Expanded bed adsorption at production scale: Scale-up verification, process example and sanitization of column and adsorbent

Expanded bed adsorption is a technique for recovery of biomolecules directly from unclarified feedstocks. The work described here demonstrates that expanded bed adsorption is a scaleable technique. The methods used to test scaleability were “determination of degree of bed expansion”, “determination of axial dispersion” and “determination of protein breakthrough capacity”. The performance of a production scale expanded bed column with 600?mm diameter was tested using these methods and the results were found to be consistent with the results obtained from lab scale and pilot scale expanded bed columns. The scaleability and function of the expanded bed technique was also tested by performing a “process example”: a purification mimicking a real process using a yeast culture spiked with bovine serum albumin as feedstock. The results show that the 600?mm diameter production scale column was as efficient as a 25?mm diameter lab scale column in recovering bovine serum albumin from the unclarified yeast culture. The production scale runs were fully automated using a software controlled system containing an adaptor position sensor and an adsorbent sensor. A cleaning study was performed which showed that after use of a proper cleaning protocol, no surviving microorganisms could be detected in the column or in the adsorbent.     

Experimental pretransfer expansion of free-flap donor sites: II. Physiology, histology, and clinical correlation

There is a statistically significant increase in blood flow into expanded cutaneous and myocutaneous flaps in a pig model over controls. The vascular trees within flaps enlarge following expansion, and pronounced neovascularization is seen in the papillary dermis and capsular layers. Experimentally, there is differential thinning of all tissue layers except the epidermis. A successful clinical application of this technique, by free-tissue transfer of an expanded fasciocutaneous scapular flap, is described. The technique is applicable when altered flap size or contour is desired.     

Increased survival and vascularity of random-pattern skin flaps elevated in controlled, expanded skin

Controlled clinical tissue expansion, a new technique of providing donor tissue, results in an increase in surface area of expanded skin. The aim of the present study was to determine the effect of controlled tissue expansion on the surviving lengths of random-pattern skin flaps elevated in expanded tissue. In five pigs the surviving lengths of flaps raised in skin expanded for 5 weeks using a 250-cc rectangular Radovan-type tissue expander were compared with the survival lengths of flaps elevated in tissue in which a similar prosthesis was not expanded, bipedicle flaps delayed for 5 weeks, and control acutely raised random-pattern flaps. The expanded flaps had a mean increase in surviving length of 117 percent over control flaps, which was statistically significant. The delay flaps had an increase in survival of 73 percent over control flaps, which was also statistically significant. There was no significant difference in survival between expanded flaps and delayed flaps. Morphologic studies using radiographic techniques on one pig demonstrated increased vascularity with tissue expansion. The results of this work demonstrate that in addition to providing increased surface area with controlled expansion, flaps raised in expanded skin have a significantly augmented surviving length. The mechanism for this increased vascularity with expansion is not known at this time, but it may be due to physical forces associated with expansion acting as a stimulus for angiogenesis.     

On-line monitoring of the purification of GST-(His)6 from an unclarified Escherichia coli homogenate within an immobilised metal affinity expanded bed

The use of a rapid chromatographic assay to monitor the level of a specific protein during its downstream processing by expanded bed adsorption is described. An expanded bed column (5 cm diameter) has been modified to allow the abstraction of liquid samples at various heights along the bed, in an automated, semi-continuous manner throughout the separation. The withdrawn samples were filtered in-line and the level of the target protein assayed by a rapid on-line chromatographic method. Using this technique it was possible to monitor the development of adsorbate profiles during the loading, washing and elution phases of the application of an unclarified feedstock. The potential of the technique is demonstrated using the separation of histidine tagged glutathione s-transferase (GST-(His)₆) from an unclarified Escherichia coli homogenate using an expanded bed of Ni²⁺ loaded STREAMLINE Chelating^TM. The level of GST-(His)₆ in the abstracted homogenate samples was measured using Zn²⁺ loaded NTA-silica as an affinity chromatographic sensor. The approach described demonstrates potential for the on-line monitoring and control of expanded bed separations and for providing a greater understanding of adsorption/desorption and hydrodynamic processes occurring within the bed.     

