Clearance of solvents and small molecule impurities in antibody drug conjugates via ultrafiltration and diafiltration operation

Ultrafiltration and diafiltration (UF/DF) processes by tangential flow filtration (TFF) are frequently used for removal of solvents and small molecule impurities and for buffer exchange for biopharmaceutical products. Antibody-drug conjugates (ADCs) as an important class of biological therapeutics, carry unique solvents and small molecule impurities into the final UF/DF step as compared to standard antibody preparation. The production process of ADCs involves multiple chemical steps, for example, reduction and conjugation. The clearance of these solvents and small molecules by UF/DF, specifically the DF step, has been assessed and described herein. The rates of clearance for all the impurities in this study are close to the ideal clearance with no apparent interaction with either the protein or the TFF membrane and system. The effect of process variables during DF, such as pH, temperature, membrane loading, transmembrane pressure, and cross flow rate, has also been evaluated and found to have minimal impact on the clearance rate. These results demonstrate efficient clearance of solvents and small molecule impurities related to the ADC process by the DF process and provide a general data package to facilitate risk assessments based on the sieving factors and program specific needs.     

Continuous ultrafiltration/diafiltration using a 3D-printed two membrane single pass module

A 3D printed ultrafiltration/diafiltration (UF/DF) module is presented allowing the continuous, simultaneous concentration of retained (bio-)molecules and reduction or exchange of the salt buffer. Differing from the single-pass UF concepts known from the literature, DF operation does not require the application of several steps or units with intermediating dilution. In contrast, the developed module uses two membranes confining the section in which the molecules are concentrated while the sample is passing. Simultaneously to this concentration process, the two membranes allow a perpendicular in and outflow of DF buffer reducing the salt content in this section. The module showed the continuous concentration of a dissolved protein up to a factor of 4.6 while reducing the salt concentration down to 47% of the initial concentration along a flow path length of only 5 cm. Due to single-pass operation the module shows concentration polarization effects reducing the effective permeability of the applied membrane in case of higher concentration factors. However, because of its simple design and the capability to simultaneously run UF and DF processes in a single module, the development could be economically beneficial for small scale UF/DF applications.     

Use of ultrafiltration/diafiltration for the processing of antisense oligonucleotides

Ultrafiltration/diafiltration (UF/DF) has been the hallmark for concentrating and buffer exchange of protein and peptide-based therapeutics for years. Here we examine the capabilities and limitations of UF/DF membranes to process oligonucleotides using antisense oligonucleotides (ASOs) as a model. Using a 3 kDa UF/DF membrane, oligonucleotides as small as 6 kDa are shown to have low sieving coefficients (<0.008) and thus can be concentrated to high concentrations (≤200 mg/mL) with high yield (≥95%) and low viscosity (<15 centipoise), provided the oligonucleotide is designed not to undergo self-hybridization. In general, the oligonucleotide should be at least twice the reported membrane molecular weight cutoff for robust retention. Regarding diafiltration, results show that a small amount of salt is necessary to maintain adequate flux at concentrations exceeding about 40 mg/mL. Removal of salts along with residual solvents and small molecule process-related impurities can be robust provided they are not positively charged as the interaction with the oligonucleotide can prevent passage through the membrane, even for common divalent cations such as calcium or magnesium. Overall, UF/DF is a valuable tool to utilize in oligonucleotide processing, especially as a final drug substance formulation step that enables a liquid active pharmaceutical ingredient.     

Effects of chemical sanitization using NaOH on the properties of polysulfone and polyethersulfone ultrafiltration membranes

Membranes used in bioprocessing applications are typically sanitized before use to insure aseptic operation. However, there is almost no information in the literature on the effects of this preuse sanitization step on the properties of the membrane. Experiments were performed with commercially available hollow fiber polysulfone (PSf) and polyethersulfone (PES) membranes with different nominal molecular weight cutoffs. Data were obtained for the membrane hydraulic permeability, dextran retention coefficients, zeta potential (surface charge), and extent of protein adsorption both before and after sanitization with 0.5 N NaOH at 45°C for 30 min. Changes in chemical composition were examined using ATR‐FT‐IR and XPS. Sanitization caused a large increase in the net negative charge for all membranes. There was a small reduction in hydraulic permeability and a significant increase in dextran retention for the polyethersulfone membranes, consistent with a reduction in the effective pore size. Spectroscopic analyses suggest that this change is likely due to the base‐catalyzed hydrolysis of the lactam ring in polyvinylpyrrolidone (PVP) that is typically is used as a wetting/pore‐forming agent in PSf and PES membranes. Preuse sanitization also appeared to have a small effect on protein adsorption, although the extent of adsorption was quite low for both the virgin and sanitized membranes. The observed changes in membrane properties could have a significant impact on the ultrafiltration performance, demonstrating the importance of standardizing the sanitization procedures even in process development and scale‐down validation studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:90–96, 2015     

