particle yield in the absence of the surfactant was <1% due to the very hydrophobic nature of the polystyrene. The LAV and Si were studied in the absence of any surfactant.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Hydrophobic Interaction Chromatography. AHIC was used to determine the relative hydrophobicity of the model proteins using a TSKgel Butyl-NPR column from Tosoh Bioscience with ∼1.0 mL volume and 10 cm bed height following the procedures originally described by Doss et al 23 The column was operated on a Vanquish Binary UHPLC system (Thermo Fisher Scientific, Waltham, MA). The column was first equilibrated with 5 column volumes (CV) of equilibration buffer containing 100 mM phosphate + 2.3 M ammonium sulfate at a pH of 7.…”

Section: ■ Methods and Materialsmentioning

confidence: 99%

Evaluating Nanoparticle Hydrophobicity Using Analytical Membrane Hydrophobic Interaction Chromatography

Taylor

Kristopeit

et al. 2022

Anal. Chem.

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Nanoparticle hydrophobicity is a key factor controlling the stability, adhesion, and transport of nanoparticle suspensions. Although a number of approaches have been presented for evaluating nanoparticle hydrophobicity, these methods are difficult to apply to larger nanoparticles and viruses (>100 nm in size) that are of increasing importance in drug delivery and gene therapy. This study investigated the use of a new analytical hydrophobic interaction chromatography method employing a 5.0 μm pore size polyvinylidene fluoride membrane as the stationary-phase in membrane hydrophobic interaction chromatography (MHIC). Experimental data obtained using a series of model proteins were in good agreement with literature values for the hydrophobicity (both experimental and computational). MHIC was then used to evaluate the hydrophobicity of a variety of nanoparticles, including a live attenuated viral vaccine, both in water and in the presence of different surfactants. This new method can be implemented on any liquid chromatography system, run times are typically <20 min, and the experiments avoid the use of organic solvents that could alter the structure of many biological nanoparticles.

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Streamlining the polishing step development process via physicochemical characterization of monoclonal antibody aggregates

Cited by 13 publications

References 45 publications

Improving viral filtration capacity in biomanufacturing processes using aggregate binding properties of polyamide‐6,6

Improving viral filtration capacity in biomanufacturing processes using aggregate binding properties of polyamide‐6,6

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

Evaluating Nanoparticle Hydrophobicity Using Analytical Membrane Hydrophobic Interaction Chromatography

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