Small molecule clearance in ultrafiltration/diafiltration in relation to protein interactions: Study of citrate binding to a Fab

Ultrafiltration/diafiltration (UFDF) is commonly utilized in the purification of recombinant proteins to concentrate and buffer exchange the product. It is often the final step in the purification process, placing the protein in its final formulation and clearing small molecules introduced in upstream purification steps. This article presents a case study of reduced small molecule clearance in ultrafiltration/diafiltration of an antigen‐binding fragment of a monoclonal antibody. Citrate, a commonly utilized small molecule in downstream processes, is shown to have reduced clearance due to specific interactions with the protein product. The study presents process solutions and utilizes a simple model to characterize clearance of small molecules which exhibit interactions with product protein. Biotechnol. Bioeng. 2009;102: 1718–1722. © 2008 Wiley Periodicals, Inc.     

Defining the mechanistic binding of viral particles to a multi‐modal anion exchange resin

A multi‐tiered approach to determine the binding mechanism of viral clearance utilizing a multi‐modal anion exchange resin was applied to a panel of four viral species that are typically used in validating viral clearance studies (i.e., X‐MuLV, MVM, REO3, and PrV). First, virus spiked buffer‐only experiments were conducted to evaluate the virus's affinity for single mode and multi‐modal chromatography resins under different buffer conditions in a chromatography column setting. From these results we hypothesize that the mechanisms of binding of the viruses involve binding to both the hydrophobic and anionic functional groups. This mechanistic view agreed with the general surface characteristics of the different virus species in terms of isoelectric point and relative hydrophobicity values. This hypothesized mechanistic binding was then tested with commercially relevant, in‐process materials, in which competitive binding occurred between the load components (e.g., viruses, target product, and impurities) and the resin. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1019–1026, 2018     

Biotransformation of Ferulic Acid to 4-Vinylguaiacol by Enterobacter soli and E. aerogenes

We investigated the conversion of ferulic acid to 4-vinylguaiacol (4-VG), vanillin, vanillyl alcohol, and vanillic acid by five Enterobacter strains. These high-value chemicals are usually synthesized by chemical methods but biological synthesis adds market value. Ferulic acid, a relatively inexpensive component of agricultural crops, is plentiful in corn hulls, cereal bran, and sugar-beet pulp. Two Enterobacter strains, E. soli, and E. aerogenes, accumulated 550–600?ppm amounts of 4-VG when grown in media containing 1,000?ppm ferulic acid; no accumulations were observed with the other strains. Decreasing the amount of ferulic acid present in the media increased the conversion efficiency. When ferulic acid was supplied in 500, 250, or 125?ppm amounts E. aerogenes converted ~72?% of the ferulic acid present to 4-VG while E. soli converted ~100?% of the ferulic acid to 4-VG when supplied with 250 or 125?ppm amounts of ferulic acid. Also, lowering the pH improved the conversion efficiency. At pH 5.0 E. aerogenes converted ~84?% and E. soli converted ~100?% of 1,000?ppm ferulic acid to 4-VG. Only small, 1–5?ppm, accumulations of vanillin, vanillyl alcohol, and vanillic acid were observed. E. soli has a putative phenolic acid decarboxylase (PAD) that is 168 amino acids long and is similar to PADs in other enterobacteriales; this protein is likely involved in the bioconversion of ferulic acid to 4-VG. E. soli or E. aerogenes might be useful as a means of biotransforming ferulic acid to 4-VG.     

Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli

Apolipoprotein A 1 Milano (ApoA‐1M), the protein component of a high‐density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co‐purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA‐1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process. Biotechnol. Bioeng. 2010; 105: 239–249. © 2009 Wiley Periodicals, Inc.     

Enzymatic Removal of Diacetyl from Beer : III. Enzyme Protection and Regeneration of Cofactor

Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at - 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors.     

Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes

For the manufacturing of recombinant protein therapeutics produced from mammalian cell culture, demonstrating the capacity of the purification process to effectively clear infectious viruses is a regulatory requirement. At least two process steps, using different mechanisms of virus removal and/or inactivation, should be validated in support of the regulatory approval process. For example, exposure of the product stream to low pH, detergents or solvent/detergent combinations is commonly incorporated in protein purification processes for the inactivation of lipid‐enveloped viruses. However, some proteins have limited stability at low pH or in the presence of the detergents, and alternative techniques for achieving the inactivation of enveloped viruses would be beneficial. We present here an alternative and novel approach for the rapid inactivation of enveloped viruses using pH‐neutral buffer solutions containing arginine. The implementation of this approach in a monoclonal antibody or Fc‐fusion protein purification process is described and illustrated with several different therapeutic proteins. The use of the neutral pH arginine solution was able to effectively inactivate two enveloped model viruses, with no measurable effect on the product quality of the investigated proteins. Thus, the use of pH‐neutral arginine containing buffer solutions provides an alternative means of virus inactivation where other forms of virus inactivation, such as low pH and/or solvent/detergent treatments are not possible or undesirable due to protein stability limitations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:108–112, 2014     

Verification of the final anion exchange chromatography in the r-hGH manufacturing process

In this study, final anion exchange chromatography in the recombinant human growth hormone (r-hGH) manufacturing process was validated using a validation protocol that was consistent with both policy and standard operation procedure (SOP). Two buffer solutions used in chromatography were first validated and were found to satisfy pre-established acceptance criteria as follows: pH: 8.2, endotoxin: < 0.6 EU/mL, bioburden test: negative. Final anion exchange chromatography was conducted using a DEAE Sepharose FF Resin and eluted with a linear gradient of 30 to 110 mM NaCl in 50 mM Tris-HCl buffer at a flow rate of 15 L/h. Three consecutive batches of hGH solutions were generated via anion exchange chromatography, which was performed within pre-established operating parameters determined through in-process control. When all three batches were assessed by the pre-established sampling plan and tested for quality control, this purification process was shown to satisfy pre-established acceptance criteria; endotoxin: ≤ 0.5 EU/mg, ECP: ≤ 1.4 ppm, IEF: same removal distance, hGH content by Native-PAGE: 100%, purity by HPLC: ≥ 99%, yield by UV scanning: 87 to 89%, hGH monomer protein content by HPLC: 99%. Therefore, the final anion chromatography process was successfully validated in this study, and this method consistently yielded hGH solutions that satisfied pre-established criteria for subsequent processing.     

Advanced protein formulations

It is well recognized that protein product development is far more challenging than that for small‐molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long‐term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site‐specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze‐dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution.     

E. coli spheroplast-mediated transfer of cloned cauliflower mosaic virus DNA into plant protoplasts

Summary Saishin (Brassica chinensis L.) mesophyll protoplasts and E. coli spheroplasts harbouring hybrid plasmid with tandemly dimerized cauliflower mosaic virus DNA were mixed in ratios of 1:1,000 and incubated for 20 min at 30° C in the presence of 20% polyvinyl alcohol. Subsequently, protoplasts/spheroplasts mixture was washed with high pH-high Ca buffer. After 3 days of culture, 8% of Saishin protoplasts were transfected as monitored by immunofluorescence technique. When plant protoplasts and bacterial spheroplasts were mixed in ratios of 1:100 or 1:2,000, 1% or 5% of protoplasts were transfected, respectively.     

Selective precipitation of DNA by spermine during the chemical extraction of insoluble cytoplasmic protein.

The direct chemical extraction of recombinant L1 protein (the major capsid protein of human papillomavirus type 16) from the cytoplasm of E. coli HMS174(DE3) has recently been demonstrated at high cell density (to OD(600) = 160) without the use of reducing agent (1). Coextraction of DNA at high concentration prevents direct coupling to postextraction recovery operations including expanded bed adsorption. In this study, spermine is used to selectively precipitate DNA during chemical extraction. Highly efficient and selective DNA precipitation was achieved. An approximate 10-fold increase in the specific spermine concentration (mg of spermine/mg of DNA) was required to precipitate DNA when 8 M urea was added to the extraction buffer. EDTA (3 mM), required for effective chemical extraction, does not significantly inhibit DNA precipitation. Precipitation selectivity was demonstrated in a bovine serum albumin spiking test, with almost complete recovery of the spiked protein. During studies on the direct extraction of L1 protein from cells at OD(600) = 80, high DNA removal efficiency (>85%) and negligible L1 protein coprecipitation were achieved. This selective precipitation technique simply requires the addition of spermine to the chemical extraction buffer and therefore does not increase technique complexity. This modification enhances the method's general applicability and enables direct coupling to downstream recovery units following chemical extraction at high cell and product concentrations.     

Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation

Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use‐period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature‐associated change in the pK_as, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well‐studied system, shows the greatest change in pH when going from +25 to ?30°C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to ?30°C, whereas Tris‐hydrochloride had an ～1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0°C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze–thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.     