Physico-Mechanical Properties of Chemically Treated Bacterial (Acetobacter xylinum) Cellulose Membrane

Bacterial cellulose obtained through fermentation by the Acetobacter xylinum is of superior functional quality in comparison to plant cellulose. Various alkali treatment methods were used to process bio-chemically complex pellicle into a clean cellulose membrane/sheet. The effect of potassium hydroxide, sodium carbonate and potassium carbonate was found to be milder on the final cellulose product in contrast to the widely used sodium hydroxide treatment. These novel treatment methods also caused improvement in the tensile strength of the membranes in comparison to sodium hydroxide. The overall quality of the 0.1 M sodium carbonate- and potassium carbonate-treated cellulose was superior, as the membranes displayed maximum tensile strength and elongation next to the native membrane. The low tensile strength of sodium hydroxide-treated membrane is attributed to its higher swelling characteristics in alkali. Further, the low swelling property of sodium carbonate- and potassium carbonate-treated membranes resulted in their high oxygen transmission rates (low oxygen barrier). Hunter lab colour parameters were determined to assess the effect of different alkali treatments on the colour characteristics of the membranes. Further, based on the high mechanical strength and comparatively low oxygen transmission rates, the processed cellulose membranes may find application as a bio- packaging material for controlled atmosphere packaging, where hydrophilic membranes with high oxygen barrier and water vapour permeation are desirable.     

Effect of biocides on<Emphasis Type="Italic">S. cerevisiae</Emphasis>: Relationship between short-term membrane affliction and long-term cell killing

The long-term action of recommended (RC) and near-recommended concentrations of several commercial biocides (Lonzabac 12.100, Genamin CS302D, benzalkonium chloride and 2-phenoxyethanol) on cells of S. cerevisiae wild-type strain DTXII was described using plating tests while short-term effects were determined using the potentiometric fluorescent probe diS-C3(3) that detects both changes in membrane potential and impairment of membrane integrity. A 2-d plating of cells exposed to 0.5xRC of benzalkonium chloride and Genamin CS302D for 15 min showed a complete long-term cell killing, with 2-phenoxyethanol the killing was complete only at 2xRC and Lonzabac caused complete killing at RC but not at 0.5xRC. The diS-C3(3) fluorescence assay performed immediately after a 10-min biocide exposure revealed several concentration-dependent modes of action: Lonzabac at 0.5xRC caused a mere depolarization, higher concentrations causing gradually increasing cell damage; benzalkonium chloride and Genamin CS302D rapidly damaged the membrane of some cells and depolarized the rest whereas 2-phenoxyethanol, which had the lowest effect in the plating test, produced a concentration-dependent fraction of cells with impaired membranes. Cell staining slightly increased during the diS-C3(3) assay; addition of a protonophore showed that part of the remaining undamaged cells retained their membrane potential. Comparison of short-term and long-term data implies that membrane depolarization alone is not sufficient for complete long-term killing of yeast cells under the action of a biocide unless it is accompanied by perceptible impairment of membrane integrity. The results show that the diS-C3(3) fluorescence assay, which reflects the short-term effects of a biocide on cell membranes, can be successfully used to assess the microbicidal efficiency of biocides.     

Changes in the proteins of the vitelline membrane of hens' eggs during storage

Vitelline membranes from fresh and stored eggs were treated with either 0.5 M sodium chloride or 1% sodium dodecyl sulphate (SDS) and the resulting solutions examined by polyacrylamide gel electrophoresis. Several differences between the fresh and stored membranes were evident, the most noticeable being the loss of protein VMO I (vitelline membrane outer I) and the formation of a dimer of lysozyme during storage.     

Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification

Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.     

