Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models

This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies. This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied. An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery. The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance. The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted.     

The use of laboratory centrifugation studies to predict performance of industrial machines: studies of shear-insensitive and shear-sensitive materials

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.     

The performance of a scaled down industrial disc stack centrifuge with a reduced feed material requirement

A method is described for the scale-down of a disc stack centrifuge which reduces the number of separating discs and also the liquid and solid hold-up of the centrifuge bowl. This is to enable a reduced volume of process material to be used for study of clarification. Scale-down is achieved in stages using a series of interlocking inserts to suit particular applications. Maximum scale-down gives a 76% reduction in the separation area and a bowl volume reduction of 70%. Separation performance of the full stack machine and scale-down variants is compared using the grade efficiency concept. Polyvinyl acetate and bakers' yeast homogenate particle suspensions are used for the comparison. The grade efficiency curves produced by the scale-down variants closely follow the curves for the full stack machine. This resulted in supernatants of the same volume size distribution and concentration when using scale-down methodology to mimic the full scale operation.

     

Comparison of the Baker's yeast process performance in laboratory and production scale

A 215 m³ industrial bubble column reactor for fedbatch production of Baker's yeast was sampled for sugar, to investigate the extent of concentration gradients. The results verify that such gradients exist: the concentration is higher closer to the feeding point. Effects of sugar heterogeneities at different scales were studied by 1)?performing a volumetric scale-down of the industrial process in a laboratory stirred tank reactor (STR); 2) performing the same scaled down process in a Scale-Down Reactor (SDR) with repeated short term exposure of the cells to high sugar concentrations. In this reactor about 10% of the Baker's yeast culture was intermittently exposed to high (0.45–1.9?g?l^?1) concentrations of sugar, for periods of 60 seconds. It was found that physiological parameters of glycolysis and respiration were affected by environmental heterogeneities: 1) A biomass yield reduction of about 6–7% was found, with both the production reactor and the SDR, as compared to the homogeneous reactor. The loss of yield is interpreted in terms of a metabolic by-pass via ethanol, where cells are consuming and producing ethanol with different yields. 2) The maximum respiration rate was higher in cells produced in the production unit and in the SDR. 3) The product quality, expressed as gassing power of the yeast in a dough, was increased for sweet and non-sugar doughs in the SDR, and for sweet doughs in the production reactor. Thus, the SDR, when run with defined glucose gradients, in some aspects resembles the large reactor. It could be regarded as a tool for scale-down and scale-up studies and may be useful in investigations on the scale-up sensitivity of a process.     

Basic concepts in Q membrane chromatography for large-scale antibody production

The large-scale production of recombinant human monoclonal antibodies demands economical purification processes with high throughputs. In this article we briefly describe a common antibody process and evaluate the Q membrane adsorber for process-scale antibody production as an alternative to a Q-packed-bed column in a flow-through mode. The scientific concepts underlining Q membrane technology and its application are reviewed. The disadvantages and advantages of using Q membrane chromatography as a purification unit in large-scale production are discussed, including problems initially seen with the Q membrane scale-down model but solved with the invention of a new scale-down model. The new Q-membrane unit operation has a process capacity greater than 3,000 g/m(2) or 10.7 kg/L with a LRV over 5 for four model viruses. In this Review, a cost analysis illustrates that Q membrane chromatography is a viable alternative to Q column chromatography as a polishing step in a flow-through mode for process-scale antibody production.     

Ultra scale-down to define and improve the relationship between flocculation and disc-stack centrifugation

The recovery of intracellular recombinant proteins produced in microbial systems typically requires physical, chemical or thermal treatment of the cells post-harvest to release the product into the broth, followed by removal of the cell debris using centrifugation or tangential flow filtration. Often a precipitation or flocculation step is introduced to facilitate the liquid-solid separation. Due to the complex nature of the cell materials and the unit operations, it is difficult to obtain data at laboratory scale that closely reflect the performance of these operations on larger scales (pilot or manufacturing). This study uses a predictive scale-down model that enables rapid optimization of the operating conditions for a flocculation followed with a centrifugation step using only small volumes (20 mL) of a high solids ( approximately 20% w/w) E. coli heat extract. Results obtained show that, with proper theoretical and experimental consideration to account for high cell density, conditions could be found that improve the beneficial interaction between flocculation and centrifugation. These experiments suggested that adding a higher level of a cationic polymer could substantially increase the strength of the flocculated particles produced, thereby enhancing overall clarification performance in a large scale centrifuge. This was subsequently validated at pilot scale.     

A systematic approach for scale-down model development and characterization of commercial cell culture processes

The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.     

