A microscale bacterial cell disruption technique as first step for automated and miniaturized process development

Cell disruption is crucial during recovery of biopharmaceuticals overexpressed in E. coli, which tend to be produced intracellularly as insoluble inclusion bodies. Miniaturized high-throughput systems can accelerate the laborious downstream protocol for such biopharmaceuticals and enable integrated process-development. A fast and robust cell disruption method reflecting the protein and impurity profile of homogenates obtained by large-scale methods is required for such an approach. We established a miniaturized bead mill for parallel mechanical cell disruption at the microscale. Its total protein and impurity release, protein pattern, and particle size distribution were compared to results from microscale enzymatic digestion and referred to laboratory-scale high-pressure homogenization. Bead mill disruption led to equivalent protein and impurity release as well as to the same particle size profile as the large-scale reference. In contrast, lysates obtained by enzymatic digestion contained only 30–47% of overall protein, 17% of dsDNA, and 7–10% of endotoxin compared to those obtained by high-pressure homogenization; also larger debris was present in lysates after enzymatic digestion. The established method is fast, efficient, robust and comparable to current large-scale standards, allowing for parallelization of experiments. Thus, it is the method of choice for rapid integrated process development at the microscale.     

Cumulative sedimentation analysis of Escherichia coli debris size

A new method to measure Escherichia coli cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 mum to 0.3 mum as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or 10 passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioang 55: 556-564, 1997.     

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release.     

A novel technique for the measurement of disruption in high-pressure homogenization: Studies on E. coli containing recombinant inclusion bodies

The high-pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20 degrees C to 5 degrees C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4 degrees C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.     

A novel method to recover inclusion body protein from recombinant <Emphasis Type="Italic">E. coli</Emphasis> fed-batch processes based on phage <Emphasis Type="Italic">Φ</Emphasis>X174-derived lysis protein E

Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ΦX174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h^?1 and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of ?0.8 ± 0.3 h^?1. This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body processes to produce technical enzymes and biopharmaceutical products.     

An ultra scale‐down approach to study the interaction of fermentation,homogenization, and centrifugation for antibody fragment recovery from rec E. coli

Escherichia coli is frequently used as a microbial host to express recombinant proteins but it lacks the ability to secrete proteins into medium. One option for protein release is to use high‐pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale‐down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high‐pressure homogenization for recovery of an antibody fragment (Fab′) and the impact of fermentation harvest time. This approach is followed by microwell‐based USD centrifugation to study the removal of the resultant cell debris. Successful verification of this USD approach is achieved using pilot scale high‐pressure homogenization and pilot scale, continuous flow, disc stack centrifugation comparing performance parameters such as the fraction of Fab′ release, cell debris size distribution and the carryover of cell debris fine particles in the supernatant. The integration of fermentation and primary recovery stages is examined using USD monitoring of different phases of cell growth. Increasing susceptibility of the cells to disruption is observed with time following induction. For a given recovery process this results in a higher fraction of product release and a greater proportion of fine cell debris particles that are difficult to remove by centrifugation. Such observations are confirmed at pilot scale. Biotechnol. Bioeng. 2013 9999:XX–XX. © 2013 Wiley Periodicals, Inc. Biotechnol. Bioeng. 2013; 110: 2150–2160. © 2013 Wiley Periodicals, Inc.     

Measurements of multiply scattered light for on-line monitoring of changes in size distribution of cell debris suspension

Dual wavelength frequency-domain measurements of photon migration (FDPM) are conducted on filtrate samples obtained from an industrial centrifugation process designed to separate Escherichia coli cell debris from the inclusion bodies. FDPM measurements consist of detecting phase delay of intensity-modulated light at 670 and 820 (or 830) nm. Optical properties of isotropic scattering and absorption are obtained from the regression of phase delay data to the optical diffusion equation. We show that the corresponding intensity-based measurements alone cannot provide accurate and independent estimates for these optical properties. However, FDPM-derived scattering coefficients of filtrate solutions (primarily consisting of 0.1-0.2 micrometer E. coli cell debris) are sensitive to approximately 1 vol % of added inclusion bodies (of 1-2 micrometer size). The technique, theory, and future adaptation of FDPM as an on-line monitor to detect the loss of inclusion bodies in centrifugation following homogenization are presented and contrasted to conventional, intensity-based measurements.     