Streamlining process characterization efforts using the high throughput ambr® crossflow system for ultrafiltration and diafiltration processing of monoclonal antibodies

Commercial process development for biopharmaceuticals often involves process characterization (PC) studies to gain process knowledge and understanding in preparation for process validation. One common approach to conduct PC activities is by using design-of-experiment, which can help determine the impact process parameter deviations may have on product quality attributes. Qualified scale-down systems are typically used to conduct these studies. For an ultrafiltration/diafiltration (UF/DF) application, however, a traditional scale-down still requires hundreds of milliliters of material per run and can only conduct one experiment at a time. This poses a challenge in resources as there could be 20+ experiments required for a typical UF/DF PC study. One solution to circumvent this is the use of high-throughput systems, which enable parallel experimentation by only using a fraction of the resources. Sartorius Stedim Biotech has recently commercialized the ambr® crossflow high-throughput system to meet this need. In this study, the performance of this system during a monoclonal antibody UF/DF step was first compared with a pilot- and a manufacturing-scale tangential flow filtration (TFF) system at a single operating condition. Due to material limitations, it was then compared to only the pilot-scale TFF system across wider ranges of transmembrane pressure; crossflow rate; and diafiltration concentration in a PC study. Permeate flux, aggregate content, process yield, pH/conductivity traces, retentate concentration, axial pressure drop, and turbidity values were measured at both scales. A good agreement was attained across scales, further supporting its potential use as a scale-down system.     

Identification of specific proteins and glycoproteins associated with membrane fractions isolated from Zea mays L. coleoptiles

The aim of this work was to identify proteins specific for plant cell membranes which could then be used as unique markers. A crude membrane fraction was isolated from corn coleoptiles and separated on non-linear sucrose density gradients. Separation of endoplasmic reticulum (NADH-cytochrome c reductase), mitochondria (cytochrome c oxidase), golgi (inosine diphosphatase), and plasma membranes (N-1-naphthylphthalamic acid-binding) was achieved. The membrane proteins from the gradient fractions were separated using sodium dodecyl sulphate-poly-acrylamide gel electrophoresis and the gels stained with coomassie blue or with concanavalin A/peroxidase to detect glycoproteins. Proteins specific for the various membranes were identified. Five proteins including two glycoproteins were plasma membrane markers. Protoplasts were isolated and iodinated using lactoperoxidase/glucose oxidase covalently attached to beads. Eleven iodinated proteins were found and three of these corresponded to proteins specifically associated with plasma membranes in the density gradients. Two methods for detecting Ca²⁺-binding proteins following sodium dodecylsulphate polyacrylamide gel electrophoresis were employed. The majority of such proteins were found in the endoplasmatic reticulum and one was specific for plasma membranes. In vitro and in vivo phosphorylation of membrane proteins was examined and the majority of proteins phosphorylated were glycoproteins. Two of the phosphorylated proteins (M_r=110,000 and 20,000) were also iodinated on protoplasts and may be part of the plasma membrane ATPases.Abbreviations ER endoplasmic reticulum - IDP inosine diphosphate - NPA N-1-naphthylphthalamic acid     

A mechanistic model to account for the Donnan and volume exclusion effects in ultrafiltration/diafiltration process of protein formulations

Ultrafiltration/diafiltration (UF/DF) is a typical step in protein drug manufacturing process to concentrate and exchange the protein solution into a desired formulation. However, significant offset of pH and composition from the target formulation have been frequently observed after UF/DF, posing challenges to the stability, performance, and consistency of the final drug product. Such shift can often be attributed to the Donnan and volume exclusion effects. In order to predict and compensate for those effects, a mechanistic model is developed based on the protein charge, mass and charge balances, as well as the equilibrium condition across the membrane. The integrated UF/DF model can be used to predict both the dynamic behavior and the final outcome of the process. Examples of the modeling results for the pH and composition variation during the UF/DF operations are presented for two monoclonal antibody proteins. The model predictions are in good agreement with a comprehensive experimental data set that covers different process steps, protein concentrations, solution matrices, and process scales. The results show that significant pH and excipient concentration shifts are more likely to occur for high protein concentration and low ionic strength matrices. As a special example, a self-buffering protein formulation shows unique pH behavior during DF, which could also be captured with the dynamic model. The capability of the model in predicting the performance of UF/DF process as a function of protein characteristics and formulation conditions makes it a useful tool to improve process understanding and facilitate process development.     

