Methods for addressing host cell protein impurities in biopharmaceutical product development.

The high demand for monoclonal antibody (mAb) therapeutics in recent years has resulted in significant efforts to improve their costly manufacturing process. The high cost of manufacturing mAbs derives mainly from the purification process, which contributes to 50%-80% of the total manufacturing cost. One of the main challenges facing industry at the purification stage is the clearance of host cell proteins (HCPs) that are produced and often co-purified with the desired mAb product. One of the issues HCPs can cause is the degradation of the final mAb protein product. In this review, techniques are considered that can be used at different stages (upstream and downstream) of mAb manufacture to improve HCP clearance. In addition to established techniques, many new approaches for HCP removal are discussed that have the potential to replace current methods for improving HCP reduction and thereby the quality and stability of the final mAb product.

Interaction between preservatives and a monoclonal antibody in support of multidose formulation development.

Multidose formulations have patient-centric advantages over single-dose formats. A major challenge in developing multidose formulations is the prevention of microbial growth that can potentially be introduced during multiple drawings. The incorporation of antimicrobial preservatives (APs) is a common approach to inhibit this microbial growth. Selection of the right preservative while maintaining drug product stability is often challenging. We explored the effects of three APs, 1.1 % (w/v) benzyl alcohol, 0.62 % (w/v) phenol, and 0.42 % (w/v) m-cresol, on a model immunoglobulin G1 monoclonal antibody, termed the "NIST mAb." As measured by hydrogen exchange-mass spectrometry (HX-MS) and differential scanning calorimetry, conformational stability was decreased in the presence of APs. Specifically, flexibility (faster HX) was significantly increased in the CH2 domain (HC 238-255) across all APs. The addition of phenol caused the greatest conformational destabilization, followed by m-cresol and benzyl alcohol. Storage stability studies conducted by subvisible particle (SVP) analysis at 40 °C over 4 weeks further revealed an increase in SVPs in the presence of phenol and m-cresol but not in the presence of benzyl alcohol. However, as monitored by size exclusion chromatography, there was neither a significant change in the monomeric content nor an accumulation of soluble aggregate in the presence of APs.

Magnetically induced pattern formation in phase separating polymer-solvent-nanoparticle mixtures.

Permanent magnetic structures with controlled dimension and architecture (labyrinthine, hexagonal, or dispersed columnar) are formed in a partially miscible ferrofluid-nonferrofluid mixture under the influence of a perpendicular magnetic field. The origin of the permanent structures, which have characteristic lateral dimensions ranging from 1 to 10 µm, is the repartitioning of the ferrofluid carrier solvent into the nonferrofluid polymeric phase. This polymer-solvent phase separation under a magnetic field leads to departures from the expected final dimension of the magnetically stabilized ferrofluid droplet sizes.

Simple magnetic cell patterning using streptavidin paramagnetic particles.

A simple methodology for cell patterning has been developed that can potentially be used to position different types of mammalian cells with high precision. In this method, cell membrane proteins were first biotinylated and then bound to streptavidin paramagnetic particles. The magnetically labeled cells were then seeded onto culture dishes and patterned using low magnetic fields. Highly defined cell patterns were achieved using HeLa, TE671 cells and human monocytes. HeLa and TE671 cells were also sequentially patterned and successfully co-cultured on the same plate using this technique. Cell viability studies proved that this magnetic labeling method was not toxic to cells. Transmission electron microscopy showed that the magnetically labeled HeLa and TE671 cells internalized some of the paramagnetic particles after two days of culture, while the labeled human monocytes did the same after only one hour. Uptake of these particles did not affect the cell patterning and cell viability. This magnetic labeling process is fast, as it involves affinity-based attachment of paramagnetic particles and does not rely on cellular uptake of magnetic materials. It may be adaptable and scalable for various applications.

The in-flow capture of superparamagnetic nanoparticles for targeting therapeutics.

Superparamagnetic nanoparticles have been synthesized that could potentially be used to magnetically target therapeutics within the body. The magnetic targeting and successful in-flow capture of 330-nm and 580-nm agglomerates of these magnetite nanoparticles was performed using a 0.5-T magnet. Optical observation of magnetic nanoparticle capture in microcapillary flow provides a useful preliminary way of establishing conditions for the magnetic capture of nanoparticles with direct relevance to blood vessels for magnetically directed therapy. A stable nanoparticle layer of 580-nm agglomerates could be formed at mean capillary flow velocities of up to 2.5 cm s(-1) and for the 330-nm agglomerates at velocities up to 4.4 cm s(-1). These data show that smaller nanoparticle agglomerates form a layer that is impervious to erosion by fluid shear. Capillary blocking by nanoparticles, analogous to an embolism, was not detected in these experiments.

Biopharmaceutical formulations for pre-filled delivery devices.

INTRODUCTION: Pre-filled syringes are becoming an increasingly popular format for delivering biotherapeutics conveniently and cost effectively. The device design and stable liquid formulations required to enable this pre-filled syringe format are technically challenging. In choosing the materials and process conditions to fabricate the syringe unit, their compatibility with the biotherapeutic needs to be carefully assessed. The biothereaputic stability demanded for the production of syringe-compatible low-viscosity liquid solutions requires critical excipient choices to be made. AREAS COVERED: The purpose of this review is to discuss key issues related to the stability aspects of biotherapeutics in pre-filled devices. This includes effects on both physical and chemical stability due to a number of stress conditions the product is subjected to, as well as interactions with the packaging system. Particular attention is paid to the control of stability by formulation. EXPERT OPINION: We anticipate that there will be a significant move towards polymer primary packaging for most drugs in the longer term. The timescales for this will depend on a number of factors and hence will be hard to predict. Formulation will play a critical role in developing successful products in the pre-filled syringe format, particularly with the trend towards concentrated biotherapeutics. Development of novel, smart formulation technologies will, therefore, be increasingly important.

