European Journal of Pharmaceutics and Biopharmaceutics (v.78, #2)
Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors
by Maria De Jesus; Florian M. Wurm (pp. 184-188).
Mammalian cells in bioreactors as production host are the focus of this review. We wish to briefly describe today’s technical status and to highlight emerging trends in the manufacture of recombinant therapeutic proteins, focusing on Chinese hamster ovary (CHO) cells. CHO cells are the manufacturing host system of choice for more than 70% of protein pharmaceuticals on the market . The current global capacity to grow mammalian cells in bioreactors stands at about 0.5 million liters, whereby the largest vessels can have a working volume of about 20,000l.We are focusing in this article on the upstream part of protein manufacturing. Over the past 25years, volumetric yields for recombinant cell lines have increased about 20-fold mainly as the result of improvements in media and bioprocess design. Future yield increases are expected to come from improved gene delivery methods, from improved, possibly genetically modified host systems, and from further improved bioprocesses in bioreactors. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight here current research in our own laboratories.
Keywords: CHO; Bioreactor; Orbital shaking; Recombinant protein; Transient gene expression; Disposables
New method to produce equine antirabies immunoglobulin F(ab′)2 fragments from crude plasma in high quality and yield
by Sukanda Kittipongwarakarn; Andrea Hawe; Ruedeeporn Tantipolphan; Kornvika Limsuwun; Sumana Khomvilai; Satit Puttipipatkhachorn; Wim Jiskoot (pp. 189-195).
A new method consisting of protein G affinity chromatography, pepsin digestion, followed by a combination of protein A affinity chromatography and ultrafiltration, was proposed to produce equine antirabies immunoglobulin F(ab′)2 fragments in higher yield and bioactivity than the current WHO method.Rabies is still a major cause of human deaths in several developing countries. According to the World Health Organization, administration of antirabies serum or antirabies immunoglobulin is recommended for patients who have experienced a category-III exposure to rabies. Improvement of antirabies immunoglobulin production is required to enhance safety and efficacy of the products. In this paper, a new method to produce equine antirabies immunoglobulin F(ab′)2 fragments from crude plasma is proposed. First, protein G affinity chromatography was used to purify IgG from equine plasma. Moreover, purification of IgG was shown to facilitate its digestion by pepsin. Compared to the direct digestion of crude plasma, a lower amount of pepsin and a shorter digestion time were required to completely digest the purified IgG to F(ab′)2. Complete digestion of purified IgG to F(ab′)2 was achieved at a pepsin/IgG (w/w) ratio of 5:45 with preservation of structure and potency. Finally, purification of F(ab′)2 was accomplished by a combination of protein A affinity chromatography and ultrafiltration with a 50-kDa nominal molecular weight cut-off membrane. The new process resulted in 68.9±0.6 (%) total recovery of F(ab′)2 and a F(ab′)2 product of high potency.
Keywords: Antirabies serum; Equine antirabies immunoglobulin; Purification; Pepsin digestion; Protein G affinity chromatography; Protein A affinity chromatography; F(ab′); 2; fragments
Addressing new analytical challenges in protein formulation development
by Henryk Mach; Tudor Arvinte (pp. 196-207).
As the share of therapeutic proteins in the arsenal of modern medicine continue increasing, relatively little progress has been made in the development of analytical methods that would address specific needs encountered during the development of these new drugs. Consequently, the researchers resort to adaptation of existing instrumentation to meet the demands of rigorous bioprocess and formulation development. In this report, we present a number of such adaptations as well as new instruments that allow efficient and precise measurement of critical parameters throughout the development stage. The techniques include use of atomic force microscopy to visualize proteinacious sub-visible particles, use of extrinsic fluorescent dyes to visualize protein aggregates, particle tracking analysis, determination of the concentration of monoclonal antibodies by the analysis of second-derivative UV spectra, flow cytometry for the determination of subvisible particle counts, high-throughput fluorescence spectroscopy to study phase separation phenomena, an adaptation of a high-pressure liquid chromatography (HPLC) system for the measurement of solution viscosity and a variable-speed streamlined analytical ultracentrifugation method. An ex vivo model for understanding the factors that affect bioavailability after subcutaneous injections is also described. Most of these approaches allow not only a more precise insight into the nature of the formulated proteins, but also offer increased throughput while minimizing sample requirements.