Substrate oscillations boost recombinant protein release from Escherichia coli

Intracellular production of recombinant proteins in prokaryotes necessitates subsequent disruption of cells for protein recovery. Since the cell disruption and subsequent purification steps largely contribute to the total production cost, scalable tools for protein release into the extracellular space is of utmost importance. Although there are several ways for enhancing protein release, changing culture conditions is rather a simple and scalable approach compared to, for example, molecular cell design. This contribution aimed at quantitatively studying process technological means to boost protein release of a periplasmatic recombinant protein (alkaline phosphatase) from E. coli. Quantitative analysis of protein in independent bioreactor runs could demonstrate that a defined oscillatory feeding profile was found to improve protein release, about 60 %, compared to the conventional constant feeding rate. The process technology included an oscillatory post-induction feed profile with the frequency of 4 min. The feed rate was oscillated triangularly between a maximum (1.3-fold of the maximum feed rate achieved at the end of the fed-batch phase) and a minimum (45 % of the maximum). The significant improvement indicates the potential to maximize the production rate, while this oscillatory feed profile can be easily scaled to industrial processes. Moreover, quantitative analysis of the primary metabolism revealed that the carbon dioxide yield can be used to identify the preferred feeding profile. This approach is therefore in line with the initiative of process analytical technology for science-based process understanding in process development and process control strategies.     

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.     

Improvement of robustness and ethanol production of ethanologenic Saccharomyces cerevisiae under co-stress of heat and inhibitors

Bioethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, yeast cells are challenged by various environmental stresses during the industrial process of ethanol production. The robustness under heat, acetic acid, and furfural stresses was improved for ethanologenic S. cerevisiae in this work using genome shuffling. Recombinant yeast strain R32 could grow at 45°C, and resist 0.55% (v/v) acetic acid and 0.3% (v/v) furfural at 40°C. When ethanol fermentation was conducted at temperatures ranging from 30 to 42°C, recombinant strain R32 always gave high ethanol production. After 42 h of fermentation at 42°C, 187.6 ± 1.4 g/l glucose was utilized by recombinant strain R32 to produce 81.4 ± 2.7 g/l ethanol, which were respectively 3.4 and 4.1 times those of CE25. After 36 h of fermentation at 40°C with 0.5% (v/v) acetic acid, 194.4 ± 1.2 g/l glucose in the medium was utilized by recombinant strain R32 to produce 84.2 ± 4.6 g/l of ethanol. The extent of glucose utilization and ethanol concentration of recombinant strain R32 were 6.3 and 7.9 times those of strain CE25. The ethanol concentration produced by recombinant strain R32 was 8.9 times that of strain CE25 after fermentation for 48 h under 0.2% (v/v) furfural stress at 40°C. The strong physiological robustness and fitness of yeast strain R32 support its potential application for industrial production of bioethanol from renewable resources such as lignocelluloses.     

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large‐scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale‐up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha‐1‐antitrypsin (rAAT) by co‐infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD₆₀₀ of 0.5 and a 0.25‐min vacuum infiltration, and multiple 1‐min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post‐induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Combined chemical and mechanical processes for the disruption of bacteria.

Mechanical cell disruption by high pressure homogenisation or high speed bead mills is currently the general method of choice for the large scale disruption of micro-organisms; however, deleterious effects include the high energy requirement, the need for efficient cooling to prevent the excessive heating of the product and the micronisation of cell debris. Certain chemical treatments for microbial cell disruption alter the permeability of bacteria and yeasts, allowing partial release of soluble products. Such treatments are insufficient for the recovery of granular intracellular products. As cell wall strength has been cited as a major factor influencing the requirements for efficient mechanical disruption, the use of chemical pretreatment to decrease cell wall strength prior to mechanical breakage by homogenisation has been considered. The following treatments were shown to increase the sensitivity of the Gram-negative bacterium, Alcaligenes eutrophus, to disruption: alkaline pH shock, the addition of an anionic detergent, increase of the monovalent cation concentration, the addition of EDTA and enzymic lysis by lysozyme. These pretreatments allow equivalent disruption to be achieved at lower operating pressures or fewer passes through the homogeniser. Alkaline pH pretreatment at pH 10.5 allowed a 37.5% increase in soluble protein release on subsequent homogenisation. An increase of some 30% in soluble protein release was found following prior addition of 0.137 M monovalent cations (Na+ or K+) at 60 degrees C. Treatment with an anionic detergent showed a considerable decrease in the number of passes required through the homogeniser. Maximum cell rupture can thus be accomplished at reduced energy inputs.     

