Characterization of DNA in cell culture supernatant by fluorescence-detection size-exclusion chromatography.

A fluorescence-detection size-exclusion chromatography (FSEC) method was developed to characterize DNA in cell culture supernatant. Samples stained with Picogreen were fractionated by size-exclusion chromatography (SEC) and monitored simultaneously by UV absorbance and fluorescence. SEC provided a size-characterization capability absent from bulk fluorescent assays, and was also free from interference from other fluorescent and UV-absorbing small-molecule cell culture components. FSEC revealed that DNA in mammalian cell culture supernatant exists mostly in the form of nucleosomal arrays. FSEC combined with agarose electrophoresis revealed spontaneous degradation of DNA in mammalian cell culture supernatant over a 30 day period at 4 °C: from arrays containing up to ~40 nucleosomes, down to arrays containing three or fewer nucleosomes. It also detected nucleosomal DNA in wheat, soy, and yeast hydrolysates commonly used to enhance cell culture productivity.

Host cell proteins in monoclonal antibody processing: Control, detection, and removal.

Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of "high-risk" HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.

Affinity analysis of DNA aptamer-peptide interactions using gold nanoparticles.

Gold nanoparticles (AuNPs) were used as colorimetric probe and fluorescence quencher for affinity analysis of DNA aptamers toward their target mucin 1 (MUC1) peptide. Single-stranded DNA (ssDNA) aptamer-coated AuNPs showed increased stability (i.e., more resistant to aggregation induced by NaCl) in the presence of their target peptide due to the increase in steric protection conferred by the ssDNA-peptide complexes formed on the AuNPs. Based on changes in the UV-vis extinction spectrum of AuNPs (a measure of AuNPs aggregation) and fluorescence restoration of CY5-ssDNA upon ssDNA-peptide complex formation, the formation of the complexes and ssDNA sequence-dependent dissociation constant (K(d)) were determined. Besides the UV-vis and fluorescence measurements, the hydrodynamic diameters, zeta potential measurements, and transmission electron microscopy (TEM) images of AuNPs after various coatings supported the assay principle. The methodology presented herein provides a rapid and sensitive alternative solution for the identification of high affinity binders from systematic evolution of ligands by exponential enrichment (SELEX).

Chaperone-assisted column refolding of gloshedobin with the use of refolding cocktail.

Gloshedobin, a recently isolated thrombin-like enzyme from the snake venom of Gloydius shedaoensis, is expressed mainly in the form of inclusion bodies (IBs) in Escherichia coli due to its cysteine-rich nature. Following extraction and solubilization of the IBs, one-step immobilized metal affinity chromatography purification produces highly purified (>99%) denatured-solubilized gloshedobin ready to enter the subsequent refolding process. However, the traditional dilution or column refolding strategy, based on gradual denaturant removal, is found to be inefficient for the recovery of protein activity. In this study, a new refolding strategy harnessing the ClpB and DnaK/DnaJ/GrpE bichaperone system is demonstrated to be superior to the conventional refolding methods in either batch dilution or column refolding mode. It is noted that the efficacy of bichaperone-mediated column refolding strategy is further highlighted especially when refolding reaction is attempted at a higher protein concentration with the recirculation of the refolding cocktail containing the bichaperone system. This is evidenced by an uncompromised refolding efficiency (ca. 21.4%) achieved at 2000 microg/mL of initial protein concentration, which is comparable to the refolding efficiency (ca. 22.5%) obtained at 20 times lower protein concentration (i.e. 100 microg/mL) in the conventional batch dilution refolding technique. The demonstrated chaperone-assisted column refolding strategy thus provides an effective tool for refolding-recalcitrant proteins whose reactivation is otherwise difficult to achieve.

Polyethyleneimine-mediated chemical extraction of cytoplasmic His-tagged inclusion body proteins from Escherichia coli.

The selectivity of polyethyleneimine (PEI) in DNA precipitation during chemical extraction was investigated. Chemical extraction was used to recover two His-tagged model proteins: gloshedobin, a thrombin-like enzyme from snake venom, and IbpA, a molecular chaperone, which were expressed mainly in the form of inclusion bodies. High DNA removal efficiency (more than 90%) was achieved at various cell densities (with OD600 ranging from 30 to 150) without affecting the solubility of host cell proteins. Compared to spermine-induced precipitation method reported elsewhere, PEI provided a higher DNA precipitation efficiency at a significantly lower cost. Moreover, PEI obviated the use of EDTA, which has been reported to be essential for the chemical extraction methods, hence exhibiting dual roles in replacing cost-prohibitive spermine and EDTA. The residual PEI in the post-extraction mixture was efficiently counteracted by addition of Mg2+, allowing the streamlined application of the extraction broth to immobilized metal affinity chromatography. Taken together, the PEI-mediated chemical extraction method provides a simpler and more economically viable processing route for the production of recombinant proteins whose expression is hampered by IB formation.

Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions.