Comparative evaluation of different cell disruption methods for the release of recombinant hepatitis B core antigen from <Emphasis Type="Italic">Escherichia coli</Emphasis>

A comparative evaluation of five different cell-disruption methods for the release of recombinant hepatitis B core antigen (HBcAg) from Escherichia coli was investigated. The cell disruption techniques evaluated in this study were high-pressure homogenization, batch-mode bead milling, continuous-recycling bead milling, ultrasonication, and enzymatic lysis. Continuous-recycling bead milling was found to be the most effective method in terms of operating cost and time. However, the highest degree of cell disruption and amounts of HBcAg were obtained from the high-pressure homogenization process. The direct purification of HBcAg from the unclarified cell disruptate derived from high-pressure homogenization and bead milling techniques, using batch anion-exchange adsorption methods, showed that the conditions of cell disruption have a substantial effect on subsequent protein recovery steps.     

Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover

Biological processing of cellulosic biomass to fuels and chemicals would open up major new agricultural markets and provide powerful societal benefits, but pretreatment operations essential to economically viable yields have a major impact on costs and performance of the entire system. However, little comparative data is available on promising pretreatments. To aid in selecting appropriate systems, leading pretreatments based on ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime were evaluated in a coordinated laboratory program using a single source of corn stover, the same cellulase enzyme, shared analytical methods, and common data interpretation approaches to make meaningful comparisons possible for the first time. Each pretreatment made it possible to subsequently achieve high yields of glucose from cellulose by cellulase enzymes, and the cellulase formulations used were effective in solubilizing residual xylan left in the solids after each pretreatment. Thus, overall sugar yields from hemicellulose and cellulose in the coupled pretreatment and enzymatic hydrolysis operations were high for all of the pretreatments with corn stover. In addition, high-pH methods were found to offer promise in reducing cellulase use provided hemicellulase activity can be enhanced. However, the substantial differences in sugar release patterns in the pretreatment and enzymatic hydrolysis operations have important implications for the choice of process, enzymes, and fermentative organisms.     

Critical analysis of quantitative indicators of cell disruption applied to Saccharomyces cerevisiae processed with an industrial high pressure homogenizer

A comparison of quantification techniques was performed on suspensions of Saccharomyces cerevisiae which had been disrupted with a high pressure homogenizer. The quantification techniques included cell counting, monitoring protein release, UV absorbance, turbidity, sample mass loss analysis, variations in viscosity and measuring the particle size distribution of the homogenate. It was found that all quantification techniques resulted in similar relationships between the measured extent of disruption and number of passes through the homogenizer. The data from all techniques (except particle sizing) could be fitted to simple exponential decay models at various homogenization pressures. Turbidity, particle sizing and UV absorbance generally gave more conservative estimates of the extent of cell disruption compared to protein release and cell counting. Measuring both the turbidity and monitoring the release of cellular metabolites using UV absorbance gave simple, reliable and reproducible measures of disruption and were identified as being the most applicable to on-line disruption monitoring.     

Disruption of Saccharomyces cerevisiae using enzymatic lysis combined with high-pressure homogenization