Keywords: Therapeutic proteins; Formulation; Spectroscopic methods; Phase separation; Flow cytometry; Analytical ultracentrifugation
Development of high concentration protein biopharmaceuticals: The use of platform approaches in formulation development
by Nicholas W. Warne (pp. 208-212).
The development of highly concentrated solutions of monoclonal antibodies has become increasingly popular in biotechnology firms as doses increase, and the demand for sub-cutaneous injection formulations rises for reasons of convenience and compliance. Protein concentrations often exceed 100mg/mL. Unfortunately, the reduced success rate of commercialization of biotechnology products result in a situation in which the majority of biologics that enter clinical trials have a very low probability of becoming commercial products. Under these circumstances, the formulation scientist must make judicious use of his or her available time and resources and must avoid over-investing in early phase 1 compounds. This circumstance has driven many laboratories to develop so-called “platform formulations” in which a suitable, high concentration, robust formulation, or dosage form is utilized broadly across a number of early stage biologics at a savings of time and effort. This highly pragmatic approach increases the efficiency of developing early clinical candidates with fewer resources, while maintaining clinical flexibility, thus saving their effort for later stage candidates for which proof of concept has been demonstrated and for which a more optimized and robust formulation and process, suitable for commercial application, must be developed.
Keywords: Monoclonal antibody; Platform; Efficiency; Formulation; Dosage form; Protein
Comparison of analytical methods to detect instability of etanercept during thermal stress testing
by Andreas van Maarschalkerweerd; Gert-Jan Wolbink; Steven O. Stapel; Wim Jiskoot; Andrea Hawe (pp. 213-221).
Several analytical methods were used to detect instability in three batches of etanercept showing significant differences in sensitivity in the order of: Bis-Ans fluorescence>HP-SEC>DLS>Far-UV CD spectroscopy>ELISA>Second derivative UV spectroscopy.The aim was comparing the capability of a set of analytical methods to detect physical instability (focus on aggregation and structural changes) of etanercept during thermal stress testing as early as possible. Pre-filled syringes of Enbrel® 50mg from three batches were thermally stressed for one week at 50°C. Samples were taken at days 0, 1, 2, 3, 4 and 7, and analyzed with high-performance liquid size exclusion chromatography (HP-SEC), SDS–PAGE gel electrophoresis, dynamic light scattering (DLS), light obscuration, extrinsic fluorescence (Bis-ANS), far-UV circular dichroism (CD) spectroscopy, second derivative UV spectroscopy (UV), and enzyme-linked immunosorbent assay (ELISA). Thermal stress resulted in the formation of small soluble aggregates (HP-SEC, DLS) which were in part covalent (SDS–PAGE), and conformationally changed (Bis-ANS, CD, UV). No significant increase in subvisible particles was detected by light obscuration. An apparent increase in TNF-α binding to etancercept in the stressed formulations was found by ELISA. The three batches were comparable when unstressed, but showed slight differences in aggregation tendency. Bis-ANS fluorescence was most sensitive with respect to early-stage detection of heat-induced instability of etanercept (significant changes already at day 1), followed by HP-SEC (day 2) and DLS (day 3). This points towards a degradation mechanism involving exposure of hydrophobic patches due to partial unfolding followed by aggregation.
Keywords: Abbreviations; HPE; high-performance ELISA buffer; TNF-α; tumor necrosis factor-α; MRW; mean residue ellipticity; AUC; area under the curve; HMW; molecular weight species; Bis-ANS; 4,4′-dianilino-1,1′-binapthyl-5,5′-disulfonic acid dipotassium salt; ELISA; enzyme-linked immunosorbent assay; DLS; dynamic light scattering; HP-SEC; high-performance liquid size exclusion chromatography; PDI; polydispersity indexAggregation; Extrinsic fluorescence; Bis-ANS; Enbrel; Protein stability; Conformational changes
The effect of GlycoPEGylation on the physical stability of human rFVIIa with increasing calcium chloride concentration
by Bitten Plesner; Peter Westh; Anders D. Nielsen (pp. 222-228).