Disinfection of human musculoskeletal allografts in tissue banking: a systematic review

Musculoskeletal allografts are typically disinfected using antibiotics, irradiation or chemical methods but protocols vary significantly between tissue banks. It is likely that different disinfection protocols will not have the same level of microorganism kill; they may also have varying effects on the structural integrity of the tissue, which could lead to significant differences in terms of clinical outcome in recipients. Ideally, a disinfection protocol should achieve the greatest bioburden reduction with the lowest possible impact on tissue integrity. A systematic review of three databases found 68 laboratory and clinical studies that analyzed the microbial bioburden or contamination rates of musculoskeletal allografts. The use of peracetic acid–ethanol or ionizing radiation was found to be most effective for disinfection of tissues. The use of irradiation is the most frequently published method for the terminal sterilization of musculoskeletal allografts; it is widely used and its efficacy is well documented in the literature. However, effective disinfection results were still observed using the BioCleanse? Tissue Sterilization process, pulsatile lavage with antibiotics, ethylene oxide, and chlorhexidine. The variety of effective methods to reduce contamination rate or bioburden, in conjunction with limited high quality evidence provides little support for the recommendation of a single bioburden reduction method.     

Bioconversion of starches into maltotetraose using Pseudomonas stutzeri maltotetraohydrolase in a membrane recycle bioreactor: Effect of multiple enzyme systems and mass balance study

A simplified single-step method involving simultaneous production and purification of maltotetraose (G₄) by employing ultrafiltration (UF) membranes was previously proposed. The addition of a pretreatment step using pullulanase and then the G₄-amylase was expected to increase the yield of G₄. The single-enzyme system, however, showed 0.42 g higher total product output than the successive dual-enzyme system throughout 6 h reaction. The G₄ yield using the successive dual-enzyme system could be improved after removing the unwanted side product with UF. Experiments were conducted with membranes of larger pore size, but this did not significantly increase the total product output. The membrane unit with a molecular weight cutoff of 1,000 was the most appropriate membrane pore size for the G₄-exo--amylase membrane recycle bioreactor system. The total amount of substrate fouled in the membrane during a 6-h reaction was estimated as 69 mg glucose equivalent when substrate concentration was 0.25% (w/v). The mass balance equation indicated that the percent conversion of soluble starch to G₄ at steady state was 65%.     

Chitosan membranes modified by contact with poly(acrylic acid)

In this work chitosan membranes modified by contact with poly(acrylic acid) (PAA) aqueous solution at two different temperatures (25 °C and 60 °C) were obtained. The pure chitosan (CS) membranes, as well as those treated with PAA (CSPAA_25 and CSPAA_60) were characterized by FTIR-ATR, water sorption capacity, thermal analysis (TG/DTG), and scanning electron microscopy (SEM). In addition, in vitro permeation experiments were carried out using metronidazol and sodium sulfamerazine aqueous solutions at 0.1% and 0.2% as model drugs. FTIR-ATR results showed the presence of absorption bands of and COO⁻ indicating the formation of a polyelectrolyte complex between chitosan and poly(acrylic acid). The results also indicated that PAA penetrates deeper into the membrane at higher temperature (60 °C), forming a thicker complex layer. Polyelectrolyte complex formation as well as the influence of treatment temperature was confirmed by lower hydrophilicity, higher thermal stability, and lower permeability of the treated membranes. The results show that the methodology used is a simple and very efficient way to drastically change some membrane properties, especially their permeability.     

Osmotic properties of spermatozoa from felids producing different proportions of pleiomorphisms: influence of adding and removing cryoprotectant

The spermatozoon of felids (cats) survives cryopreservation inconsistently. Using ejaculates from three species (domestic cat [normospermic versus teratospermic], the normospermic serval and the teratospermic clouded leopard), this study (1) determined the influence of adding and removing two permeating cryoprotectants (glycerol and dimethylsulfoxide) and (2) assessed the impact of one-step versus multi-step cryoprotectant removal on sperm motility and membrane integrity. Spermatozoa were exposed in a single step to various anisotonic solutions or to 1M solutions of glycerol or dimethylsulfoxide. In both cases, sperm then were returned to near isotonic conditions in a single or multi-step with de-ionized water, Ham's F10 medium or saline. Percentage of sperm motility was measured subjectively, and plasma membrane integrity was assessed using a dual fluorescent stain and flow cytometry. Sperm motility was more sensitive to anisotonic conditions than membrane integrity. Rapid dilution into various test solutions and removal of cryoprotectant with de-ionized water reduced (P<0.01) sperm motility compared to control spermatozoa maintained in Ham's F10. Exposing sperm from all species to a 1M solution of either cryoprotectant resulted in >85% spermatozoa retaining intact membranes. However, return to isotonicity with de-ionized water in a single step or multiple steps always caused severe plasma membrane disruption. In contrast, sperm motility and membrane integrity in all species and populations remained unaffected (P>0.05) when spermatozoa were returned to isotonicity in multiple steps with Ham's F10 medium or 0.9% sodium chloride. Results demonstrate that: (1) felid spermatozoa are resistant to hypertonic stress; (2) sperm motility is more sensitive to changes in osmolality than membrane integrity; and (3) removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption in both normospermic and teratospermic males.     