Separation of negatively charged molecules, such as plasmid DNA (pDNA), RNA and endotoxin forms a bottleneck for the development of pDNA vaccine production process. The use of affinity interactions of transition metal ions with these molecules may provide an ideal separation methodology. In this study, the binding behaviour of pDNA, RNA and endotoxin to transition metal ions, either in immobilised or free form, was investigated. Transition metal ions: Cu2+, Ni2+, Zn2+, Co2+ and Fe3+, typically employed in the immobilised metal affinity chromatography (IMAC), showed very different binding behaviour depending on the type of metal ions and their existing state, i.e. immobilised or free. In the alkaline cell lysate, pDNA showed no binding to any of the IMAC chemistries tested whereas RNA interacted significantly with Cu2+-iminodiacetic acid (IDA) and Ni2+-IDA but showed no substantial binding to the rest of the IMAC chemistries. pDNA and RNA, however, interacted to varying degrees with free metal ions in the solution. The greatest selectivity in terms of pDNA and RNA separation was achieved with Zn2+ which enabled almost full precipitation of RNA while keeping pDNA soluble. For both immobilised and free metal ions, ionic strength of solution affected the metal ion-nucleic acid interaction significantly. Endotoxin, being more flexible, was able to interact better with the immobilised metal ions than the nucleic acids and showed binding to all the IMAC chemistries. The specific interactions of immobilised and/or free metal ions with pDNA, RNA and endotoxin showed a good potential, by selectively removing RNA and endotoxin at high efficiency, to develop a simplified pDNA purification process with improved process economics.

Advance chromatin extraction improves capture performance of protein A affinity chromatography.

Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%.

Chromatin-mediated depression of fractionation performance on electronegative multimodal chromatography media, its prevention, and ramifications for purification of immunoglobulin G.

Chromatin heteroaggregates comprising nucleosomal arrays, DNA, histone and non-histone host cell proteins contributed 28% of the host contaminant mass in an IgG cell culture harvest. A subset comprising soluble particles of 50-400nm was retained through its histone component by an electronegative multimodal chromatography adsorbent. Accumulated heteroaggregates hindered pore access and depressed IgG capacity by 69% compared with capacity determined with purified IgG. Heteroaggregate retention persisted in 2M NaCl but some associations within them were dissociated. This manifested during IgG elution as contaminant leaching from the retained heteroaggregate mass, which contributed more than 95% of the contaminants in the IgG. Retained heteroaggregates also interfered with pore egress of eluted IgG, increasing peak width ∼3-fold. IgG recovery was 80%. Remaining heteroaggregates were removed with 1M NaOH. Advance reduction of heteroaggregates with a hybrid fatty acid-solid phase clarification method reduced DNA more than 3 logs, histones beneath the level of detectability, and non-histone proteins by 90% while conserving 90% of the IgG. Non-lipid-enveloped virus was reduced by 5 logs and lipid-enveloped virus by 9 logs. IgG capacity on the multimodal adsorbent was 94mg/mL, compared with 95mg/mL when loaded with protein A-purified IgG. Non-histone host protein reduction increased to 98%. IgG eluted in a sharp concentrated peak and step recovery was >95%. Residual fatty acids did not bind the adsorbent and were mostly eliminated during sample loading. A single polishing step with an electropositive multimodal adsorbent reduced non-histone host proteins to <100ppm, DNA to <10ppb, and aggregates to <0.05%. IgG recovery across the entire process was >80%. These results qualify electronegative multimodal adsorbents as an IgG capture option, but more importantly reveal chromatin heteroaggregate content as a keystone process variable at all stages of purification process development.

Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance.

Chromatin released from dead host cells during in vitro production of IgG monoclonal antibodies exists mostly in complex hetero-aggregates consisting of nucleosomal arrays (DNA+histone proteins), non-histone proteins, and aberrant forms of IgG. They bind immobilized protein A more aggressively than IgG, through their nucleosomal histone components, and hinder access of IgG to Fc-specific binding sites, thereby reducing dynamic binding capacity. The majority of host cell contaminants in eluted IgG are leachates from chromatin hetero-aggregates that remain bound to protein A. Formation of turbidity in eluted IgG during pH titration is caused by neutral-pH insolubility of chromatin hetero-aggregates. NaOH is required at 500 mM to remove accumulated chromatin. A chromatin-directed clarification method removed 99% of histones, 90% of non-histone proteins, achieved a 6 log reduction of DNA, 4 log reduction of lipid-enveloped virus, and 5 log reduction of non-enveloped retrovirus, while conserving 98% of the native IgG. This suspended most of performance compromises imposed on protein A. IgG binding capacity increased ~20%. Host protein contamination was reduced about 100-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Aggregates were reduced to less than 0.05%. Turbidity of eluted IgG upon pH neutralization was nearly eliminated. Column cleaning was facilitated by minimizing the accumulation of chromatin.

Designer tridentate mucin 1 aptamer for targeted drug delivery.

