Article

Non-Specific Binding and Saturation of Polysorbate-20 with Aseptic Filter Membranes for Drug Substance and Drug Product during mAb Production

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Abstract

An issue identified during filtration of mAb drug substance is Polysorbate-20 absorption by polyethersulfone filter membranes. Experiments determined that both polyvinylidene fluoride filter membranes and polyethersulfone filter membranes bind Polysorbate-20. Saturation point, bound surfactant amount per square cm of membrane, and non-specific binding mechanisms are described in this report. An appropriate approach for preventing Polysorbate-20 loss in drug substance and drug product is presented.

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... For filtration of parenteral products, hydrophilic polyvinylidene difluoride (PVDF) and polyethersulfone (PES) filters are widely used during the fill-finish process. 6,10,11 The filtration process can be influenced by different parameters including solution viscosity and formulation properties besides filter area, filtration pressure, pore size, and surface properties of the filter material. 6,11,12 To protect the protein from interfacial stress occurring during manufacture (e.g., filtration, freeze/thawing) and storage (e.g., glass as primary packaging, shaking stress), protein formulations often require the addition of stabilizers like surfactants. ...
... 6,11,12 To protect the protein from interfacial stress occurring during manufacture (e.g., filtration, freeze/thawing) and storage (e.g., glass as primary packaging, shaking stress), protein formulations often require the addition of stabilizers like surfactants. [13][14][15][16][17][18][19][20][21] Currently, most commonly used surfactants of marketed protein therapeutics are polysorbate (PS) 20 or 80. 10,11,22,23 These nonionic surfactants bind with a higher affinity to interfaces than proteins, such as to the air-liquid or ice-liquid interface, thus preventing interfacial protein adsorption and protein aggregation. 20,24 It was recently reported that PS 80 significantly adsorbs to PES sterilizing-grade filters. ...
... 20,24 It was recently reported that PS 80 significantly adsorbs to PES sterilizing-grade filters. 11 Furthermore, Zhou et al. 10 have recently shown that PS 20 also adsorbs to filter material (PES and PVDF) suggesting a non-specific hydrophobic binding mechanism. The authors reported that PES filters were adsorbing PS 20 to a much greater extent than the PVDF membranes. ...
Article
Differences in filtration behavior of concentrated protein formulations were observed during aseptic drug product manufacturing of biologics dependent on formulation composition. The present study investigates filtration forces of monoclonal antibody formulations in a small-scale set-up using polyvinylidene difluoride (PVDF) or polyethersulfone (PES) filters. Different factors like formulation composition and protein concentration related to differences in viscosity, as well as different filtration rates were evaluated. The present study showed that filtration behavior was influenced by the presence or absence of a surfactant in the formulation, which defines the interaction between filter membrane and surface active formulation components. This can lead to a change in filter resistance (PES filter) independent on the buffer system used. Filtration behavior was additionally defined by rheological non-Newtonian flow behavior. The data showed that high shear rates resulting from small pore sizes and filtration pressure up to 1.0 bar led to shear-thinning behavior for highly concentrated protein formulations. Differences in non-Newtonian behavior were attributed to ionic strength related to differences in repulsive and attractive interactions. The present study showed that the interplay of formulation composition, filter material, and filtration rate can explain differences in filtration behavior/filtration flux observed for highly concentrated protein formulations thus guiding filter selection. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.
... Examples of commonly used stabilizers include polysorbate 20 (PS20) and polysorbate 80 (PS80). These excipients can present a challenge in sterile filtration operations because they can adsorb to filter material (Mahler et al., 2010;Zhou et al., 2008). This can be mitigated through preconditioning of the filters to saturate the binding sites with formulation buffer or protein solution, or evaluation of different filter types. ...
... Adjust pH (Sharma et al., 2008), operate away from protein pI (Wu et al., 2010) Adjust excipients in formulation (Allmendinger et al., 2015) Membrane selectionmore hydrophilic membranes may reduce hydrophobic fouling/aggregation (Tang et al., 2019) Adsorption of excipients to sterile filter (Mahler et al., 2010;Zhou et al., 2008) Precondition the filter to saturate binding sites (Mahler et al., 2010;Zhou et al., 2008) Stability of high-concentration drug substance ...