Cryostat Microtomy of Lung Tissue in an Expanded State

A technique is reported for cryostat sectioning of lung tissue in an expanded state for use in viral immunofluorescence studies. A 1: 2 mixture of O.C.T. embedding compound and phosphate-buffered saline is injected intratracheally into fresh lung tissne. The lung tissue is frozen in liquid nitrogen and sectioned with a cryostat. Compared to other published reports of lung sectioning for immunofluorescence miscroscopy, this method has the advantages of bekg easy and quick, maintaining the lung sectiom in an expanded rather than collapsed state and avoiding contact with chemicals potentially capable of altering sensitive viral antigens.     

Survival of island flaps after tissue expansion: a pig model

Survival of island flaps after tissue expansion has been studied. Expanders were placed under each buttock flap of six minipigs and one side was expanded while the other was left empty as a control. Both flaps were then raised and isolated on their vascular pedicles in order to compare flap survival 7 days later. It was found that the survival lengths of the expanded flaps were approximately 50 percent greater than those of the delayed controls. Microangiography suggested that the diameter of the axial artery increased following expansion. In clinical practice this technique would provide a larger flap for reconstruction and the possibility of direct closure of the donor site. In addition, the observed increase in vessel caliber should facilitate the free tissue transfer of expanded flaps.     

Specular reflection of neutrons at phospholipid monolayers. Changes of monolayer structure and headgroup hydration at the transition from the expanded to the condensed phase state.

The specular neutron reflection technique has been applied for the first time to study the structure and head group hydration of a phospholipid monolayer (dimyristoylphosphatidylcholine containing negatively charged phospholipids) in the condensed and expanded state. By variation of the contrast of the subphase to that of the air it is shown that at the transition from the condensed to the expanded state the carboxyl bonds of the glycerol backbone are hydrated leading to a strong structural change of the headgroup. The total monolayer thickness in the condensed state is 22.5 +/- 1 A (tilt angle 32 +/- 6 degrees) and decreases to 19.5 +/- 1 A in the expanded state. The mean molecular volume increases from 1190 +/- 50 A3 to 1250 +/- 50 A3.     

Successful embryo transfer in a small dasyurid marsupial, Sminthopsis crassicaudata

A technique for the transfer of early embryos from the uterus of donors to recipient females of a small dasyurid marsupial, Sminthopsis crassicaudata , is described. When the recipients were killed 3 to 5 d later, a few expanded blastocysts were found to be present in the uteri of most of the females provided that corpora lutea were present in the ovaries. This study demonstrates successful embryo transfer in a small marsupial species and supports the view that secretion of the endometrium due to luteal activity may be necessary for development of the expanded unilaminar blastocyst stage.     

Surface plasmon resonance characterization of specific binding of polyglutamine aggregation inhibitors to the expanded polyglutamine stretch

Proteins with an abnormally expanded polyglutamine (polyQ) stretch are prone to change their conformations, leading to their aggregation, and cause inherited neurodegenerative diseases called the polyQ diseases. Although screening for polyQ aggregation inhibitors has been extensively performed, many common false-positive hits have been identified so far. In this study, we employed surface plasmon resonance (SPR) to characterize the binding specificities and affinities of polyQ aggregation inhibitors to the expanded polyQ stretch. SPR successfully detected specific binding of polyQ binding peptide 1 (QBP1) to the expanded polyQ stretch (K_d = 5.7 μM), and non-specific binding of Congo red to polyQ proteins independent of their polyQ-length. Binding affinities of polyQ aggregation inhibitors to the expanded polyQ stretch were correlated with their inhibitory effects on polyQ aggregation. We therefore conclude that SPR is a useful technique for screening for specific polyQ aggregation inhibitors as promising therapeutic candidates for the currently untreatable polyQ diseases.     

On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser

A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques.Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei.     

Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography

A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.     

High gradient magnetic separation versus expanded bed adsorption: a first principle comparison

A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the loaded adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase® from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h^–1. Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h^–1 resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h^–1 raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption.     