GlycoPEGylation postpones the calcium induced thermal destabilisation of recombinant human factor VIIa. Increasing the CaCl2 concentration from 10mM to 35mM resulted in a decrease in the apparent unfolding temperature, Tm, of rFVIIa, whereas the concentration of CaCl2 has to be raised to 100mM in order to see the same effect on the GlycoPEGylated rFVIIa compounds.The effects of calcium chloride on the structural, kinetic and thermal stability of recombinant human factor VIIa (rFVIIa) were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10kDa PEG and a branched 40kDa PEG, respectively. Three different CaCl2 concentrations were used: 10mM, 35mM and 100mM. The secondary structure and tertiary structure of rFVIIa at 25°C, measured by circular dichroism (CD), were maintained upon GlycoPEGylation as well as CaCl2 content. In contrast, the thermal stability of the three rFVIIa compounds, measured by differential scanning calorimetry (DSC) and circular dichroism (CD), and aggregation behaviour, measured by light scattering (LS), were affected by the increasing calcium concentration. Increasing the CaCl2 concentration from 10mM to 35mM resulted in a decrease in the apparent unfolding temperature, Tm, of rFVIIa, whereas the concentration of CaCl2 has to be raised to 100mM in order to see the same effect on the GlycoPEGylated rFVIIa compounds. The temperature of aggregation of rFVIIa, Tagg, increased as the CaCl2 concentration increased from 35mM to 100mM, while Tagg for the GlycoPEGylated rFVIIa compounds was practically independent of the CaCl2 concentration. From the obtained results, it is concluded that GlycoPEGylation postpones the calcium induced thermal destabilisation of rFVIIa, and a much higher calcium concentration also postpones the thermally induced aggregation of rFVIIa. The thermally induced aggregation of the GlycoPEGylated rFVIIa compounds is unaffected by an increasing calcium chloride concentration.
Keywords: rFVIIa; GlycoPEGylation; Protein aggregation; Protein unfolding; Salt effects; Ca; 2+
Enhanced physical stability of human calcitonin after methionine oxidation
by Filippo Mulinacci; Emilie Poirier; Martinus A.H. Capelle; Robert Gurny; Tudor Arvinte (pp. 229-238).
The current work describes the stabilizing effect of methionine oxidation on the physical stability of hcT.Calcitonin is a blood-calcium-lowering peptide, present in different species, which inhibits the resorption of bone by osteoclasts. Human calcitonin (hCT) is one of the few calcitonin peptides, which contains a methionine residue; this residue is in position 8. Methionines are known to be readily oxidized to sulfoxides both in vivo and in vitro. The current work describes the effect of methionine oxidation on the physical stability of hCT. Aggregation kinetics of human calcitonin were studied at different pH values by intrinsic fluorescence spectroscopy, turbidity at 350nm, microscopy analyses, Nile Red, and 1,8-ANS fluorescence emission. In all the experiments, methionine oxidation reduced the aggregation rate of human calcitonin. The effect of methionine oxidation was independent of pH. Fluorescence lifetime data also showed that the conformation of hCT in the aggregated state can be influenced by methionine oxidation. A hypothesis for the enhanced physical stability of oxidized hCT is presented and discussed.
Keywords: Aggregation; Methionine oxidation; Human calcitonin; Stability; Aggregation kineticsAbbreviations; hCT; human calcitonin; sCT; salmon calcitonin; MS; mass spectrometry; ESI; electrospray ionization; Da; dalton; 1,8-ANS; 1-anilinonaphthalene-8-sulfonic acid; TFA; trifluoroacetic acid
Influence of pH and ionic strength on IgG adsorption to vials
by Johannes Mathes; Wolfgang Friess (pp. 239-247).