... Adjust pH (Sharma et al., 2008), operate away from protein pI (Wu et al., 2010) Adjust excipients in formulation (Allmendinger et al., 2015) Membrane selectionmore hydrophilic membranes may reduce hydrophobic fouling/aggregation (Tang et al., 2019) Adsorption of excipients to sterile filter (Mahler et al., 2010;Zhou et al., 2008) Precondition the filter to saturate binding sites (Mahler et al., 2010;Zhou et al., 2008) Stability of high-concentration drug substance ...
Article
In order to achieve the high protein concentrations required for subcutaneous administration of biologic therapeutics, numerous manufacturing process challenges are often encountered. From an operational perspective, high protein concentrations result in highly viscous solutions, which can cause pressure increases during ultrafiltration. This can also lead to low flux during ultrafiltration and sterile filtration, resulting in long processing times. In addition, there is a greater risk of product loss from the hold‐up volumes during filtration operations. From a formulation perspective, higher protein concentrations present the risk of higher aggregation rates as the closer proximity of the constituent species results in stronger attractive intermolecular interactions and higher frequency of self‐association events. There are also challenges in achieving pH and excipient concentration targets in the ultrafiltration/diafiltration (UF/DF) step due to volume exclusion and Donnan equilibrium effects, which are exacerbated at higher protein concentrations. This paper highlights strategies to address these challenges, including the use of viscosity‐lowering excipients, appropriate selection of UF/DF cassettes with modified membranes and/or improved flow channel design, and increased understanding of pH and excipient behavior during UF/DF. Additional considerations for high concentration drug substance manufacturing such as appearance attributes, stability, and freezing and handling are also discussed. These strategies can be employed to overcome the manufacturing process challenges and streamline process development efforts for high concentration drug substance manufacturing. This article is protected by copyright. All rights reserved.
... Another study, also found adsorption of polysorbate to sterilizing-grade filters. 36 Zhou et al. reported specific binding of polysorbate 20 of $70 mg/cm 2 to PES membranes (Sartopore 2). Converting this specific binding value to our set-up (6000 cm 2 membrane area, 0.01% polysorbate 80 concentration), leads to a calculated filtrate volume of 4.2 L until the complete surface area is saturated with polysorbate. ...
... However, different adsorption behavior of different filter material can also be expected, based on the lab-scale experiments. Zhou et al. 36 have also suggested that PES filters adsorbed much more polysorbate 20 compared to PVDF filters. ...
... This behavior is in good agreement with previously reported polysorbate binding values. 36 Depending on the setup of the aseptic filtration and filling process, this observation may have significant impact on the definition of the discarded preflush. ...
Article
Formulations of therapeutic proteins usually contain a surfactant such as polysorbate 80 to protect them against interfacial stresses. Since surfactants may interact with surfaces, the aim of the present work was to study the adsorption behavior of low concentrations of polysorbate 80 and of a monoclonal antibody during sterile filtration. Lab-scale tests were performed to study the adsorption behavior of a monoclonal antibody to different filter materials (PVDF, PES, CA, and Nylon) from different suppliers. Subsequently, protein and polysorbate 80 adsorption were tested in manufacturing scale experiments. It was found that the extent of protein adsorption differed with filter materials, but also with different suppliers. Prominently, Nylon filters showed the highest degree of protein adsorption. In manufacturing-scale filtration experiments, significant adsorption of polysorbate 80 to sterilizing-grade filters was found. Thus, the adsorption of both protein and polysorbate to filters should be taken into consideration in the formulation and manufacturing process and assessed on a case-by-case basis depending on the manufacturing process set-up.
... It has been reported that PS-20 will be adsorbed on the sterilizing filters, and the amount adsorbed is dependent on the membrane material. 8,15 However, neither the amount of adsorption nor composition of PS-20 after sterilizing filtration has been reported. In the cases where PS-20 is present in bulk drug substance (e.g. ...
... The amount of adsorbed PS-20 is in the same scale as published data. 15 According to the manufacturer, Fluorodyne V R EX is a combination of an upstream layer of PES over a PVDF membrane, but its behavior resembles hydrophilized PVDF material. Overall, less PS-20 was adsorbed per unit membrane area for hydrophilized PVDF material than PES material. ...
