Minimising biomass/adsorbent interactions in expanded bed adsorption processes: a methodological design approach

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from crude feedstock. Interactions between solid matter in the feed suspension and fluidised adsorbent particles influence bed stability and therefore have a significant impact on protein adsorption in expanded beds. In order to design efficient and reliable EBA processes a strategy is needed, which allows to find operating conditions, where these adverse events do not take place. In this paper a methodological approach is presented, which allows systematic characterisation and minimisation of cell/adsorbent interactions with as little experimental effort as possible. Adsorption of BSA to the anion exchanger Streamline Q XL from a suspension containing S. cerevisiae cells was chosen as a model system with a strong affinity of the biomass towards the stationary phase. Finite bath biomass adsorption experiments were developed as an initial screening method to estimate a potential interference. The adhesiveness of S. cerevisiae to the anion exchanger could be reduced significantly by increasing the conductivity of the feedstock. A biomass pulse response method was used to find optimal operation conditions showing no cell/adsorbent interactions. A good correlation was found between the finite bath test and the pulse experiment for a variety of suspensions (intact yeast cells, E. coli homogenate and hybridoma cells) and adsorbents (Streamline Q XL, DEAE and SP), which allows to predict cell/adsorbent interactions in expanded beds just from finite bath adsorption tests. Under the optimised operating conditions obtained using the prior methods, the stability of the expanded bed was investigated during fluidisation in biomass containing feedstock (up to 15% yeast on wet weight basis) employing residence time distribution analysis and evaluation by an advanced model. Based on these studies threshold values were defined for the individual experiments, which have to be achieved in order to obtain an efficient EBA process. Breakthrough experiments were conducted to characterise the efficiency of BSA adsorption from S. cerevisiae suspensions in EBA mode under varying operating conditions. This allowed to correlate the stability of the expanded bed with its sorption efficiency and therefore could be used to verify the threshold values defined. The approach presented in this work provides a fast and simple way to minimise cell/adsorbent interactions and to define a window of operation for protein purification using EBA.     

Dye–ligand expanded bed adsorption of G6PDH from highly dense unclarified yeast extract

The application of dye–ligand expanded bed chromatography adsorption (EBA) of glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast extract was undertaken by using a commercially available expanded bed column (20 mm i.d.) and UpFront adsorbent (ρ = 1.5 g/mL) from UpFront Chromatography. The influence of biomass concentration on the adsorption capacity was explored by employing yeast extracts containing various biomass concentrations (5–30%, w/v). It was demonstrated that the biomass concentration had little effect on G6PDH adsorption performance. Feedstock containing 15% (w/v) biomass gave a relatively high recovery yield (>90%) of G6PDH compared to feedstock containing 30% (w/v) biomass, which gave a recovery of 75% G6PDH. Nevertheless, the enzyme specific activity of 7 U mg⁻¹ with a purification factor of 6 was achieved in the feedstock containing biomass concentration of 30% (w/v). The generic applicability of dye–ligand as an affinity tool in expanded bed chromatography is discussed.     

Non-electrostatic interactions between cultured Saccharomyces cerevisiae yeast cells and adsorbent beads in expanded bed adsorption: Influence of cell wall properties

The present work focused on the importance of performing EBA experiments under real process conditions. To this end, a high ionic strength medium was used. The influence of cultivating Saccharomyces cerevisiae yeast cells on their subsequent adsorption on a Q HyperZ anion-exchanger was investigated. Two types of industrial yeast cells were used. Once cultured, both types of cells presented similar hydrophilic surface properties and identical adsorption profiles on the anion-exchanger. This was significantly different from the results obtained in previous work on the same yeast cells, just rehydrated in saline buffer, a biological model widely used in the literature. It was postulated that unavoidable “sticky” compounds, initially present in the culture medium or formed during the drying process, were strongly adsorbed on the cell wall and could not be completely removed during the successive washings of the rehydrated cell suspension before use. This could dramatically alter the yeast surface properties and modify the biomass/adsorbent interactions and the bed hydrodynamics, thus demonstrating the necessity to work with yeast coming from fresh cultures. Using biologically active yeast cells allowed to really elucidating the main physico-chemical mechanisms involved in cell adsorption by focusing on the role of the non-electrostatic interactions. Two laboratory mutant yeast strains in which the protein-related cell wall biogenesis was affected, were tested. No significant differences were observed between hydrophilic and hydrophobic yeast cells: the bed remained stable at its initial value and there was a low adsorption ratio in a narrow range between 10% and 17%. This clearly demonstrated that non-electrostatic interactions play a minor role on the affinity of yeast to anion-exchanger adsorbents.     