IgG adsorption to borosilicate glass vials is mediated by electrostatic interactions and the charge of both sorbent surface and protein, together with the charge screening by ions, is of fundamental importance.It was the aim of this work to investigate the influence of the formulation parameters pH and ionic strength on the adsorption of IgG to borosilicate glass vials. The charge characteristics of IgG and glass surface were determined by isoelectric focusing and electrokinetic measurements. It could be shown that IgG adsorption highly depends on formulation pH and ionic strength. The amount of IgG adsorbed results from an interplay of attractive and repulsive electrostatic interactions between protein molecules and the glass surface as well as among adsorbed protein molecules. The pH value where the ion uptake in the adsorption boundary was minimal coincided well with the pH of maximum adsorption. At pH 4, the presence of Na2SO4 gave rise to a stronger increase in adsorption than NaCl at equal ionic strength, whereas we observed no differences at e.g. pH 7.2 and 8.6. In summary, it can be stated that IgG adsorption on borosilicate glass is to a large extent mediated by electrostatic interactions. Other driving forces like hydrophobic interactions or surface-induced structural alterations contribute to a much lesser extent.
Keywords: Protein formulation; Protein adsorption; Vials; Borosilicate glass; Ionic strength; Adsorption isotherm
The freezing step in lyophilization: Physico-chemical fundamentals, freezing methods and consequences on process performance and quality attributes of biopharmaceuticals
by Julia Christina Kasper; Wolfgang Friess (pp. 248-263).
Understanding how the freezing step in lyophilization influences process performance and quality attributes of the product helps to develop more efficient lyophilization cycles and to improve product quality and stability.Lyophilization is a common, but cost-intensive, drying process to achieve protein formulations with long-term stability. In the past, typical process optimization has focused on the drying steps and the freezing step was rather ignored. However, the freezing step is an equally important step in lyophilization, as it impacts both process performance and product quality.While simple in concept, the freezing step is presumably the most complex step in lyophilization. Therefore, in order to get a more comprehensive understanding of the processes that occur during freezing, the physico-chemical fundamentals of freezing are first summarized. The available techniques that can be used to manipulate or directly control the freezing process in lyophilization are also reviewed. In addition, the consequences of the freezing step on quality attributes, such as sample morphology, physical state of the product, residual moisture content, reconstitution time, and performance of the primary and secondary drying phase, are discussed. A special focus is given to the impact of the freezing process on protein stability.This review aims to provide the reader with an awareness of not only the importance but also the complexity of the freezing step in lyophilization and its impact on quality attributes of biopharmaceuticals and process performance. With a deeper understanding of freezing and the possibility to directly control or at least manipulate the freezing behavior, more efficient lyophilization cycles can be developed, and the quality and stability of lyophilized biopharmaceuticals can be improved.
Keywords: Lyophilization; Freeze-drying; Modifications of the freezing step; Influence on process performance; Influence on product quality; Influence on protein stability
The aggregative stability of β-lactoglobulin in glassy mixtures with sucrose, trehalose and dextran
by N. Perez-Moral; C. Adnet; T.R. Noel; R. Parker (pp. 264-270).
Thermally induced aggregation in glassy β-lactoglobulin-carbohydrate mixtures is slowed by increasing dilution with carbohydrate though at concentrations of 50 and 75 wt.% dextran the aggregation rate is enhanced suggesting protein-polymer phase separation has occurred.The aim of this study was to investigate the effect of the addition of different carbohydrates on the thermally induced aggregation of a model globular protein, β-lactoglobulin (BLG), in the glass state. Amorphous mixtures of BLG with trehalose, sucrose and dextran were prepared by freeze-drying, their glass behaviour was characterised using calorimetry and thermally induced aggregation was measured using size exclusion chromatography. Pure BLG shows increasing levels of aggregation when heated in the temperature range 70–100°C for 48–144h. The addition of the disaccharides sucrose and trehalose both resulted in a decrease in aggregation rate which approached negligible rates at 50wt.% carbohydrate. The effect of dextran addition was similar to that of the disaccharides when preparations containing 9wt.% carbohydrate were heated at 70°C for 2days. However, when the concentration exceeded 23wt.%, the reaction temperature was 70°C or above or the reaction time was longer than 48h, the addition of the polysaccharide did not protect the protein from thermally induced aggregation, suggesting that protein–polymer phase separation could have occurred during freeze-drying. Overall the results support the proposal that one aspect of carbohydrate additive functionality is as a diluent with the added condition that the carbohydrate remains miscible with the protein during processing.