... These findings agreed with published reports that more PS-20 (or PS-80) was adsorbed by PES material than PVDF material. 15,27 According to these reports, the presence of protein at common concentrations (2-150 mg/mL) does not affect the adsorption of PS-20 or PS-80 on sterilizing filters. 15,27 The differences among different types of filters are possibly related to the differences in the hydrophobicity of membrane materials. ...
Article
Polysorbate 20 (PS-20) is often included in the formulation for therapeutic proteins to reduce protein aggregation and surface adsorption. During the production process of therapeutic proteins, various membrane filters are used to filter product pools containing PS-20. The purpose of this study is to quantify the effects of these membrane filtration processes on the concentration and composition of PS-20. A quantitative understanding of this process provides the knowledge base for better controlling the consistency of formulation excipients in drug products. PS-20 solutions (without protein) were filtered through either 0.2 µm sterilizing filters or membrane filters with 30kDa MWCO. The concentration of PS-20 was measured by a mixed-mode chromatography method and a nuclear magnetic resonance spectroscopy (NMR) assay. The composition of PS-20 was characterized by (1) H-NMR and a reverse-phase chromatography method. Non-specific adsorption of PS-20 on both the sterilizing filter and 30 kDa MWCO membrane filter was quantified. Composition of PS-20 was altered after 30 kDa MWCO membrane filtration, possibly because the different interactions between heterogeneous PS-20 components and the 30 kDa MWCO membrane were not uniform. As a result, the retentate after the 30 kDa MWCO membrane filtration step contains no POE sorbitan and increased amount of POE sorbitan di-esters and tri-esters. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
... These interactions are influenced by the solvent pH, ionic strength and type of ions (Mahler et al., 2010). In a similar study of PS20, Zhou et al. (2008) showed that both polyvinyldifluoride and polyethersulphone filters bind PS20, but that the amount bound to the polyethersulphone filters was much greater. Adsorption depends on the filter material used, although even the same filter materials provided by different suppliers can show different adsorptions (Zhou et al., 2008). ...
... In a similar study of PS20, Zhou et al. (2008) showed that both polyvinyldifluoride and polyethersulphone filters bind PS20, but that the amount bound to the polyethersulphone filters was much greater. Adsorption depends on the filter material used, although even the same filter materials provided by different suppliers can show different adsorptions (Zhou et al., 2008). ...
Article
Formulation development is an essential part of any biopharmaceuticals development programme, and this will affect quality, safety and efficacy of the final drug product. The vast majority of biopharmaceuticals on the market are therapeutic proteins; however, these are less stable compared to conventional pharmaceuticals. To counter aggregation, denaturation and surface adsorption of proteins in solution, surfactants are added to the formulations; however, the choice of the best formulation is a challenge that is faced during formulation development. Polysorbates are the most widely used surfactants in the pharmaceutical industry, and are presented in >80% of commercial monoclonal antibody formulations. In this review, we provide a general overview of polysorbates and their issues, and the characteristics that have to be taken into account during formulation development. Degradation of polysorbates, namely by hydrolysis and/or oxidation, is one of the main concerns associated with their use. Furthermore, degradation of polysorbates is determined by formulation composition, pH and storage conditions, therefore underlining the importance and complexity of protein formulation development using polysorbates. A need-based approach should be used for correct selection of excipients in protein formulations that contain polysorbates.
... 117 An additional concern during the DP manufacturing process is loss of surfactant to interfaces, specifically filter membranes. 174,175 The risk of protein and/or surfactant loss to filters is minimized by flushing of filters with buffer or product prior to the filtration step, and this approach is often an acceptable mitigation even though flushing can result in wasted product. More recently, as discussed above, some alternative surfactants have been identified that are more compatible with various manufacturing unit operations. ...
Article
Biologics may be subjected to various destabilizing conditions during manufacturing, transportation, storage, and use. Therefore, biologics must be appropriately formulated to meet their desired quality target product profiles. In the formulations of protein-based biologics, one critical component is surfactant. Polysorbate 80 and Polysorbate 20 remain the most commonly used surfactants. Surfactants can stabilize proteins through different mechanisms and help the proteins withstand destabilization stresses. However, the challenges associated with surfactants, for instance, impurities, degradation, and potential triggering of adverse immune responses, have been encountered. Therefore, there are continued efforts to develop novel surfactants to overcome these challenges associated with traditional surfactants. Meanwhile, surfactants have also found their use in formulations of newer and novel modalities, namely, antibody-drug conjugates, bispecific antibodies, and adeno-associated viruses (AAV). This review provides an updated in-depth discussion of surfactants in the above-mentioned areas, namely mechanism of action of surfactants, a critical review of challenges with surfactants and current mitigation approaches, and emerging technologies to develop novel surfactants. In addition, gaps, current mitigations, and future directions have been presented to trigger further discussion and research to facilitate the use and development of novel surfactants.