In situ fabrication of a microfluidic device for immobilised metal affinity sensing.

In this work a novel microfluidic device was constructed in situ containing the smallest microscopic co-polymeric immobilised metal affinity (IMA) adsorbent yet documented. This device has for the first time allowed the microlitre scale chromatographic assay of histidine-tagged proteins in a biological sample. To enable this approach, rather than using a high capacity commercial packed bed column which requires large sample volumes and would be susceptible to occlusion by cell debris, a microgram capacity co-polymeric chromatographic substrate suitable for analytical applications was fabricated within a microfluidic channel. This porous co-polymeric IMA micro-chromatographic element, only 27µl in volume, was assessed for the analytical capture of two different histidine-tagged recombinant fusion proteins. The micro-chromatographic adsorber was fabricated in situ by photo-polymerising an iminodiacetic acid (IDA) functionalised polymer matrix around a template of fused 100µm diameter NH(4)Cl particles entirely within the microfluidic channel and then etching away the salt with water to form a network of interconnected voids. The surface of the micro-chromatographic adsorber was chemically functionalised with a chelating agent and loaded with Cu(2+) ions. FTIR and NMR analysis verified the presence of the chelating agent on the adsorbent surface and its Cu(2+) ion binding capacity was determined to be 2.4µmol Cu(2+) (ml of adsorbent)(-1). Micro-scale equilibrium adsorption studies using the two different histidine-tagged proteins, LacI-His(6)-GFP and α-Synuclein-His(8)-YFP, were carried out and the protein binding capacity of the adsorbent was determined to be 0.370 and 0.802mg(g of adsorbent)(-1), respectively. The dynamic binding capacity was determined at four different flow rates and found to be comparable to the equilibrium binding capacity at low flow rates. The sensing platform was also used to adsorb LacI-His(6)-GFP protein from crude cell lysate. During adsorption, laser scanning confocal microscopy identified locations within the adsorbent where protein adsorption and desorption occurred. The findings indicate that minimal channelling, selective product capture and near quantitative elution of the captured (adsorbed) product could be achieved, supporting the application of this new device as a high-throughput process analytical tool (PAT) for the in-process monitoring of histidine-tagged proteins in manufacturing.

The precise control of cell labelling with streptavidin paramagnetic particles.

A previously developed cell labelling methodology has been evaluated to assess its potential to precisely control the degree of magnetic labelling. The two-step method provides a quick way of labelling cells by first biotinylating the cell membrane proteins and then binding streptavidin paramagnetic particles onto the biotinylated proteins. Characterisation studies on biotinylated HeLa cells have revealed that the biotin concentration on the cell surface can be varied by changing the biotinylating reagent concentration. At the optimal concentration (750 microm), a substantial surface biotin density (approximately 10(8) biotin per cell) could be achieved within 30 min. The degree of magnetic labelling could be altered by adjusting the concentration of paramagnetic particles added to the cells and the binding of the particles onto the cell surface was not considerably affected by the biotin density on the cell surface. The magnetic moment of the labelled cells was measured and correlated well with the degree of magnetic labelling. Cell viability studies indicated that the magnetic labelling was not cytotoxic. Magnetically labelled cells were then successfully targeted and manipulated by magnetic fields to form three dimensional multicellular structures.

Effect of magnetite nanoparticle agglomerates on ultrasound induced inertial cavitation.

High intensity focused ultrasound (HIFU) induced inertial cavitation has been shown to improve release and cellular uptake of drugs. The effects of magnetite nanoparticle agglomerates (290+/-10nm diameter), silica coated magnetite nanoparticle agglomerates (320+/-10nm diameter) and silica particles (320+/-10nm diameter) suspended in MilliQ water on the degree of inertial cavitation due to HIFU were investigated. The HIFU transducer was operated at a frequency of 1.1 MHz, 1.67 kHz pulse repetition frequency, with applied duty cycles (DC) between 0% and 5% and different peak negative focal pressures (PNFPs) applied up to 7.2 MPa. The inertial cavitation dose (ICD: time averaged root-mean-squared broadband noise amplitude in the frequency domain) was measured in the presence and absence of nanoparticles when subjected to HIFU. Magnetite nanoparticle agglomerates caused a significant increase in the ICD above 2.7 MPa PNFP compared with MilliQ water, silica coated magnetite agglomerates and silica particles. With the dramatic increase in ICD on introduction of these magnetite agglomerates, this technique could provide a method of HIFU triggered drug delivery by enhancing inertial cavitation. The superparamagnetic properties of these particles offer the possibility of magnetic targeting to the site of disease.

Manipulation and tracking of superparamagnetic nanoparticles using MRI.

The use of magnetic fields in magnetic resonance imaging (MRI) for the tracking and delivery of chemotherapeutics bound to superparamagnetic nanoparticles offers a promising method for the non-invasive treatment of inoperable tumours. Here we demonstrate that superparamagnetic magnetite nanoparticles fabricated by an easily scalable method can be driven and tracked in real time at high velocities in vitro using MRI hardware. Force balance calculations are consistent with the magnetic properties of individual 10 nm diameter particles that move collectively as micron sized agglomerates with hydrodynamic diameter similar to that inferred from zero-magnetic-field dynamic light scattering measurements.