Efficiency of ultrafiltration/diafiltration in removing organic and elemental process equipment related leachables from biological therapeutics

In the production of biological therapeutics such as monoclonal antibodies (mAbs), ultrafiltration and diafiltration (UF/DF) are widely regarded as effective downstream processing steps capable of removing process equipment related leachables (PERLs) introduced upstream of the UF/DF step. However, clearance data available in the literature are limited to species with low partition coefficients (log P) such as buffer ions, hydrophilic organic compounds, and some metal ions. Additional data for a wide range of PERLs including hydrophobic compounds and elemental impurities are needed to establish meaningful, comprehensive safety risk assessments. Herein, we report the results from studies investigating the clearance of seven different organic PERLs representing a wide range of characteristics (i.e., log P (−0.3 to 18)), and four model elements with different chemical properties spiked into a mAb formulation at 10 ppm and analyzed during clearance using gas chromatography–mass spectrometry (GC–MS), liquid chromatography-photodiode-array-mass spectrometry (LC-PDA-MS), and inductively coupled plasma mass spectrometry (ICP-MS). The clearance data showed ideal clearance and sieving of spiked organic PERLs with log P < 4, partial clearance of PERLs with 4 < log P < 9, and poor clearance of highly hydrophobic PERLs (log P > 9) after nine diafiltration volumes (DVs). Supplemental clearance studies on seven additional PERLs present at much lower concentration levels (0.1–1.5 ppm) in the mAb formulation upstream of UF/DF and three PERLs associated with the tangential flow filtration (TFF) equipment also demonstrated the similar correlations between log P and % clearance. For model elements, the findings suggest that UF/DF in general provides ideal clearance for elements. Evidence showed that the UF/DF process does not only help mitigate leachables risk from PERLs introduced upstream of UF/DF, but also from the TFF operation itself as all three TFF-related PERLs were effectively cleared. Overall, the UF/DF clearance presented in this work demonstrated whereas highly hydrophobic PERLs and elements that exist as charged species, particularly transition metal ions, may not be as effectively cleared and thus warrant further risk assessment; hydrophilic and some hydrophobic PERLs (log P < 4) are indeed well-cleared and thus present a lower overall safety risk.     

Multi‐criteria manufacturability indices for ranking high‐concentration monoclonal antibody formulations

The need for high‐concentration formulations for subcutaneous delivery of therapeutic monoclonal antibodies (mAbs) can present manufacturability challenges for the final ultrafiltration/diafiltration (UF/DF) step. Viscosity levels and the propensity to aggregate are key considerations for high‐concentration formulations. This work presents novel frameworks for deriving a set of manufacturability indices related to viscosity and thermostability to rank high‐concentration mAb formulation conditions in terms of their ease of manufacture. This is illustrated by analyzing published high‐throughput biophysical screening data that explores the influence of different formulation conditions (pH, ions, and excipients) on the solution viscosity and product thermostability. A decision tree classification method, CART (Classification and Regression Tree) is used to identify the critical formulation conditions that influence the viscosity and thermostability. In this work, three different multi‐criteria data analysis frameworks were investigated to derive manufacturability indices from analysis of the stress maps and the process conditions experienced in the final UF/DF step. Polynomial regression techniques were used to transform the experimental data into a set of stress maps that show viscosity and thermostability as functions of the formulation conditions. A mathematical filtrate flux model was used to capture the time profiles of protein concentration and flux decay behavior during UF/DF. Multi‐criteria decision‐making analysis was used to identify the optimal formulation conditions that minimize the potential for both viscosity and aggregation issues during UF/DF. Biotechnol. Bioeng. 2017;114: 2043–2056. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Perodicals, Inc.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard^®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard^® stimulated CRP and complement responses to A. salmonicida infection in common carp.     

Effect of dietary β-glucan on growth performance,fecal microbial shedding and immunological responses after lipopolysaccharide challenge in weaned pigs