Recent advances in the application of expressed protein ligation to protein engineering

Expressed protein ligation is a technique for joining recombinantly expressed proteins to polypeptides containing biophysical probes, post-translational modifications or unnatural amino acids. Recent advances have expanded the scope of expressed protein ligation and have allowed the approach to be applied to the study of basic biological questions.     

Repair of scalp defects using a tissue expander and Marlex mesh.

A simple technique using Marlex mesh and a tissue expander to cover scalp defects is described and two patients are presented. This technique is suitable for medium-sized defects that cannot be closed primarily. Marlex mesh is sutured to the wound edges in lieu of a temporary skin graft and to prevent enlargement of the defect during tissue expansion. The tissue expander is placed under adjacent normal scalp in a subgaleal pocket developed through the scalp defect. The scalp defect is closed secondarily using the expanded scalp flap. This technique was performed in two patients with satisfactory results. Marlex mesh obviates the need for a temporary skin graft to cover the scalp defect.     

Cryogel biocomposite containing chitosan-gelatin/cerium–zinc doped hydroxyapatite for bone tissue engineering

The present examination includes manufacture and portrayal of cryogel bio-composite implants containing chitosan-gelatin (CS-GT), cerium–zinc doped hydroxyapatite (CS-GT/Ce-Zn-HA) by cryogelation technique. The prepared cryogel biocomposites (CS-GT/HA and CS-GT/Ce-Zn-HA) were described by scanning electron microscope (SEM) and X-Ray diffraction (XRD) contemplates. The expansion of Ce-Zn in the CS-GT implants essentially expanded growing, diminished swelling, expanded protein sorption, and expanded bactericidal movement. The CS-GT/Ce-Zn-HA biocomposite had non-toxic towards rodent osteoblast cells. So the created CS-GT/Ce-Zn-HA biocomposite has favorable and potential applications over the CS-GT/HA platforms for bone tissue engineering.     

Cell separation by expanded bed adsorption: use of ion exchange chromatography for the separation of <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">S. cerevisiae</Emphasis>

In a wide variety of biotechnological and medical applications it is necessary to separate different cell populations from one another. A promising approach to cell separations is demonstrated to be the adoption of chromatographic techniques conducted in expanded beds. The high voidage between the adsorbent beads in an expanded bed allows for the efficient capture of particulate entities such as cells together with washing and subsequent elution without entrapment and loss. In addition, the combination of a gentle hydrodynamic environment, a high surface area and low mixing within the expanded bed make this technique highly favourable. A model system for the separation of two types of microbial cells using STREAMLINE DEAE adsorbent in expanded bed procedures has been investigated. The use of a less selective ligand such as an ion exchange group, which is often characterised by gentle elution procedures, has been investigated as an alternative to affinity ligands whose strong binding characteristics can result in harsh elution procedures with consequent loss of yield and cell viability. Expanded bed experiments have demonstrated selective and high capacity capture of cells from feedstocks containing either a single type of cell or as a mixture of cells of Saccharomyces cerevisiae and Eschericia coli. The capture, washing and elution phases of the separation have been studied with respect to capacity, selectivity and yield of released cells. In these procedures, separation of cell types is achieved by the presence of multiple equilibrium stages within the expanded bed. The results show the potential for carrying out cell separations in expanded beds as an alternative to immunomagnetic cell separations. The combination of these recently developed technologies promises to be a powerful, but economic technique for cell separations involving simple equipment that can readily be scaled up.     

Osseous expansion of the cranial vault by craniotasis.

A study of cranial vault lengthening using a custom expandable fixation-distraction (craniotatic) appliance was performed in the young-adult rabbit model. Ten 24-week-old rabbits underwent circumferential suturectomy plus expansion (expanded group), 10 underwent circumferential suturectomy without expansion (sham control group), and 10 served as normal controls. The appliance was lengthened at a rate of 2.5 mm per week for 5 weeks. Serial lateral cephalometry, comparative dry-skull anthropometric measurements, and histologic examinations were performed. The expanded group demonstrated a significantly longer skull, cranial vault, anterior cranial base, posterior face, and orbit as compared with the control groups (p less than 0.05). Callus bone filled the distracted suturectomy and united the frontofacial complex to the posterior cranium. In conclusion, skull lengthening by distraction osteogenesis is possible in the rabbit model and offers a new technique for future investigation in the treatment of coronal synostosis.     

