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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.