... Hydrophilic and hydrophobic surfaces are present inside the filter cartridge that may become a contributing factor to the adsorption of polysorbates. Non-specific bindings of PS20 have been reported with 0.22 mm hydrophilic filters [30,31]. Unlike other stress conditions, polysorbate was unlikely to be consumed in the absence of stress and could interact more with the filter. ...
Article
This study aimed to explore the suitability of flow injection spectrophotometry (FIS) to analyze three degraded therapeutic monoclonal antibodies (bevacizumab, nivolumab, and rituximab). For this purpose, aggregates were generated with stirring, freeze-thaw, and heat stresses. The intact and stressed mab samples were filtered with 0.22 µm hydrophilic filters and analyzed by size exclusion chromatography (SEC), cation-exchange chromatography (CEX), and FIS. In terms of quantitative and qualitative analysis, protein loss and structural changes were assessed. Various aggregates profiles were obtained according to the mabs and the stresses. FIS allowed performing very satisfactory quantifications for each mab with intermediate precision RSD < 3.0 % and recovery between 97.9 – 102.0 %. From the protein loss measurements, it appears that SEC underestimates the mab aggregate proportions up to two times less as compared with FIS since the latter avoids any non-specific interactions (electrostatic or hydrophobic interactions). Using second derivative spectroscopy and multivariate data analysis, we noticed apparent structural differences, located in the regions 245 – 265 nm for rituximab and nivolumab and 280 - 300 nm for bevacizumab, depending on the stress. The FIS complementarity with the other techniques used in this study allowed us to demonstrate that the three mabs behave differently for a given stress condition. While extreme mechanical stress formed large aggregates irrespective of the mabs, rituximab showed to be less stable and more sensitive than the two other mabs under freeze-thaw and heat stresses, generating large aggregates (> 200 nm) and partial unfolding. Nivolumab tends to form small aggregates less than 50 nm when heated and freeze-thawed. Moreover, freeze-thaw seems to generate native IgG-1 aggregates with rituximab. Similarly, bevacizumab showed to form these IgG-1 aggregates and was resistant to freeze-thaw, likely thanks to trehalose cryoprotectant from its formulation. Finally, FIS associated with multivariate analysis is able to provide rich information in one single run and appears to be a fast, simple, and reliable method to set complementary and orthogonal approaches for protein aggregate monitoring.
... Consequently, we hypothesized the adsorption of the surfactants, which are more available outside stress conditions, at the solid or liquid interfaces, and the formation of a layer inside the filter impacting the filtration of low volumes of mAb solutions. The studies of Mahler et al. 39 and Zhou et al. 40 have reported the adsorption of polysorbates 80 and 20 to a polyethylsulfonate filter. However, a thorough analysis is needed to provide a better understanding in our case. ...
Article
Compounded therapeutic mAbs used in a hospital require quality control (QC). In our hospital, analytical QC process intended to mAbs identification and quantification is based on flow injection analysis associated with second-derivative UV spectroscopy and matching method algorithm. We studied the influence of degraded mAbs after compounding on this validated QC. Three forced stress conditions including mechanical, thermal, and freeze-thawing stresses were studied to yield degraded mAbs from 2 model compounds, that is, bevacizumab (IgG1) and nivolumab (IgG4). Different degraded mAbs were generated and were analyzed in terms of turbidity, the percentage of aggregation, size distribution, and changes in tertiary structure. Stresses showed to be mAb-dependent in terms of aggregation. Tertiary structural changes were observed in most of the stressed samples by principal component analysis of the UV second-derivative data. The structural and physicochemical modifications conducted to mismatch depending on the nature of the stress. The mismatch ranged from 17% to 72% for the mAbs, except for freeze-thawed bevacizumab for which a perfect match (100%) was reached. The quantification with an unfulfilled relative error of the concentration (i.e., > ±15%) was detected only for mechanically stressed mAbs. In conclusion, the study revealed that the influence of the mAbs and the type of stress impact on the QC of compounded mAbs.