Auxin-stimulated ATPase in membrane fractions from pumpkin hypocotyls (Cucurbita maxima L.)

Membrane fractions from Cucurbita maxima hypocotyls were isolated in a medium which inhibits the action of endogenous phospholipases. After removal of soluble phosphatases by Sepharose 2B-CL column chromatography, an auxin-stimulated ATPase activity was found in membrane fractions from linear sucrose gradients. In the presence of 10^-4 M phenylacetic acid (PAA), the stimulation by indol-3-acetic acid (IAA) exhibited a bimodal concentration dependence with maximal stimulation of about 50% at 10^-6 M IAA. Without PAA, only a high concentration of 10^-4 M IAA was stimulatory, whereas 10^-6 M IAA had no apparent effect and 10^-8 M IAA exhibited weak inhibition. PAA alone had only weak or no effects. The effects of IAA must be considered as hormone-specific. The ATPase activity in the presence of 10^-4 M PAA was activated only by 2,4-dichlorophenoxyacetic acid (2,4-D), an active auxin analogue, but not by the inactive stereoisomers, 2,3-D and 3,5-D. Comparison with marker enzyme profiles suggested that part of the auxin-stimulated ATPase was localized on plasma membranes as well as other compartments. Thus, the auxin-stimulated ATPase may become a useful tool in the investigation of the mechanism of action of auxin.Abbrevations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 3,5-D 3,5-dichlorophenoxyacetic acid - IAA indol-3-acetic acid - PAA phenylacetic acid - MES (2-(N-morpholino))-ethanesulfonic acid - EDTA ethylenediamine tetraacetic acid     

Liquid chromatographic determination of penicillins by postcolumn alkaline degradation using a hollow-fiber membrane reactor

A high-performance liquid chromatographic method using a hollow-fiber membrane reactor is described for the determination of penicillins. This method involves separation of penicillins on a C18 column, postcolumn reaction with sodium hydroxide and mercury (II) chloride introduced into the main flow stream using sulfonated hollow-fiber membrane reactors immersed in each solution (4 M sodium hydroxide and 3 X 10(-2) M mercury (II) chloride plus 10(-2) M nitric acid), and detection at 290 nm based on the uv absorbance of the degradation products. At penicillin concentrations of 5 micrograms/ml, within- and between-run precisions (relative standard deviation) were 0.24-2.39 and 1.19-4.13%, respectively. The detection limits of the proposed method were 1-5 ng at a signal-to-noise ratio of 3. The method was applied to assays of ampicillin and its metabolites in human serum and urine.     

Preparation and characterisation of surfaces properties of poly(hydroxyethylmethacrylate-co-methacrylolyamido-histidine) membranes: application for purification of human immunoglobulin G

In this study, an affinity membrane containing L-histidine as an amino acid ligand was used in separation and purification of human immunoglobulin G (HIgG) from solution and human serum. The polarities and the surface free energies of the affinity membranes were determined by contact angle measurements. HIgG adsorption and purification onto the affinity membranes from aqueous solution and human serum were investigated in a batch and a continuous system. Effect of different system parameters such as ligand density, adsorbent dosage, pH, temperature, ionic strength and HIgG initial concentration on HIgG adsorption were investigated. The maximum adsorption capacity of p(HEMA-MAAH-4) membranes for HIgG was 13.06 mgml(-1). The reversible HIgG adsorption on the affinity membrane obeyed both the Langmuir and Freundlich isotherm models. The adsorption data was analysed using the first- and second-order kinetic model and the experimental data was well described by the first-order equations. In the continuous system, the purity of the eluted HIgG, as determined by HPLC, was 93% with recovery 58% for p(HEMA-MAAH-4) membrane. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.     

Strategies for high-concentration drug substance manufacturing to facilitate subcutaneous administration: A review

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.