Operational intensification by direct product sequestration from cell disruptates: application of a pellicular adsorbent in a mechanically integrated disruption-fluidised bed adsorption process

A novel prototype adsorbent, designed for intensified fluidised bed adsorption processes, was assembled by the emulsification coating of 4% (w/v) porous agarose upon a zirconia-silica solid core. The adsorbent, designated ZSA (particle density 1.75 g/ml, maximum pellicle depth 40 microm), was subjected to physical and biochemical comparison with the performance of two commercial adsorbents (Streamline and Macrosorb K4AX). Bed expansion qualities and hydrodynamic characteristics (N, D(axl) and B(o)) of ZSA demonstrated a marked robustness in the face of elevated velocities (up to 550 cm/h) and biomass loading (up to 30% (ww/v)) disrupted yeast cells. Cibracron Blue derivatives of the pellicular prototype (ZSA-CB), evaluated in the batch and fluidised bed recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from unclarified yeast disruptates, exhibited superior capacities and adsorption/desorption performance to the commercial derivatives. These advanced physical and biochemical properties facilitated a demonstration of the direct, mechanical coupling of bead-milling and fluidised bed adsorption in a fully integrated process for the accelerated recovery of G3PDH from yeast. The generic application of such pellicular adsorbents and integrated processes to the recovery of labile, intracellular products is discussed.     

The performance of anion exchange expanded bed adsorption chromatography on the recovery of G6PDH from unclarified feedstock with high biomass concentration

The bed stability of Streamline DEAE (p=1.2 g/mL) in a 20 mm (i.d.) glass expanded bed contactor, and its performance on the recovery of glucose 6-phosphate dehydrogenase (G6PDH) from unclarified yeast homogenate were investigated. A residence time distribution study showed that a stable expanded bed was achieved. The theoretical plate and Bodenstein numbers determined were 25 and 53, respectively. A recovery yield of 87% and purification factor of 4.1 were achieved in the operation using 5% (w/v) biomass concentration feedstock. The performance of the anion exchange EBAC was still considerable good at a biomass concentration as high as 15% (w/v).     

Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption

The use of expanded beds of STREAMLINE ion exchange adsorbents for the direct extraction of an intracellular enzyme glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast cell homogenates has been investigated. It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps. The purification of G6PDH from an unclarified yeast homogenate was chosen as a model system containing the typical features of a direct extraction technique. Optimal conditions for the purification were determined in small scale, packed bed experiments conducted with clarified homogenates. Results from these experiments were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 EBA apparatus. The use of an on-line rotameter for measuring and controlling the height of the expanded bed when operated in highly turbid feedstocks was demonstrated. STREAMLINE DEAE has been shown to be successful in achieving isolation of G6PDH from an unclarified homogenate with a purification factor of 12 and yield of 98% in a single step process. This ion exchange adsorbent is readily cleaned using simple cleaning-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent after many cycles of operation. The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. (c) 1996 John Wiley & Sons, Inc.     