... Only 55 ± 28% of genomic particles were recovered when using Triton X-100 for concentration, while 92 ± 13% was recovered when using Polysorbate 20. The fact that this detergent is known by the non-specific binding to the PES membranes [29], might contribute to it having the highest recovery yield when it is used in the tangential flow filtration. The detergent binds to the membrane surface, reducing the membrane area available for unspecific binding of viral particles, thus improving the recovery yield. ...
Article
Full-text available
Introduction Oncolytic virus therapy is currently considered as a promising therapeutic ap-proach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer prepara-tions. The addition of this surfactant in the list of substances with the very high concern of the Registra-tion, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives. Methods In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorb-ate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impu-rities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell pro-teins and ds-DNA was 99.9% and 97.1%, respectively. Results & Conclusion The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the devel-oped process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.
... However, in real applications, considerable amount of the filtered solutes can be adsorbed during the initial filtration stages, predetermined by the membrane (material, presence/absence of wetting agents, pore size and distribution), solute type, pH, ionic strength, and even filtration flow rate [1]. A range of separations have been studied in the scope of this phenomenon: from individual compounds such as toluidine blue [1], benzalkonium chloride, chlorhexidine acetate, phenylmercuric nitrate and phenylethyl alcohol [2][3], estrogen 17β-estradiol [4], triethanolamine and sodium benzoate [5], a gamut of sixteen neutral, acidic and basic compounds [6], surfactants [7], to viruses [8] and bovine serum albumin (BSA) [9] and other peptides (luteinizing hormone releasing hormone, gamma globulin and BSA) [10], monoclonal antibodies and surfactant influence thereon [11]; with membranes of different materials being mainly of microfiltration types (sterilising grade) though. ...
Article
Full-text available
Depot effects were found to be accompanying phenomena of membrane separation processes. Accumulation of target species in the membrane matrix during feasibility tests can hamper proper conclusions or compromise the filtration results. Therefore, we investigated the effects of delayed membrane release of chlorogenic acid and caffeine, considered as key compounds of interest in spent coffee products' recovery treatment. Permeate fluxes and key components release were studied in course of 24 hours via nanofiltration of pure solvent, both immediately after the mock solution filtration and after idle stay. Conclusions are drawn and recommendations advised for proper analysis of experimental data on membrane screening.
... Saturation of the non-specific binding sites using PS-20 present in the formulation buffer or in the product is possible and most feasible approach determined was preconditioning the filters prior to the protein preparation, the pre-condition was found not to be affected by temperature or the flow rate. The authors warn again usage of a over-sized filter in such applications [12]. ...
Article
Filters are widely used in the pharmaceutical industry and hospital care for multiple applications such as API processing and purification, pharmaceutical and bio-pharmaceutical operations such as sterile filtration and protein purification, analysis of drug products, while administration to the patients and so on. A multitude of filters with different pore ratings and material of construction are available for use in various applications. The wide variety and type of filters available today also mean that we do not completely understand the nature of such filters and random or un-informed usage could introduce unwanted alterations in the quality or quantity of the product. Thus, one needs to be aware that filters could alter the stability, quality and safety of the product if the right choices are not made. Various mechanisms and examples are described wherein the final quality of the product was affected. Adsorption is the major mechanism of interaction between filters and drug product. Filters can adsorb formulation components such as active pharmaceutical ingredient, preservatives and other excipients which could lead to therapeutic failure or toxicity. Other mechanisms include leaching of filter materials into product during filtration which could adversely affect the product by changing the safety aspect of the product. Despite certain challenges involved while working with filter, usage of filters is the pharmaceutical and biopharmaceutical industry is increasing day by day and many crucial operations such as meeting sterility in aseptic operations, purification of peptides/proteins/Active pharmaceutical ingredients, dissolution analysis etc rely heavily on the quality and right usage of the appropriate filters. It is necessary to minimize the adverse interaction between the filter and the drug product and thus deliver the best to the patient and health care for which the end user needs to thoroughly understand their application, nature of the material being processed and details of the filter to better understand the outcome of the filtration process.