The disruption of commercially-available pressed Bakers' yeast (Saccharomyces cerevisiae) was studied using a relatively new high-pressure homogenizer (the Microfluidizer). Initial experiments using only mechanical disruption generally gave low disruption yields (i.e., less than 40% disruption in 5 passes). Consequently combinations of two disruption methods, namely enzymatic lysis and subsequent homogenization, were tested to identify achievable levels of disruption. The enzyme preparation employed was Zymolyase, which has been shown to effectively lyse the walls of viable yeast. Yeast cell suspensions ranging in concentration from 0.6 to 15 gDW/L were disrupted with and without enzymatic pre-treatment. Final total disruption obtained using the combined protocol approached 100% with 4 passes at a pressure of 95 MPa, as compared to only 32% disruption with 4 passes at 95 MPa using only homogenization. A model is presented to predict the fraction disrupted while employing this novel enzymatic pretreatment.Nomenclature a exponent of pressure (-) - b exponent of number of passes (-) - K disruption constant (MPa^-a) - N number of passes (-) - P pressure (MPa) - R total fraction of cells disrupted (-) - Ro fraction of cells disrupted after enzymatic pre-treatment (-) - X cell concentration (dry weight) (gDW/L) abbreviation DW dry weight     

Evaluation of Escherichia coli cell disruption and inclusion body release using nucleic acid binding fluorochromes and flow cytometry

Many types of commercially valuable recombinant proteins produced by fermentation are expressed at high levels in Escherichia coli. Often, high-level expression in the host results in the formation of insoluble inclusion bodies. The release of these intracellular inclusion bodies from E. coli following cell disruption is a requirement for further downstream recovery. The ability to discern between intact unruptured cells and granules released from broken cells can provide valuable information for improving recovery yields in downstream purification. This paper describes a rapid and sensitive cytometry-based method that allows the simultaneous measurement of intact heat-killed E. coli and inclusion bodies using staining with nucleic acid binding fluorochromes.     

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.     

Enhanced disruption of Candida utilis using enzymatic pretreatment and high-pressure homogenization

The enhancement of the overall disruption of a native strain of Candida utilis (ATCC 9226) was studied using a combination of two methods, namely, pretreatment in the form of partial enzymatic lysis by Zymolyase followed by mechanical disruption in a Microfluidizer high-pressure homogenizer. The cells were grown in both batch and continuous cultures to examine the effect of specific growth rate on disruption. Cell suspensions ranging in concentration from 7 to 120 g DW/L were disrupted with and without enzymatic pretreatment. For yeast grown in batch culture, final total disruption obtained using the combined protocol approached 95% with four passes at a pressure of 95 MPa, as compared with only 65% disruption using only mechanical homogenization. A modified model was developed to predict the fraction disrupted by the enzymatic pretreatment-mechanical homogenization two-stage process. Predicted disruptions agreed favorably with experimental observations (maximum deviation of 20%) over a wide range of operating conditions. (c) 1994 John Wiley & Sons, Inc.     

A microscale yeast cell disruption technique for integrated process development strategies

Miniaturizing protein purification processes at the microliter scale (microscale) holds the promise of accelerating process development by enabling multi-parallel experimentation and automation. For intracellular proteins expressed in yeast, small-scale cell breakage methods capable of disrupting the rigid cell wall are needed that can match the protein release and contaminant profile of full-scale methods like homogenization, thereby enabling representative studies of subsequent downstream operations to be performed. In this study, a noncontact method known as adaptive focused acoustics (AFA) was optimized for the disruption of milligram quantities of yeast cells for the subsequent purification of recombinant human papillomavirus (HPV) virus-like particles (VLPs). AFA operates by delivering highly focused, computer-controlled acoustic radiation at frequencies significantly higher than those used in conventional sonication. With this method, the total soluble protein release was equivalent to that of laboratory-scale homogenization, and cell disruption was evident by light microscopy. The recovery of VLPs through a microscale chromatographic purification following AFA treatment was within 10% of that obtained using homogenization, with equivalent product purity. The addition of a yeast lytic enzyme prior to cell disruption reduced processing time by nearly 3-fold and further improved the comparability of the lysate to that of the laboratory-scale homogenate. In addition, unlike conventional sonication methods, sample heating was minimized (< =8 degrees C increase), even using the maximum power settings required for yeast cell disruption. This disruption technique in combination with microscale chromatographic methods for protein purification enables a strategy for the rapid process development of intracellularly expressed proteins.     