Physicochemical parameters involved in the interaction of Saccharomyces cerevisiae cells with ion-exchange adsorbents in expanded bed chromatography

Expanded bed adsorption (EBA) is an interesting primary technology allowing the adsorption of target proteins from unclarified feedstock in order to combine separation, concentration, and purification steps. However, interactions between cells and adsorbent beads during the EBA process can strongly reduce the performance of the separation. So, to minimize these interactions, the mechanisms of cell adsorption on the support were investigated. Adsorption kinetics of the baker's yeast Saccharomyces cerevisiae on the anion exchanger Q Hyper Z were directly performed under real EBA operating conditions, in a lab-scale UpFront 10 column. The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast). For both types, a complete series of experiments for determining the adsorption profile versus time was performed, varying the superficial velocity or the pH. In parallel, the surface physicochemical properties of the cells (surface charge and electron-donor and electron-acceptor components) and of the support were determined. First of all, whatever the yeast types, the relation between cell adsorption and bed expansion has been highlighted, demonstrating the important role of hydrodynamic. However, for the type II yeast cells, adsorption increased dramatically, compared to the type I, even though it was shown that both types exhibited the same surface charge. In fact, there were strong differences in the Lewis acidic and basic components of the two yeasts. These differences explain the variable affinity toward the support, which was characterized by a strong electron-donor and a weak electron-acceptor component. These observed behaviors agreed with the colloidal theory. This work demonstrates that all kinds of interaction between the cells and the support (electrostatic, Lifshitz-van der Waals, acid/base) have to be taken into account together with hydrodynamic characteristics inside the bed.     

A study of the influence of yeast cell debris on protein and alpha-glucosidase adsorption at various zones within the expanded bed using in-bed sampling

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.     

Interaction of mammalian cell culture broth with adsorbents in expanded bed adsorption of monoclonal antibodies

The interaction of a mammalian cell culture broth with two commercially available adsorbents for the use in expanded bed adsorption (EBA) has been studied. A cation exchange resin (Streamline SP) and an affinity adsorbent (Streamline rProtein A) were compared with regard to adsorption of hybridoma cells during sample application as well as potential cell damage. The results showed that hybridoma cells interact significantly with an expanded bed of cation exchange adsorbents but not with the Protein A adsorbent. After application of 17–20 sedimented bed volumes a saturation of the Streamline SP resin with cells was noted. With both adsorbents no measurable cell damage was found and IgG₁ was recovered in approximately 95% yield. The capacity for IgG₁ adsorption at 3% breakthrough was 2.7 mg IgG₁/ml Streamline rProtein A at a constant fluid velocity of 380 cm/h and 1.0 mg IgG_l/ml Streamline SP at 215–240 cm/h fluid velocity.     

Purification of recombinant nucleocapsid protein of Newcastle disease virus from unclarified feedstock using expanded bed adsorption chromatography

In the present work, a single-step purification of recombinant nucleocapsid protein (NP) of the Newcastle disease virus (NDV) directly from unclarified feedstock using an expanded bed adsorption chromatography (EBAC) was developed. Streamline 25 column (ID = 25 mm) was used as a contactor and Streamline chelating adsorbent immobilized with Ni2+ ion was used as affinity adsorbent. The dynamic binding capacity of Ni2+ -loaded Streamline chelating adsorbent for the NP protein in unclarified feedstock was found to be 2.94 mg ml(-1) adsorbent at a superficial velocity of 200 cm h(-1). The direct purification of NP protein from unclarified feedstock using expanded bed adsorption has resulted in a 31% adsorption and 9.6% recovery of NP protein. The purity of the NP protein recovered was about 70% and the volume of processing fluid was reduced by a factor of 10. The results of the present study show that the IMA-EBAC developed could be used to combine the clarification, concentration and initial purification steps into a single-step operation.     

Direct process integration of cell disruption and fluidised bed adsorption in the recovery of labile microbial enzymes