... Multiple studies have reported PS adsorption and loss with various in-process components (e.g., filters) used in the drug product manufacturing. [27][28][29] However, data for the loss of PS in drug products, observed over time and under real-time storage conditions, were not readily available until recently. 18,30 PS degradation seems to be a frequently encountered problem in the biopharmaceutical industry, for which not much is published. ...
Article
This study investigated the root cause behind an observed free fatty acid particle formation and resulting Polysorbate 20 (PS20) loss for a sulfatase drug product upon long-term storage at 5 ± 3°C. Reversed- phase chromatography with mass spectrometric analysis as well as charged aerosol detection was used to characterize the peaks associated with the intact and degraded PS20. Additionally, a proteomics study was undertaken to identify the residual host cell proteins in the sulfatase drug substance. PS20 stability studies were conducted in the presence of sulfatase, a sulfatase inhibitor, putative phospholipase B-like 2, and mock drug substance produced using a null cell line vector under experimental conditions optimized for PS20 degradation. This study provides the first published evidence where the residual host cell protein present in the drug substance was identified and experimentally shown to catalyze the breakdown of PS20 in a protein formulation over time, resulting in free fatty acid particles and PS20 loss. This study demonstrates the importance of early detection of potential impurities in the protein drug substance that may contribute to polysorbate degradation to make a judicious selection of the surfactant and its optimized concentration for the final drug product.
Article
A spectrophotometric method was developed to quantify low polysorbate (PS) levels in biopharmaceutical formulations containing high protein concentrations. In the method, Oasis HLB solid phase extraction (SPE) cartridge was used to extract PS from high protein concentration formulations. After loading a sample, the cartridge was washed with 4M guanidine HCl and 10% (v/v) methanol, and the retained PS was eluted by acetonitrile. Following the evaporation of acetonitrile, aqueous cobalt-thiocyanate reagent was added to react with the polyoxyethylene oxide chain of polysorbates to form a blue colored PS-cobaltothiocyante complex. This colored complex was then extracted into methylene chloride and measured spectrophotometrically at 620nm. The method performance was evaluated on three products containing 30-40mgL(-1) PS-20 and PS-80 in ≤70gL(-1) protein formulations. The method was specific (no matrix interference identified in three types of protein formulations), sensitive (quantitation limit of 10mgL(-1) PS) and robust with good precision (relative standard deviation ≤6.4%) and accuracy (spike recoveries from 95% to 101%). The linear range of the method for both PS-20 and PS-80 was 10 to 80mgL(-1) PS. By diluting samples with 6M guanidine HCl and/or using different methylene chloride volumes to extract the colored complexes of standards and samples, the method could accurately and precisely quantify 40mgL(-1) PS in up to 300gL(-1) protein formulations.
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Chapter
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The absorptiometric determination of polyethyleneglycol mono-oleate in aqueous solution is described. The procedure depends on the formation under controlled conditions of a blue complex between the compound and ammonium cobaltothiocyanate, this complex being extracted into chloroform. Measurement of the optical density of the blue chloroform solution at either 318·5 or 620 mμ then gives the concentration of the polyethyleneglycol mono-oleate, and the procedure is applicable at concentrations from 0 up to 1 g per litre. The method is applicable in the presence of low molecular weight polyethyleneglycol monoesters, triethyleneglycol monostearate having been studied in detail in this connection. If necessary, the method can be adapted to a Spekker absorptiometer, when the complete determination takes about 45 minutes. The qualitative application of the procedure to other compounds is briefly considered.
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The Horwitz curve is a simple exponential relationship between the relative standard deviation among laboratories to concentration, C, expressed in mass/mass units. Examination of almost 10 000 interlaboratory data sets shows that the curve is more or less independent of analyte, matrix, method, and time of publication, over the range from pure materials, C = 1 (100%), to trace polychlorinated aromatic contaminants (PCCs), C ≈ 10-12. The functional relationship can be derived simply by assuming that the infinitesimal fractional change in standard deviation is proportional to the infinitesimal fractional change in C, by integrating, and by determining the constant of integration from empirical results. Mycotoxin and PCC data show that the limit of measurement in the interlaboratory environment is C ≈ 10-9, where results become uninterpretable because of the appearance of excessive numbers of false positive and false negative values. Lower values are possible only because of the extraordinary specifications for quality control for these analyses.