Keywords: Freeze-drying; Glass; Protein; Aggregation; Anti-viral dry heat processing; Spray-drying
Association of ranibizumab (Lucentis®) or bevacizumab (Avastin®) with dexamethasone and triamcinolone acetonide: An in vitro stability assessment
by Marieke Veurink; Cinzia Stella; Cyrus Tabatabay; Constantin J. Pournaras; Robert Gurny (pp. 271-277).
The in vitro association of either ranibizumab or bevacizumab with dexamethasone sodium phosphate or triamcinolone acetonide suspension does not decrease the stability of these antibodies.The in vitro stability of monoclonal antibodies used for age-related macular degeneration, ranibizumab and bevacizumab, was investigated. The aggregation profile of the antibodies was compared, alone and after association with dexamethasone sodium phosphate or triamcinolone acetonide. Commercial formulations of ranibizumab and bevacizumab were dialysed into three different buffers. After dialysis, samples were stored at 4°C, 25°C and 40°C during 35days, alone and in combination with dexamethasone sodium phosphate, triamcinolone acetonide phosphate solution or triamcinolone acetonide suspension. Combined formulations based on both commercial formulations were investigated as well. The aggregation state of the antibodies was measured by multi-angle light scattering (MALS) after separation by asymmetrical flow field-flow fractionation (AFFF) or size-exclusion chromatography (SEC). Ranibizumab results to be more stable than bevacizumab, alone and in combination with dexamethasone sodium phosphate or triamcinolone acetonide. Elevation in concentration, pH and temperature causes a decrease in stability of both antibodies. The association of triamcinolone acetonide phosphate solution with either ranibizumab or bevacizumab is observed to be the least stable combination of all samples tested. Dexamethasone sodium phosphate was shown to have a stabilizing effect on bevacizumab, although this is not the case for its combination with the commercial formulation Avastin®. The results demonstrate that the in vitro association of either ranibizumab or bevacizumab with dexamethasone sodium phosphate or triamcinolone acetonide suspension does not decrease the stability of these antibodies. Although ranibizumab is more stable than bevacizumab in vitro, further research has to point out how this affects their mechanism of action in vivo.
Keywords: Monoclonal antibodies; Asymmetrical flow field-flow fractionation; Aggregation state; Anti-inflammatory drugs; Neovascular age-related macular degeneration
Human growth hormone: New delivery systems, alternative routes of administration, and their pharmacological relevance
by J. Cázares-Delgadillo; A. Ganem-Rondero; Y.N. Kalia (pp. 278-288).
Human growth hormone is a 191 amino acid non-glycosylated protein hormone. It has been used as a therapeutic agent for more than two decades. Many different formulation and delivery strategies have been explored to improve efficacy and patient compliance.The availability of recombinant human growth hormone (GH) has broadened its range of clinical applications. Approved indications for GH therapy include treatment of growth hormone deficiency (in children and in adults), Turner syndrome, Prader–Willi syndrome, chronic renal insufficiency and more recently, idiopathic short stature in children, AIDS-related wasting and fat accumulation associated with lipodystrophy in adults. Therapy with GH usually begins at a low dose and is gradually titrated to obtain optimal efficacy while minimizing side effects. It is usually administered on a daily basis by subcutaneous injection, since this was considered to impact upon patient compliance, extended-release GH preparations were developed and new delivery platforms – e.g., auto-injectors and needle-free devices – were introduced in order to improve not only compliance and convenience but also dosing accuracy. In addition, alternative less invasive modes of administration such as the nasal, pulmonary and transdermal routes have also been investigated. Here, we provide an overview of the different technologies and routes of GH administration and discuss the principles, limitations and pharmacological profiles for each approach.
Keywords: Human growth hormone; Therapeutics; Delivery; Pharmacology
Sustained release formulations of rhVEGF165 produce a durable response in a murine model of peripheral angiogenesis
by Ann L. Daugherty; Linda K. Rangell; Renee Eckert; Jose Zavala-Solorio; Frank Peale; Randall J. Mrsny (pp. 289-297).