Scale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according to substrate heterogeneities

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane permeability and the synthesis of several outer-membrane components allowing to cope with substrate limitation commonly found in high cell density culture. A comparative analysis of protein leakage has thus been performed in well-mixed bioreactors and in scale-down devices. The extracellular proteome of E.coli has been investigated by 2D-gel electrophoresis and identified by subsequent MALDI-TOF analysis. On 110 picked spots, 67 proteins have been identified and the sub-localisation and the molecular function of these proteins have been determined. A majority of the extracellular proteome was composed of outer-membrane and periplasmic proteins (64 %) confirming the fact that leakage is involved in high cell density cultures. About 50 % of this extracellular proteome was composed of transport and binding proteins. Furthermore, the more abundant spots on the gel corresponded to porin proteins and periplasmic transporters. In particular, the OmpC porin was found to be very abundant. Moreover, the scale-down effect on this extracellular proteome has been investigated by two-dimensional differential in-gel electrophoresis analysis (2D-DIGE), and significant differences have been observed by comparison with culture carried out in well-mixed systems. Indeed, since substrate limitation signal is alleviated in this kind of apparatus, cell permeability was lowered as shown by flow cytometry. In scale-down conditions, protein leakage was thus less abundant.     

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.     

Development and qualification of a representative scale-down model of automated Ficoll-based processing of a cell-based therapeutic according to quality by design principles

Background aimsThis article describes the development of a small-scale model for Ficoll-based cell separation as part of process development of an advanced therapy medicinal product and its qualification. Because of the complexity of biological products, their manufacturing process as well as characterization and control needs to be accurately understood. Likewise, scale-down models serve as an indispensable tool for process development, characterization, optimization and validation. This scale-down model represents a cell processor device widely used in advance therapies. This approach is inteded to optimise resources and to focus its use on process characterisation studies under the paradigm of the Quality by design. A scale-down model should reflect the large manufacturing scale. Consequently, this simplified system should offer a high degree of control over the process parameters to depict a robust model, even considering the process limitations. For this reason, a model should be developed and qualified for the intended purpose.MethodsProcess operating parameters were studied, and their resulting performance at full scale was used as a baseline to guide scale-down model development. Once the model was established, comparability runs were performed by establishing standard operating conditions with bone marrow samples. These analyses showed consistency between the bench and the large scale. Additionally, statistical analyses were employed to demonstrate equivalence.ResultsThe process performance indicators and assessed quality attributes were equivalent and fell into the acceptance ranges defined for the large-scale process.ConclusionsThis scale-down model is suitable for use in process characterization studies.     

Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment

The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.     

A fast and simple approach to optimize the unit operation high pressure homogenization - a case study for a soluble therapeutic protein in E. coli

Escherichia coli is one of the most commonly used host organisms for the production of recombinant biopharmaceuticals. E. coli is usually characterized by fast growth on cheap media and high productivity, but one drawback is its intracellular product formation. Product recovery from E. coli bioprocesses requires tedious downstream processing (DSP). A typical E. coli DSP for an intracellular product starts with a cell disruption step to access the product. Different methods exist, but a scalable process is usually achieved by high pressure homogenization (HPH). The protocols for HPH are often applied universally without adapting them to the recombinant product, even though HPH can affect product quantity and quality. Based on our previous study on cell disruption efficiency, we aimed at screening operational conditions to maximize not only product quantity, but also product quality of a soluble therapeutic protein expressed in E. coli. We screened for critical process parameters (CPPs) using a multivariate approach (design of experiments; DoE) during HPH to maximize product titer and achieve sufficient product quality, based on predefined critical quality attributes (CQAs). In this case study, we were able to gain valuable knowledge on the efficiency of HPH on E. coli cell disruption, product release and its impact on CQAs. Our results show that HPH is a key unit operation that has to be optimized for each product.     

An effective extracellular protein secretion by an ABC transporter system in <Emphasis Type="Italic">Escherichia coli</Emphasis>: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.     

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.     

Preparation and characterization of poly(D,L-lactide-co-glycolide) microspheres for controlled release of human growth hormone

The purpose of this research was to assess the physicochemical properties of a controlled release formulation of recombinant human growth hormone (rHGH) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) composite microspheres. rHGH was loaded in poly(acryloyl hydroxyethyl) starch (acHES) microparticles, and then the protein-containing microparticles were encapsulated in the PLGA matrix by a solvent extraction/evaporation method. rHGH-loaded PLGA microspheres were also prepared using mannitol without the starch hydrogel microparticle microspheres for comparison. The detection of secondary structure changes in protein was investigated by using a Fourier Transfer Infrared (FTIR) technique. The composite microspheres were spherical in shape (44.6±2.47 μm), and the PLGA-mannitol microspheres were 39.7±2.50 μm. Drug-loading efficiency varied from 93.2% to 104%. The composite microspheres showed higher overall drug release than the PLGA/mannitol microspheres. FTIR analyses indicated good stability and structural integrity of HGH localized in the microspheres. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.