Increased disruption resistance of Candida utilis grown from survivors of enzymatic lysis combined with high-pressure homogenization

The resistance of Candida utilis (ATCC 9226) to disruption as a result of enzymatic pretreatment combined with high-pressure homogenization was found to increase when the yeast was grown from an inoculum which had previously been subjected to enzymatic pretreatment combined with high-pressure homogenization. The inoculum thus consisted of a mixture of undisrupted, viable cells and non-viable cells. The enzyme preparation employed was Zymolyase, which depolymerizes various components of the cell walls of viable yeast. A Microfluidizer was used for the high-pressure homogenization step. In order to obtain the 'disruption-resistant' cell fraction for use as an inoculum, 'normal' C. utilis was enzymatically pretreated, and subsequently homogenized (herein referred to as Microfluidization) using either three or 10 passes through the Microfluidizer at an operating pressure of 95 MPa. Yeast grown from the survivors of the enzyme/3-pass treatment were found to be somewhat more resistant to disruption by either enzymatic pretreatment alone or to enzymatic pretreatment followed by Microfluidization. Cells grown from enzyme/ 10-pass treated inocula exhibited the highest resistance to disruption. The 'disruption-resistant' fraction exhibited this characteristic through three serial re-cultivations. Possible mechanisms for the increased 'disruption-resistance' of this isolated population of C. utilis are presented.     

Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography

The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.     

Development of a high‐throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high‐throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high‐throughput disruption methods exist. The development of an automated, miniaturized, high‐throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high‐pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA‐based methods to mimic large‐scale homogenization processes. These results demonstrate that AFA‐mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130–140, 2018     

High throughput inclusion body sizing: Nano particle tracking analysis

The expression of pharmaceutical relevant proteins in Escherichia coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In the scientific literature, substantial effort has been devoted to the quantification of IB size. However, particle‐based methods used up to this point to analyze the physical properties of representative numbers of IBs lack sensitivity and/or orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells. TEM imaging in combination with manual grey scale image segmentation allowed the quantification of relative areas covered by the inclusion body within the cytosol. As a high throughput method nano particle tracking analysis (NTA) enables one to derive the diameter of inclusion bodies in cell homogenate based on a measurement of the Brownian motion. The NTA analysis of fixated (glutaraldehyde) and non‐fixated IBs suggests that high pressure homogenization annihilates the native physiological shape of IBs. Nevertheless, the ratio of particle counts of non‐fixated and fixated samples could potentially serve as factor for particle stickiness. In this contribution, we establish image segmentation of TEM pictures as an orthogonal method to size biologic particles in the cytosol of cells. More importantly, NTA has been established as a particle‐based, fast and high throughput method (1000–3000 particles), thus constituting a much more accurate and representative analysis than currently available methods.     

Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars

Miscanthus x giganteus is a tall perennial grass whose suitability as an energy crop is presently being appraised. There is very little information on the effect of pretreatment and enzymatic saccharification of Miscanthus to produce fermentable sugars. This paper reports sugar yields during enzymatic hydrolysis from ammonia fiber expansion (AFEX) pretreated Miscanthus. Pretreatment conditions including temperature, moisture, ammonia loading, residence time, and enzyme loadings are varied to maximize hydrolysis yields. In addition, further treatments such as soaking the biomass prior to AFEX as well as washing the pretreated material were also attempted to improve sugar yields. The optimal AFEX conditions determined were 160 degrees C, 2:1 (w/w) ammonia to biomass loading, 233% moisture (dry weight basis), and 5 min reaction time for water-soaked Miscanthus. Approximately 96% glucan and 81% xylan conversions were achieved after 168 h enzymatic hydrolysis at 1% glucan loading using 15 FPU/(g of glucan) of cellulase and 64 p-NPGU/(g of glucan) of beta-glucosidase along with xylanase and tween-80 supplementation. A mass balance for the AFEX pretreatment and enzymatic hydrolysis process is presented.