Chapter
IntroductionClinical ApplicationPharmaceutical Product DevelopmentStability IssuesFormulation and Manufacturing of Parenteral Delivery SystemsFormulation and Manufacturing of Local Delivery SystemsOutlook
Article
Microfiltration of a γ-globulin solution has been investigated through the virus removal membranes having different pore sizes (i.e. r=15, 35 and 75 nm) and a dialysis membrane (r=3.4 nm), which were all made of the same regenerated cellulose material. The addition of NaCl in the γ-globulin feed solution was effective to enhance the flux and transmission through the membranes having a pore size ranging from 15 to 75 nm. DNase treatment of a γ-globulin solution with Micrococcal nuclease enhanced the flux and transmission of γ-globulin through the membranes either with or without NaCl. The membranes having a pore size of 35 nm showed dramatically enhanced flux in the microfiltration of a γ-globulin solution containing NaCl and/or being treated with Micrococcal nuclease. This can be explained as a DNase treatment and NaCl addition in the protein solution dissociate protein aggregates of DNA–γ-globulin complex, which plugs the pores in the microfiltration membranes.
Article
To retain biological activity, proteins generally must be maintained in a specific, three-dimensional conformation. This conformation is only marginally stable, and thus relatively minor perturbing forces can disrupt protein structure, causing loss of biological activity, as well as formation of non-native protein aggregates. Such perturbations are commonly encountered as proteins are produced, stored, transported, and delivered to patients. For example, it is well known that during common industrial processes such as filtering (Maa and Hsu, 1998), storage (Mcleod et al., 2000), agitation (Thurow and Geisen, 1984; Maa and Hsu, 1997) freeze/thawing (Eckhardt, Oeswein et al., 1991; Nema and Avis, 1993; Izutsu et al., 1994), lyophilization (Carpenter and Chang, 1996; Carpenter et al., 1997), nebulization (Ip et al., 1995) and spray-drying (Broadhead et al., 1994; Mumenthaler et al., 1994; Maa et al., 1998; Adler and Lee, 1999; Millqvist-Fureby, Malmsten et al., 1999; Tzannis and Prestrelski, 1999) proteins can suffer damage to their native conformation. Further, delivery of protein pharmaceuticals to patients may also provoke losses of conformational integrity via unfavorable interactions of proteins with surfaces (e.g., inner walls of catheter tubing or syringes (Tzannis et al., 1996)).
Article
The CMC of polysorbate 20 was determined using a surface tension method; the concentration (C) of polysorbate 20 studied varied from 0.001 to 10.000 mg./ml. The results show clearly that the surface tension (γ) decreases linearly with log C up to a concentration of 0.06 mg./ml. and is practically constant for more concentrated solutions. This suggests that the CMC of polysorbate 20 is in the vicinity of 0.06 mg./ml., which is in excellent agreement with the values obtained by other methods.
Article
The concept of a weighted pool for estimating the area under the curve (AUC) is presented and set in relationship to the trapezoidal rule. An example from a pharmacokinetic study on ethinyl estradiol is used to demonstrate the use of variance component analysis for relating the intraindividual variance of the AUC, trapezoidal rule and weighted pool to the variance of the determination process. Depending on the sampling times, the theoretical variance of the weighted pool is greater than the theoretical variance of the trapezoidal rule. In the example presented, it was shown that this difference is of no importance in relation to the interindividual variance of the AUC, which dominates the total variance. In the example study, routine quality control samples were also determined in each assay, which allowed independent confirmation of the discussed results on the intraindividual variance of the AUCs.
Article
Pulmonary delivery is an attractive alternative route to deliver protein drugs that are currently delivered by injection. Inhalation therapy via nebulizers is a well accepted way for pulmonary application of proteins considering the formulation difficulties of MDIs or DPIs. This research presents the effect of variable excipients on the stability and aerosol performance of freeze-dried aviscumine after reconstitution and nebulization. Aviscumine formulations containing 100 mmol/L Tris buffer, 0.1% (w/v) Polysorbate 80, 0.01% (w/v) Na(2)-EDTA and 8% (w/v) Hydroxyethyl starch have been lyophilized and reconstituted with a buffered isotonic solution pH 8. The aviscumine activity was determined by a binding assay directly after reconstitution and after nebulization with a PariBoy air-jet nebulizer, a Multisonic and a Systam ultrasonic nebulizer. The stabilization of aviscumine by the addition of variable buffer salts to the reconstitution medium, such as 50, 100, and 200 mmol/L Tris buffer, 20 and 100 mmol/L phosphate buffer, and 20 and 100 mmol/L Tricine buffer, was studied. About 50% of aviscumine activity was lost after 20 min nebulization time without any additives. Nevertheless, higher buffer concentrations confer greater stability. About 70% of the aviscumine activity could be retained by the addition 0.03% octanoyl-N-methylglucamide and 100 mmol/L Tricine to the reconstitution medium.