PLGA polymer microspheres and a gel of PLGA solubilized in N-methyl pyrrolidone formulations of VEGF were tested in vitro and in vivo following hind-limb intramuscular (IM) injection. Three-dimensional resin castings of vascular architecture visualized by scanning electron microscopy along with histological and immunohistochemical parameters demonstrated profound vascular changes within three days of dosing that persisted for two months.Local delivery of therapeutic angiogenic agents that stimulate blood vessel formation represents a promising strategy for the treatment of peripheral vascular disease (PVD). At present, requirements for temporal and spatial parameters for localized delivery are unclear, with a variety of sustained delivery approaches being examined. Two polymer-based sustained formulations containing the 165 amino acid isoform of human recombinant vascular endothelial growth factor-A (rhVEGF165) were evaluated for their potential application in the treatment of PVD following intramuscular injection. Microspheres prepared from a 50:50 ratio of polylactic-co-glycolic acid (PLGA) and a gel of PLGA polymer solubilized in N-methyl pyrrolidone (PLGA:NMP) were each loaded with rhVEGF165 and tested in vitro and in vivo. PLGA microspheres averaged ∼30μm in diameter and contained 8.9% ( w/ w) rhVEGF165, while the PLGA:NMP gel was formulated with varying amounts of spray freeze-dried rhVEGF165 to result in final gel formulations having concentrations of 0.36, 0.72, or 3.6mg/mL rhVEGF165. In vitro release of rhVEGF165 from PLGA microspheres showed ∼10% cumulative release by day 6, whereas the cumulative release of rhVEGF165 from the PLGA:NMP gel matrices (0.65% w/ w loading) was less than 0.25% at this same time point. While the in vitro release characteristics of these two sustained release formulations were broadly different, the plasma rhVEGF165 concentration–time profiles following hind-limb intramuscular (IM) injection of these formulations in non-compromised rats revealed similar in vivo pharmacokinetics. Three-dimensional resin casts of vascular architecture were prepared at days 3, 7, 14, 21, 28, 60, and 75 following a single IM dosing of these sustained release microsphere and gel matrix formulations in the gastrocnemius muscle of immune-compromised mice. Scanning electron microscopic visualization of these vascular casts demonstrated spatial arrangement of capillary sprouts and vessel enlargement consistent with profound vascular changes occurring within 3days of dosing that persisted for 2months, approximately 1month beyond the anticipated completion of rhVEGF165 release from these sustained delivery formulations. Vascular re-modeling events were correlated with histological and immunohistochemical parameters attributed to known biological actions of rhVEGF165 signaling. Together, these pharmacokinetic and pharmacodynamic results support the use of sustained release PLGA-based formulations for the local delivery of rhVEGF165 to achieve a durable vascular re-modeling response.
Keywords: Angiogenesis; Vascular endothelial growth factor; Peripheral vascular disease; PLGA microspheres; PLGA:NMP gels; Vascular casts
Integrity characterization of myoglobin released from poly(ε-caprolactone) microspheres using two analytical methods: UV/Vis spectrometry and conductometric bi-enzymatic biosensor
by M. Hnaien; E. Ruffin; C. Bordes; O. Marcillat; F. Lagarde; N. Jaffrezic-renault; S. Briançon (pp. 298-305).
A bi-enzymatic biosensor was developped to analyse myoglobin encapsulated in polymeric microspheres. It was associated with a UV/Vis spectrometry analysis method. Both revealed that only low molecular weight polymer and solvent elimination under gentle conditions can preserve myoglobin native conformation.Myoglobin (Mb)-loaded poly(ε-caprolactone) (PCL) microparticles were prepared by multiple emulsion with solvent extraction/evaporation method under more or less deleterious operating conditions. The protein integrity was monitored using both UV/Vis absorbance ratio method at specific wavelengths and a conductometric bi-enzymatic biosensor based on proteinase K and pronase. Under standard operating conditions, Mb remained in native conformation, while different degrees of protein denaturation were observed by changing the encapsulation conditions. It was shown that solvent elimination under reduced pressure and in a lower extent addition of a higher molecular weight PCL led to protein alteration. In the first case, the loss of protein integrity can be attributed to residual solvent entrapped in particles whose solidification was accelerated. In the second case, denaturation may be explained by an increase in the protein exposure time at water/organic solvent interface due to an increase in organic phase viscosity.
Keywords: Myoglobin; Multiple emulsion; Protein integrity; Conductometric biosensor; UV/Vis spectrometry; Microparticles
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