Influence of Incubation Solution on the Rate of Recovery of Pratylenchus brachyurus from Cotton Roots

The rate of recovery of Pratylenchus brachyurus from cotton roots was enhanced when the tissue was incubated in solutions containing 10 ppm ethoxyethyl mercuric chloride, 50 ppm dihydrostreptomycin sulfate, 50, 100, or 1,000 ppm diisobutylphenoxethyl dimethyl benzyl ammonium chloride, or mixtures of these compounds. Incubation in 10 or 100 ppm zinc sulfate, zinc chloride, or magnesium chloride also enhanced the rate of recovery. Incubation solutions containing 1 or 1,000 ppm zinc chloride or magnesium chloride had no influence on this phenomenon, whereas, 10,000 ppm zinc sulfate, zinc chloride, or magnesium chloride retarded the rate of recovery. A t all incubation intervals during the first 21 days after the roots were removed from soil, the P. brachyurus population consisted of approximately 25% second-stage juveniles, 44% third and fourth-stage juveniles, and 31% females. At least 88% of the second-stage juveniles and 51% of the third and fourth-stage juveniles passed through a single 325-mesh sieve, whereas, 84% of the females collected were retained on a sieve of this mesh.     

Process‐relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics

The biopharmaceutical industry has invested considerably in the implementation of single‐use disposable bioreactors in place of or in addition to their stainless steel‐counterparts. This new wave of construction materials for disposable bioprocess containers encompass a plethora of uncharacterized secondary compounds that, when in contact with the culture media, can leach, contaminating the bioprocess. One such cytotoxic leachable already receiving attention is bis(2,4‐di‐tert‐butylphenyl)‐phosphate (bDtBPP), a breakdown product of the secondary antioxidant Irgafos 168 in polyethylene‐film based bags. This compound has been demonstrated to inhibit cell growth at concentrations ranging from 0.12 to 0.73 mg/L across an array of cell lines. Here we demonstrate that a further two CHO cell lines exhibit sensitivity to bDtBPP exposure at concentrations lower than that previously reported (0.035–0.1 mg/L). Furthermore, these inhibitory concentrations reflect bDtBPP levels found to leach early into the bioprocess, exposing reactor inoculums to serious risk. Quantitative label‐free LC‐MS/MS revealed that irrespective of cell line or concentration of bDtBPP, 8 proteins were found to be commonly differentially expressed in response to exposure to the compound highlighting biological processes related to cellular stress. Although the glycoprofile of the recombinant antibody remains primarily unchanged, we demonstrate that this compound when spiked at meaningful concentrations 72 h into culture considerably reduces the maximum cell density achieved. Studies like this reinforce the requirement for the complete characterization of all potential leachable compounds from disposable materials to assess their risk not only to the patient but also to the production pipeline itself. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1547–1558, 2016     

Virus filtration of high‐concentration monoclonal antibody solutions

The ability to process high‐concentration monoclonal antibody solutions (> 10 g/L) through small‐pore membranes typically used for virus removal can improve current antibody purification processes by eliminating the need for feed stream dilution, and by reducing filter area, cycle‐time, and costs. In this work, we present the screening of virus filters of varying configurations and materials of construction using MAb solutions with a concentration range of 4–20 g/L. For our MAbs of interest—two different humanized IgG1s—flux decay was not observed up to a filter loading of 200 L/m² with a regenerated cellulose hollow fiber virus removal filter. In contrast, PVDF and PES flat sheet disc membranes were plugged by solutions of these same MAbs with concentrations >4 g/L well before 50 L/m². These results were obtained with purified feed streams containing <2% aggregates, as measured by size exclusion chromatography, where the majority of the aggregate likely was composed of dimers. Differences in filtration flux performance between the two MAbs under similar operating conditions indicate the sensitivity of the system to small differences in protein structure, presumably due to the impact of these differences on nonspecific interactions between the protein and the membrane; these differences cannot be anticipated based on protein pI alone. Virus clearance data with two model viruses (XMuLV and MMV) confirm the ability of hollow fiber membranes with 19 ± 2 nm pore size to achieve at least 3–4 LRV, independent of MAb concentration, over the range examined. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Antimicrobial Activity of Essential Oil Components Against Potential Food Spoilage Microorganisms