Article
The objective of this study was to compare different agitation stress methods (stirring in Reacti Vials versus horizontal shaking) in their effect on protein destabilization, to assess several analytical techniques (light obscuration, turbidimetric and light scattering analysis) for detection of aggregates of various sizes and to evaluate the protecting effect of polysorbate 80 on protein aggregation. A monoclonal IgG1 antibody was used as model protein. Both mechanical stress methods can provoke aggregate formation. The method of stirring induces particles in the range of 10-25 microm comparable to shaking stress. However, stirred samples show a much higher absorbance and reveal a second particle species in DLS analysis, suggesting that stirring stress induces a higher amount of smaller protein aggregates. Addition of polysorbate 80 protects the antibody against aggregation. Only in stirred samples a slight increase in sub-visible particles and turbidity was noted. However, a greater extent of aggregation products was detected by DLS as compared to surfactant-free formulations. Thus, polysorbate 80 appears to stabilise small aggregates and prevents further proceeding of the aggregation process. The induction of aggregates by stirring stress in Reacti Vials analysed by absorbance measurement seems to be a good combination for high-throughput formulation studies.
Article
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.
Article
Process-scale antibody production requires polishing steps with extremely high product throughput and robust operation. In this communication, the Sartobind Q membrane adsorber for process-scale antibody production is evaluated as an alternative to Q column chromatography. Although the capacity seen with large-scale membrane adsorbers is competitive with column chromatography, the same throughput is not achieved with the current scale-down models. The operational issues currently found in membrane scale-down models, including backpressure, which significantly compromises the membrane's capacity, were examined. A new scale-down model was designed to mimic the liquid flow path found in the large-scale capsule, and a new process capacity equivalent at both small and large scale was successfully achieved. Results of a 4-model virus study with a redesigned Sartobind Q absorber scale-down model at the new process capacity are presented.
Article
This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized.
Article
Polysorbates 20 and 80 (Tween 20 and Tween 80) are used in the formulation of biotherapeutic products for both preventing surface adsorption and as stabilizers against protein aggregation. The polysorbates are amphipathic, nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan being polyoxyethylene sorbitan monolaurate for polysorbate 20 and polyoxyethylene sorbitan monooleate for polysorbate 80. The polysorbates used in the formulation of biopharmaceuticals are mixtures of different fatty acid esters with the monolaurate fraction of polysorbate 20 making up only 40-60% of the mixture and the monooleate fraction of polysorbate 80 making up >58% of the mixture. The polysorbates undergo autooxidation, cleavage at the ethylene oxide subunits and hydrolysis of the fatty acid ester bond. Autooxidation results in hydroperoxide formation, side-chain cleavage and eventually formation of short chain acids such as formic acid all of which could influence the stability of a biopharmaceutical product. Oxidation of the fatty acid moiety while well described in the literature has not been specifically investigated for polysorbate. This review focuses on the chemical structure of the polysorbates, factors influencing micelle formation and factors and excipients influencing stability and degradation of the polyoxyethylene and fatty acid ester linkages.
Article
The commercial production of recombinant human monoclonal antibody therapeutics demands robust processes. In this article we describe the development of a pH-conductivity hybrid gradient for a cation-exchange chromatography step to obtain high binding capacity and consistent purification resolution in scale process. Operational parameters and their ranges were characterized with DOE statistical method. Aggregate, DNA and leached protein A removal were examined during development. The advantages and disadvantages of hybrid gradient elution compared to sodium chloride gradient elution were explored. As this step was designed as a good fit for the compatibility of the feed and operating pH/conductivity conditions for next step, the effects of elution by either changing sodium chloride concentration or changing pH of elution buffers on overall separation efficiency were compared. The operation condition was further confirmed in six 2000 L scale runs. The thorough evaluation demonstrated process reliability of hybrid gradient cation-exchange chromatography with high step purity and yield.