Implications of the Fast-Evolving Scale-Up of Adult Voluntary Medical Male Circumcision for Quality of Services in South Africa

Background

The scale-up of voluntary medical male circumcision (VMMC) services in South Africa has been rapid, in an attempt to achieve the national government target of 4.3 million adult male circumcisions for HIV prevention by 2016. This study assesses the effect of the scale-up on the quality of the VMMC program.

Methods and Findings

This analysis compares the quality of services at 15 sites operational in 2011 to (1) the same 15 sites in 2012 and (2) to a set of 40 sites representing the expanded program in 2012. Trained clinicians scored each site on 29 items measuring readiness to provide quality services (abbreviated version of the WHO Quality Assessment [QA] Guide) and 29 items to assess quality of surgical care provided (pre-op, surgical technique and post-op) based on the observation of VMMC procedures at each site. Declines in quality far outnumbered improvements. The negative effects in terms of readiness to provide quality services were most evident in expanded sites, whereas the declines in provision of quality services tended to affect both repeat sites and expanded sites equally. Areas of notable concern included the monitoring of adverse events, external supervision, post-operative counselling, and some infection control issues. Scores on quality of surgical technique tended to be among the highest across the 58 items observed, and the South Africa program has clearly institutionalized three “best practices” for surgical efficiency.

Conclusions

These findings demonstrate the challenges of rapidly developing large numbers of new VMMC sites with the necessary equipment, supplies, and protocols. The scale-up in South Africa has diluted human resources, with negative effects for both the original sites and the expanded program.     

Effect of radiation on skin expansion and skin flap viability in pigs

The aim of the present study was to investigate the effect of radiation treatment both on skin tissue expansion with the chronic inflation of subcutaneous expanders and on skin flap viability in surgically delayed and expanded skin in the pig. One flank in each of six pigs (initially weighing 17 +/- 1.8 kg) was randomly assigned for radiation treatment, and the contralateral flank served as a nonirradiated control. Three mirror-image, 8 x 10 cm, rectangular templates were marked on each flank; these templates were randomly assigned to the construction of a delayed skin flap (group A), a skin flap raised on expanded skin (group B), or a skin flap raised on expanded skin with a capsulectomy before flap surgery (group C). Radiation treatment was performed using sequential radiation with three fractions per week (810 cGy/fraction) for 2 weeks, with a total dose of 4,860 cGy. Twelve weeks after radiation treatment, skin expanders (8 x 10 cm) were installed subcutaneously in the locations assigned for skin expansion. Skin expansion by the inflation of subcutaneous skin expanders with saline twice weekly was started 8 weeks later and lasted for 3 weeks. Two weeks after surgical delay and the last skin expansion, 8 x 20 cm skin flaps were raised on the locations assigned for delayed skin flaps, expanded skin flaps, and expanded skin flaps with a capsulectomy. Skin flap viability was assessed 24 hours later using a fluorescein dye-staining technique. Skin expansion by the inflation of subcutaneous expanders with saline was slower (p < 0.05) in the radiated skin (39 +/- 6 ml/filling) than in the nonirradiated control skin (51 +/- 6 ml/filling). Radiation reduced the overall area of expanded skin by 23 percent (p < 0.05) compared with the control. Radiation treatment also reduced skin viability by 36 percent (p < 0.05) in the delayed skin flaps, 27 percent (p = 0.10) in the expanded skin flaps, and 36 percent (p < 0.05) in the expanded skin flaps with a capsulectomy when compared with their contralateral, nonirradiated controls. There were no significant differences in skin viability among these three types of skin flaps within the radiated and nonirradiated groups. Taken together, these observations indicate that radiation treatment reduced the effectiveness of the surgical delay procedure, the amount of subcutaneous skin expansion (by an increase in skin area), and skin flap viability. However, a capsulectomy alone did not affect the viability of skin flaps raised on expanded skin.