The antimicrobial activity of six essential oil components against the potential food spoilage bacteria Aeromonas (A.) hydrophila, Escherichia (E.) coli, Brochothrix (B.) thermosphacta, and Pseudomonas (P.) fragi at single use and in combination with each other was investigated. At single use, the most effective oil components were thymol (bacteriostatic effect starting from 40 ppm, bactericidal effect with 100 ppm) and carvacrol (50 ppm/100 ppm), followed by linalool (180 ppm/720 ppm), α-pinene (400 ppm/no bactericidal effect), 1,8-cineol (1,400 ppm/2,800 ppm), and α-terpineol (600 ppm/no bactericidal effect). Antimicrobial effects occurred only at high, sensorial not acceptable concentrations. The most susceptible bacterium was A. hydrophila, followed by B. thermosphacta and E. coli. Most of the essential oil component combinations tested showed a higher antimicrobial effect than tested at single use. Antagonistic antimicrobial effects were observed particularly against B. thermosphacta, rarely against A. hydrophila. The results show that the concentration of at least one of the components necessary for an antibacterial effect is higher than sensorial acceptable. So the use of herbs with a high content of thymol, carvacrol, linalool, 1,8-cineol, α-pinene or α-terpineol alone or in combination must be weighted against sensorial quality.     

In-line monitoring of protein concentration with MIR spectroscopy during UFDF

Rapid increase of product titers in upstream processes has presented challenges for downstream processing, where purification costs increase linearly with the increase of the product yield. Hence, innovative solutions are becoming increasingly popular. Process Analytical Technology (PAT) tools, such as spectroscopic techniques, are on the rise due to their capacity to provide real-time, precise analytics. This ensures consistent product quality and increased process understanding, as well as process control. Mid-infrared spectroscopy (MIR) has emerged as a highly promising technique within recent years, owing to its ability to monitor several critical process parameters at the same time and unchallenging spectral analysis and data interpretation. For in-line monitoring, Attenuated Total Reflectance—Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a method of choice, as it enables reliable measurements in a liquid environment, even though water absorption bands are present in the region of interest. Here, we present MIR spectroscopy as a monitoring tool of critical process parameters in ultrafiltration/diafiltration (UFDF). MIR spectrometer was integrated in the UFDF process in an in-line fashion through a single-use flow cell containing a single bounce silicon ATR crystal. The results indicate that the one-point calibration algorithm applied to the MIR spectra, predicts highly accurate protein concentrations, as compared with validated offline analytical methods.     

Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system

Chinese hamster ovary (CHO) cells remain the default production host for many biopharmaceutical drugs, particularly monoclonal antibodies (mAb). Production of gram and kilogram quantities of protein typically requires the generation of stable CHO clones. Unfortunately, this process takes several months, significantly slowing down the drug discovery and development process. Therefore, improved technologies are needed to accelerate biopharmaceutical drug discovery and final drug substance manufacturing. In this study, we describe the generation of stable CHO pools using the piggyBac transposon system. We evaluated the system using four model antibody molecules (3 mAbs and 1 bispecific Ab). Stable CHO pools were isolated in 7–12 days. Using a simple 16‐day fed‐batch process, we measured titers ranging from 2.3 to 7.6 g/L for the four model antibodies. This represented a 4‐ to 12‐fold increase relative to the controls. Additionally, we isolated stable CHO clones. We found that the stable CHO clones isolated from the piggyBac transposon pools yielded titers two to threefold higher relative to the control clones. Taken together, these results suggest that stable CHO pool and clone generation can be significantly improved by using the piggyBac transposon system. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1301–1307, 2016     

The effects of ethyl methanesulfonate treatment on <Emphasis Type="Italic">Eucalyptus</Emphasis> pollen behaviour in vitro

Treating pollen with mutagens prior to controlled pollination may facilitate the production of mutant trees for developmental studies and eventual plantation improvement. To establish a suitable dose of the chemical mutagen ethyl methanesulfonate (EMS) for the testing of this hypothesis, pollen of Eucalyptus globulus ssp. globulus and E. grandis was studied in vitro. Pollen germination, pollen tube elongation and generative cell division were examined after 48 h of culture, following exposure to between 0 and 1,000 ppm EMS. Doses of 600 to 1,000 ppm EMS reduced pollen germination in vitro in both species. Doses of up to 1,000 ppm EMS were not observed to significantly impact on either pollen tube length, or generative cell division in vitro of either species. A dose of 600 ppm EMS in paraffin oil is predicted to induce mutation in Eucalyptus species whilst impacting minimally on seed production based on the effect on pollen germination.