The practical feasibility and generic applicability of the direct integration of cell disruption by bead milling with the capture of intracellular products by fluidised bed adsorption has been demonstrated. Pilot-scale purification of the enzyme L-asparaginase from unclarified Erwinia chrysanthemi disruptates exploiting this novel approach yielded an interim product which rivalled or bettered that produced by the current commercial process employing discrete operations of alkaline lysis, centrifugal clarification and batch adsorption. In addition to improved yield and quality of product, the process time during primary stages of purification was greatly diminished. Two cation exchange adsorbents, CM HyperD LS (Biosepra/Life Technologies) and SP UpFront (custom made SP form of a prototype stainless steel/agarose matrix, UpFront Chromatography) were physically and biochemically evaluated for such direct product sequestration. Differences in performance with regard to product capacity and adsorption/desorption kinetics were demonstrated and are discussed with respect to the design of adsorbents for specific applications. In any purification of L-asparaginase (pI = 8.6), product-debris interactions commonly diminish the recovery of available product. It was demonstrated herein, that immediate disruptate exposure to a fluidised bed adsorbent promoted concomitant reduction of product in the liquid phase, which clearly counter-acted the product-debris interactions to the benefit of product yield.     

Expanded bed affinity purification of bacterial α-amylase and cellulase on composite substrate analogue–cellulose matrices

Expanded bed purification of α-amylase and cellulase directly from unclarified fermentation broth was carried out on specially prepared composite affinity matrices. The concept used was incorporation of polymeric substrates/substrate analogue during cross-linking of cellulose to prepare rigid, porous, cross-linked composite affinity matrices for target enzymes. Of the several polymeric substrates/substrate-analogue used, alginic acid (AA) and microcrystalline cellulose (MCC) when used to prepare cross-linked composite matrices with cellulose, resulted in best affinity purification matrices for α-amylase and cellulase, respectively. These matrices were suitable for purification of the enzymes by batch, packed bed as well as expanded bed purification protocols. The optimized expanded bed protocol for α-amylase from Bacillus spp. B3 gave 51-fold purification on AA-CELBEADS with 69% recovery, whereas, cellulase from Bacillus spp. B21 was purified on MCC-CELBEADS to 18-fold purification with 97% recovery. The SDS-PAGE of both purified preparations showed single bands indicating significant purification on composite affinity adsorbents in a single step strategy.     

Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates

In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.     

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.     

Pilot scale recovery of monoclonal antibodies by expanded bed ion exchange adsorption

The aim of the investigations was to estimate the scale up properties of an efficient chromatographic first capture step for the recovery of murine IgG₁ from undiluted and unclarified hybridoma cell culture broth using an ion exchange matrix in expanded bed mode. The tested new sulfopropyl-based ion exchange matrix (Streamline^TM SP XL, Amersham Pharmacia Biotech) stands out due to its enhanced capacity compared to its precursor (Streamline^TM SP). Defining the working pH in preliminary electrophoretic analyses (titration curve, SDS-PAGE) and small-scaled chromatographic binding studies showed, that the optimal value for the IgG purification was pH 4.6, where a co-chromatography of the medium supplement albumin (500 mg l^-1, pI = 4.8) could not be avoided. Further scouting experiments dealt with the dynamic capacity of the matrix, which was evaluated by frontal adsorption analysis. In packed bed mode no break-through of the target protein was achieved even after 6.5 mg IgG per ml matrix were applied. These results could not be reproduced in expanded bed mode with cell-free supernatant, where the dynamic capacity was found to be only 1.5 mg IgG/ml SP XL. Processing cell-containing broth resulted in an additional decrease of the value down to 0.5 mg ml^-1, presumably caused by the remarkable biomass adsorption to the matrix. The search for the reasons led to the examination of the hydrodynamic conditions. Buffer experiments with a tracer substance (acetone) pointed out, that the flow in expanded bed was significantly more influenced by back-mixing effects and channel formations than in packed bed. These effects could be compensated with an enhanced viscosity of the liquid phase, which was achieved by the addition of glucose. As a result of the improved hydrodynamic conditions in the expanded bed, the dynamic capacity could be increased from 0.5 to more than 4.5 mg IgG/ml matrix for the processing of cell culture broth with 400 mM glucose. Finally, the scale up from a Streamline^TM 25 to a Streamline^TM 200 column was performed under conditions, which proved to be optimal: 100 L of unclarified hybridoma broth were concentrated with a binding rate of 95% in less than 3.5 hours. Loading the column no break-through of the target protein was achieved. However, the eluate still contained debris and cells, which points out the major disadvantage of the method: the biomass attachment to the matrix.     