A single-stranded DNA aptamer (APT) capable of targeting mucin 1 (MUC1) extracellular protein was modified to increase its drug delivery specificity toward MUC1 overexpressing cancer cell line, MCF7. The active targeting region of APT was truncated and variable repeats (one, two, or three) of this sequence were synthesized. An aptamer formed from three repeats of this active targeting region (L3) was shown to possess enhanced doxorubicin (DOX) intercalation ability, and L3-DOX complex exhibited selective cytotoxicity to MCF7 over RAW cells. Most importantly, L3 was able to evade RAW 264.7 macrophages (2-fold reduction in L3 uptake relative to APT), thus resulting in an overall 5.5-fold increase of survivability of RAW cells as compared with when free DOX was used. These results indicate that aptamer L3 has good potential for targeted drug therapeutics.

The interplay of peptide sequence and local structure in TiO2 biomineralization.

Using cyclic constrained TiO(2) binding peptides STB1 (CHKKPSKSC), RSTB1 (CHRRPSRSC) and linear peptide LSTB1 (AHKKPSKSA), it was shown that while affinity of the peptide to TiO(2) is essential to enable TiO(2) biomineralization, other factors such as biomineralization kinetics and peptide local structure need to be considered to predict biomineralization efficacy. Cyclic and linear TiO(2) binding peptides show significantly different biomineralization activities. Cyclic STB1 and RSTB1 could induce TiO(2) precipitation in the presence of titanium(IV)-bis-ammonium-lactato-dihydroxide (TiBALDH) precursor in water or tris buffer at pH 8. In contrast, linear LSTB1 was unable to mineralize TiO(2) under the same experimental conditions despite its high affinity to TiO(2) comparable with STB1 and/or RSTB1. LSTB1 being a flexible molecule could not render the stable condensation of TiBALDH precursor to form TiO(2) particles. However, in the presence of phosphate buffer ions, the structure of LSTB1 is stabilized, leading to efficient condensation of TiBALDH and TiO(2) particle formation. This study demonstrates that peptide-mediated TiO(2) mineralization is governed by a complicated interplay of peptide sequence, local structure, kinetics and the presence of mineralizing aider such as phosphate ions.

PEGylated anti-MUC1 aptamer-doxorubicin complex for targeted drug delivery to MCF7 breast cancer cells.

Targeted drug delivery is especially important in cancer treatment as many anti-cancer drugs are non-specific and highly toxic to both cancer and normal cells. The targeted drug delivery of DOX to the MUC1-expressing breast cancer cell line (MCF7) was obtained using APT as a carrier. Modification of the APT-DOX complex by PEG increases the survivability of the macrophage control (RAW 264.7) by about six-fold as compared to free DOX treatment without significantly affecting the cytotoxicity toward the target cell line. Thus, PEG-APT-DOX is potentially a new therapeutic agent for targeted drug delivery to MUC1-expressing cell lines.

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification.

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification.

Chromatographic biopanning for the selection of peptides with high specificity to Pb2+ from phage displayed peptide library.

Toxic heavy metal pollution is a global problem occurring in air, soil as well as water. There is a need for a more cost effective, renewable remediation technique, but most importantly, for a recovery method that is selective for one specific metal of concern. Phage display technology has been used as a powerful tool in the discovery of peptides capable of exhibiting specific affinity to various metals or metal ions. However, traditional phage display is mainly conducted in batch mode, resulting in only one equilibrium state hence low-efficiency selection. It is also unable to monitor the selection process in real time mode. In this study, phage display technique was incorporated with chromatography procedure with the use of a monolithic column, facilitating multiple phage-binding equilibrium states and online monitoring of the selection process in search of affinity peptides to Pb2+. In total, 17 candidate peptides were found and their specificity toward Pb2+ was further investigated with bead-based enzyme immunoassay (EIA). A highly specific Pb2+ binding peptide ThrAsnThrLeuSerAsnAsn (TNTLSNN) was obtained. Based on our knowledge, this is the first report on a new chromatographic biopanning method coupled with monolithic column for the selection of metal ion specific binding peptides. It is expected that this monolith-based chromatographic biopanning will provide a promising approach for a high throughput screening of affinity peptides cognitive of a wide range of target species.

Synergistic coordination of polyethylene glycol with ClpB/DnaKJE bichaperone for refolding of heat-denatured malate dehydrogenase.

The use of polyethylene glycol (PEG) as a refolding additive to a refolding cocktail comprising the molecular bichaperone ClpB and DnaKJE significantly enhances chaperone-mediated refolding of heat-denatured malate dehydrogenase (MDH). The critical factor to affect the refolding yield is the time point of introducing PEG to the refolding cocktail. The refolding efficiency reached approximately 90% only when PEG was added at the beginning of refolding reaction. The synergistic coordination of an inexpensive refolding additive PEG with the ClpB/DnaKJE bichaperone system may provide an economical route to further enhance the efficacy of ClpB/DnaKJE refolding cocktail approach, facilitating its implementation in large-scale refolding processes.