Two experiments (Exp.) were conducted to evaluate the effects of β-glucan inclusion in the diet on growth performance and immune function after lipopolysaccharide (LPS) challenge. In Exp. 1, a total of 40 weaned pigs (progeny of Landrace×Yorkshire sows by Duroc) with an initial body weight (BW) of 7.89 ± 0.84 kg (21 ± 2 d) of age) were used in a 28-day (d) experiment to determine the effects of dietary β-glucan on growth performance. Pigs were allotted randomly to two treatments consisting of addition of 0 or 0.1 g β-glucan/kg diet with four replicate pens per treatment and five pigs per pen. Growth performance was not affected by β-glucan supplementation throughout the experiment. However, dietary β-glucan reduced (P<0.05) the number of fecal Escherichia coli. In Exp. 2, a total of 20 weaned barrows (6.22 ± 0.25 kg of BW and 21 ± 2 d of age) individually raised in metabolic cages were used to evaluate immunological responses following LPS challenge. Pigs were fed 0 or 0.1 g β-glucan/kg diet for 42 d. At the end of the trial, half of the pigs (n = 5) from each treatment were injected intraperitoneal with E. coli LPS at a concentration of 100 μg/kg BW and the other half were injected with sterile saline solution. Treatments were arranged as a 2×2 factorial, with the main effect of LPS challenge (saline vs. LPS) and β-glucan supplementation (0 g/kg vs. 0.1 g/kg). After LPS injection, blood was taken at 0, 2, 4, 6, 8 and 12 hours (h) for the blood cell counts and blood inflammatory response. Dietary β-glucan increased (P<0.05) leukocytes counts at 4, 6 and 8 h, and blood lymphocyte concentrations at 2, 4 and 6 h and LPS challenge increased (P<0.05) counts of leukocytes at 2, 4, 6 and 8 h and blood lymphocyte at 2 and 4 h post-challenge. The rectal temperature was increased (P<0.05) at 2, 4, 6 and 8 h after LPS challenge. Dietary β-glucan reduced (P<0.05) and LPS challenge increased (P<0.05) blood plasma tumor necrosis factor-α (TNF-α) concentration at 2 and 4 h post-challenge. Dietary β-glucan increased (P<0.05) the concentration of the cluster of differentiation antigens 4 cells (CD4⁺) at 2, 4 and 6 h, and of 8 (CD8⁺) at 4 and 6 h post-challenge, respectively. The LPS challenge increased (P<0.05) CD4⁺ and CD8⁺ cell concentrations at 2, 4 and 6 h post-challenge. The CD4⁺:CD8⁺ ratio was reduced (P<0.05) by LPS challenge but was increased (P<0.05) by dietary β-glucan at 2, 4, 6 and 8 h post-challenge. In conclusion, dietary β-glucan decreased E. coli numbers but did not affect growth performance in weaned pigs and may offer benefits on immune function in weaned pigs challenged with LPS.     

Effects of dietary β-glucan and rice fermented on growth performance,fatty acids,and Newcastle disease immune response in turkey broilers

Poultry production has been developing in Vietnam with challenges of disease. Thus, feed additive should be investigated not only growth but also health enhancement. Here, we aimed to determine the effects of Saccharomyces cerevisiae-fermented rice (FR) and β-glucan on turkey’s growth performance, carcass characteristics, immune and fatty acid (FA) profiles. A total of 180 turkey chicks aged 1–56 days were randomly assigned to five sextuplicate groups and the birds had ad libitum feed and water access throughout the experiment. The five treatment groups were given the same diet with different proportions of FR and β-glucan. Broilers supplemented with 4% β-glucan and 4% FR presented the highest and second-highest growth performance, respectively. The 4% β-glucan and 4% FR treatments resulted in the highest carcass characteristic values without significantly affecting the breast or thigh meat pH or cooking loss. The 4% β-glucan and 4% FR treatments maximally increased the Newcastle disease (ND) antibody titers at 28, 42 and 56 days, respectively as well as thymus organ index. The foregoing treatments did not significantly affect the blood profiles relative to the control. However, the 4% FR treatment lowered the blood cholesterol levels (p > 0.05). The total FA profiles did not significantly differ among treatments. Nevertheless, both the β-glucan and FR treatments increased the MUFA levels compared to that of the control (p > 0.05). Hence, the dietary administration of 4% β-glucan and FR to turkey broilers could effectively improve their growth performance and immunity.     

Yeast Killer Toxin-Like Candidacidal Ab6 Antibodies Elicited through the Manipulation of the Idiotypic Cascade

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by β-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to β-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble β-1,3-glucan, but not by pustulan, a β-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.     