Surface Energetics to Assess Microbial Adhesion onto Fluidized Chromatography Adsorbents

Cell‐to‐support interaction and cell‐to‐cell aggregation phenomena have been studied in a model system composed of intact yeast cells and agarose‐based chromatography adsorbent surfaces. Biomass components and beaded adsorbents were characterized by contact angle determinations with three diagnostic liquids and, complementarily, by zeta potential measurements. Such experimental characterization of the interacting surfaces has allowed the calculation of interfacial free energy of interaction in aqueous media vs. distance profiles. The extent of biomass adhesion was inferred from calculations performed assuming standard chromatographic conditions, but different adsorption modes. Several stationary support/mobile phase systems were considered, i.e., ion exchange, hydrophobic interaction, and pseudo‐affinity. The calculated interaction energy minima revealed marginal attraction between cells and cation exchangers or agarose‐matrix beads (U ≤ |10–20| kT) but strong attraction with anion exchangers (U ≥ |200–1000| kT). Other systems including hydrophobic interaction and chelating beads showed intermediate energy minimum values (U <$>\approx<$> |40–100| kT) for interaction with biological particles. However, the calculations also showed that working conditions in the presence of salt can promote cell aggregation apart from cell‐to‐support interaction. Predictions based on the application of the XDLVO approach were confirmed by independent experimental methods, e.g., biomass deposition experiments and laser diffraction spectroscopy. The understanding of biomass attachment onto chromatographic supports can help in alleviating process limitations normally encountered during direct (primary) sequestration of bioproducts.     

Surface energetics to assess biomass attachment onto immobilized metal-ion chromatography adsorbents in expanded beds

Cell-to-support interaction and cell-to-cell agglomeration phenomena have been studied in a model system composed of intact yeast cells and Chelating-Streamline™ adsorbents. Biomass components and beaded adsorbents were mainly characterized by contact angle determinations with three diagnostic liquids. Complementarily, zeta potential measurements were performed. These experimental values were employed to calculate free energy of interaction versus distance profiles in aqueous media. The effect of immobilized metal-ion type and buffer pH on the interaction energy was evaluated. Calculations indicated that moderate interaction between cell particles and adsorbent beads can develop due to the presence of Cu²⁺ ions onto the solid phase. The strength of interaction increased with buffer pH, within the range 6.0 to 8.3 e.g. secondary energy pockets increased from |15| to |60| kT. Cell-to-cell secondary energy minimum was ≥ |14| kT showing low-to-moderate tendencies to aggregate, particularly at pH ≥ 8. Extended DLVO predictions were generally confirmed by biomass deposition experiments. However, an exception was found when working with immobilized Cu²⁺ at pH 8 since yeast cells were able to sequestrate such immobilized ions. Therefore, lower-than-expected values for the depositions coefficient (α) were observed. Understanding biomass attachment onto Chelating supports can help in better design and operate expanded bed adsorption of bioproducts.     

Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology

The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.     

The influence of biomass on the hydrodynamic behavior and stability of expanded beds

Expanded bed adsorption is an innovative chromatographic technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed. Provided a perfectly classified fluidized bed (termed expanded bed) is formed in the crude feedstock and the biomass is not influencing protein transport towards the adsorbent surface, a sorption performance comparable to packed beds is found. The influence of biomass on the hydrodynamic stability of expanded beds is essential and was investigated systematically in this article. Residence-time distribution analyses were performed using model systems and a yeast suspension under various fluid-phase conditions. It is demonstrated that three factors (biomass/adsorbent interactions, biomass concentration, and flow rate) play an interdependent role disturbing the classified fluidization of an expanded bed. A clear correlation between the degree of aggregative fluidization--obtained by PDE modeling of RTD data--and the expansion behavior of the fluidized bed has been found. Thus, combining three analytical methods, namely cell transmission index analysis, expansion analysis, and RTD analysis provides a solid base for understanding and control of the fluidization behavior and thus further process design during the initial phase of process development.