Antibody-dependent difference in biodistribution of monoclonal antibodies in animal models and humans

 The study was designed to clarify the difference in pharmacokinetics of monoclonal antibodies (mAb) in animal models and humans, and to elucidate the applicability of animal models. ^99mTc-labeled murine mAb – against carcinoembryonic antigen (designated BW431/26), and neural cell adhesion molecule (NE150) – and one chimeric mouse/human mAb against nonspecific cross-reacting antigen (chNCA) were administered i.v. to normal mice and athymic mice (370 kBq, 400 ng) xenografted with human cancer cells expressing antigens, and into patients with tumor (925 MBq, 1 mg). The biodistribution of two of the three mAb (not ^99mTc-BW431/26) differed clearly in mice and patients. ^99mTc-NE150 showed specific uptake in xenografted tumor and otherwise a normal biodistribution; however, clinical examination showed increased uptake in the liver with rapid blood clearance (mean α half-life = 31.1 min) compared with ^99mTc-BW431/26 (28.4 h). ^99mTc-chNCA demonstrated increased blood clearance and renal excretion in both normal and athymic mice, with accumulation in tumors. Clinical examination showed rapid blood clearance (mean α half-life = 6.4 min) and increased uptake in the liver. High-performance liquid chromatographic analysis of ^99mTc-chNCA revealed the immune complex in blood, suggesting uptake of the complex by the reticuloendothelial cells. The biodistribution of radiolabeled mAb in animal and human models was variable and specific for each of the three mAb. The results of animal studies with mAb should be evaluated carefully before being extrapolated to humans, on the basis of the nature of the mAb and interacting substances. Received: 9 April 1997 / Accepted 3 March 1998     

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.     

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.     

A lichenase-like family 12 endo-(1-->4)-beta-glucanase from Aspergillus japonicus: study of the substrate specificity and mode of action on beta-glucans in comparison with other glycoside hydrolases

Using anion-exchange chromatography on Source 15Q followed by hydrophobic interaction chromatography on Source 15 Isopropyl, a lichenase-like endo-(1→4)-β-glucanase (BG, 28 kDa, pI 4.1) was isolated from a culture filtrate of Aspergillus japonicus. The enzyme was highly active against barley β-glucan and lichenan (263 and 267 U/mg protein) and had much lower activity toward carboxymethylcellulose (3.9 U/mg). The mode of action of the BG on barley β-glucan and lichenan was studied in comparison with that of Bacillus subtilis lichenase and endo-(1→4)-β-glucanases (EG I, II, and III) of Trichoderma reesei. The BG behaved very similar to the bacterial lichenase, except the tri- and tetrasaccharides formed as the end products of β-glucan hydrolysis with the BG contained the β-(1→3)-glucoside linkage at the non-reducing end, while the lichenase-derived oligosaccharides had the β-(1→3)-linkage at the reducing end. The BG was characterized by a high amino acid sequence identity to the EG of Aspergillus kawachii (UniProt entry Q12679) from a family 12 of glycoside hydrolases (96% in 162 identified aa residues out of total 223 residues) and also showed lower sequence similarity to the EglA of Aspergillus niger (O74705).     

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.     

Assessing the impact of sanitization methods for regenerated cellulose ultrafiltration/diafiltration membrane on membrane integrity and protein quality

Ultrafiltration/diafiltration (UF/DF) is typically the final step in downstream processing of recombinant monoclonal antibody (mAb) products, which serves for protein concentration and buffer exchange. For UF/DF membranes composed of regenerated cellulose (RC), sanitization with 0.1 M sodium hydroxide is generally recommended by the supplier, but it may not be sufficient for reducing bioburden during large scale manufacturing. Therefore, more stringent sanitization methods for RC membranes are required. However, chemicals used in such sanitization step may disrupt membrane integrity, while the corresponding residuals may reduce product quality. Previous work has shown that high concentration of sodium hydroxide or addition of peracetic acid (PAA) can effectively reduce bioburden, but their effects on the RC membranes remain unknown. In this work, we assessed the impact of two sanitization methods, 0.5 M sodium hydroxide and 30 mM PAA in combination with 0.5 M sodium hydroxide, on membrane integrity and protein quality of Millipore and pall corporation (PALL) membranes. Both methods showed a similar impact as the control after performing 15 cycles. However, the addition of PAA may cause residual chemical concerns, therefore, 0.5 M sodium hydroxide was recommended as an effective and safe sanitization method for RC UF/DF membranes.     

Gold Immunochromatography Assay for the Rapid Detection of Spiramycin in Milk and Beef Samples Based on a Monoclonal Antibody

Spiramycin (SP) residues in food do harm to human health. It is necessary to establish rapid detection method for SP. In this work, a monoclonal antibody (mAb)‐based gold immunochromatography assay (GICA) is developed for the rapid detection of SP. Under optimum conditions, the half‐maximal inhibitory concentration of SP‐mAb is 0.43 ng mL^–1. The subtype of SP‐mAb is IgG2b. This antibody has no cross‐reactivity with other analogues and has high affinity (4.52 × 10¹⁰ L mol^–1). Qualitative results can be visualized with the naked eye, with a visual detection limit of 1.0 ng mL^–1 and cut‐off value of 10 ng mL^–1. A hand‐held strip scanner is used for the quantitative analysis, with LOD 0.43 ng mL^–1 in assay buffer. The recoveries of SP ranged from 72.3% to 112% in milk and 98.5% to 115% in beef, with variable coefficient ranging from 9.4% to 11.7% in milk and 8.14% to 15.4% in beef. Besides, the proposed GICA method for SP is confirmed by LC–MS/MS in SP‐spiked milk and beef samples. Overall, the developed GICA can be a useful tool for SP residues on‐site screening in milk and beef samples.     

Performance of the (1,3)-Beta-d-Glucan Assay on Bronchoalveolar Lavage Fluid for the Diagnosis of Invasive Pulmonary Aspergillosis

Detection of (1,3)-beta-d-glucan (BDG), a component of the cell wall of many fungi, was studied in bronchoalveolar lavage fluid (BALF) as a possible aid for the diagnosis of proven/probable invasive pulmonary aspergillosis (IPA). BDG was measured on stored BALF from 13 patients with EORTC/MSGERC defined proven/probable IPA and 26 matched control patients without IPA. The median BALF BDG was 80 pg/mL (range <?45–8240 pg/mL) in the IPA cohort and 148 pg/mL (range <?45–5460 pg/mL) in the non-IPA cohort. Using a positive cutoff of?≥?80 pg/mL, sensitivity was 54% and specificity was 38%. Higher cutoff values led to improvement in specificity but a dramatic decrease in sensitivity. ROC/AUC analysis was unable to identify an optimal cutoff value at which test performance was enhanced: AUC 0.43, 95% CI 0.24–0.63. When the BDG assay was performed on BALF, neither sensitivity nor specificity was sufficient for use in the diagnosis of IPA.

     

Development of a competitive ELISA for the detection of soybean α subunit of β-conglycinin

The alpha subunit of β-conglycinin is one of the main allergens found in soybeans. For the preparation of a specific monoclonal antibody (mAb) against the α subunit, potential epitopes were predicted using Protean and evaluated by Wu's Antigenic Index. The specific epitope ⁸⁵EQDERQFPFPR⁹⁵ was synthesized, and then conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) for use as an immunogen and the plate-coating antigen, respectively. The resulting mAb, termed mAb-60K, was characterized as belonging to the IgG1 isotype and containing the κ light chain; it exhibited high specificity for the α subunit and did not cross react with the α′ and β subunits of β-conglycinin or other proteins found within soybeans. A competitive enzyme-linked immunosorbent assay (cELISA) with high accuracy and reproducibility was developed based on mAb-60K and the plate-bound peptide. Co-incubation with the full-length α subunit showed a 50% mAb binding inhibition concentration (IC₅₀) value of 4.42 ng/mL, with a linear inhibition curve observed α subunit concentrations between 0.65 and 29.84 ng/mL. The newly developed mAb-60K and the companion cELISA could provide a valuable tool for determining sensitivity towards the α subunit of soybean β-conglycinin and for future studies on food allergies resulting from this protein.     

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.     

Potential application of two thermostable lichenases from a newly isolated Bacillus licheniformis UEB CF: Purification and characterization

Two thermostable and alkali-stable β-1,3–1,4 glucanases (EC 3.2.1.73) EG1 and EG2 from a newly isolated Bacillus licheniformis UEB CF were purified. The molecular weights of EG1 and EG2 enzymes determined by SDS–PAGE were approximately 30 kDa and 55 kDa, respectively. The N-terminal amino acid sequences of EG1 and EG2 β-glucanases were determined to be GAAPIKKGTTKLN and DINGGGATLPQK, respectively. The optimum temperature, optimum pH, k_m and V_max of EG1 were 70 °C, 5.0, 2.1 mg/ml and 21.25 μmol/min/mg, respectively. These values for EG2 were 60 °C, 7.0, 1.82 mg/ml and 18.54 μmol/min/mg, respectively.Both endoglucanases were highly active against barley β-glucan and lichenan. However, they were inactive against CMC and laminarin. The purified β-glucanases were found to be relatively stable toward non-ionic surfactants and oxidizing agents. In addition, both enzymes showed excellent stability and compatibility with a wide range of commercial solid detergents suggesting that they are a potential candidate in detergent industries formulation.