Evaluation of product comparability.
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Animals↗
Biomedical subjects
S E Builder
Publications and source records attributed to S E Builder.
Purification of insulin-like growth factor-I and related proteins using underivatized silica.
Adsorption chromatography using underivatized porous glass can be an effective capture step for the purification of recombinant proteins. Classical desorption techniques using chaotropic agents or harsh chemical solvents often result in elution of inactive material and may not be economical at the process scale. More recently, elution schemes have used tetramethylammonium chloride (TMAC) to obtain biologically active material. A TMAC elution was shown to be effective in the initial purification steps for the recovery of recombinant human insulin-like growth factor-I (rhIGF-I) from an Escherichia coli fermentation broth. However, TMAC also elutes other, more hydrophobic, proteins that are difficult to remove in subsequent purification steps. This paper describes the capture of IGF-I from a crude fermentation broth and a more specific elution using a combination of ethanol and NaCl rather than TMAC. This elution also can be used with other proteins including an IGF-I binding protein (BP3) expressed in mammalian cell culture.
Chromatography, High Pressure Liquid↗
Use of multifactorial analysis to develop aqueous two-phase systems for isolation of non-native IGF-I.
A high yield procedure was developed to solubilize and extract IGF-I from recombinant E. coli by adding chaotrope and disulfide reductant to alkaline fermentation broth. To enhance centrifugation performance and recovery yield, a salt/polymer aqueous two-phase extraction procedure was developed whereby soluble non-native IGF-I and biomass solids are enriched in separate liquid phases. To develop this extraction system a multifactorial experimental approach was used to simultaneously map the phase diagram and identify conditions to suitably partition IGF-I and cell remnants. The presence of urea in these systems tended to disrupt two-phase formation and solids sedimentation. This, in turn, constrained the concentrations of phase forming solutes which could be effectively used. Systems containing low levels of salt (less than about 4% w/w) and polymer (less than about 10% w/w) did not form two phases. Systems containing high levels of salt (greater than about 7% w/w) and polymer (greater than about 18% w/w) formed two phases with floating solids. Intermediate levels of salt (between about 4% and 7% w/w) and polymer (between about 10% and 18% w/w) formed two phases in which solids were enriched in the heavy phase. Systems in this latter desired category were produced with a variety of different salts and polymers and all enriched non-native IGF-I in the light phase. Highest recovery yield (about 90%) was obtained with systems composed of 5% sodium sulfate and 14% PEG-8000.(ABSTRACT TRUNCATED AT 250 WORDS)
Centrifugation↗
Preparative isolation of recombinant human insulin-like growth factor 1 by reversed-phase high-performance liquid chromatography.
The isolation of recombinant human insulin-like growth factor 1 (rhIGF-1) is complicated by the presence of several rhIGF-1 variants which co-purify using conventional chromatographic media. These species consist primarily of a methionine-sulfoxide variant of the properly folded molecule and a misfolded form and its respective methionine-sulfoxide variant. An analytical reversed-phase high-performance liquid chromatography procedure using a 5-micron C18 column, an acetonitrile-trifluoroacetic acid (TFA) isocratic elution, and elevated temperature gives baseline resolution of the four species. Using this analytical method as a development tool, a process-scale chromatography step was established. The 5-micron analytical packing material was replaced with a larger-size particle to reduce back-pressure and cost. Since the TFA counter-ion binds tightly to proteins and is difficult to subsequently dissociate, a combination of acetic acid and NaCl was substituted. Isocratic separations are not good process options due to problems with reproducibility and control. A shallow gradient elution using premixed mobile phase buffers at the same linear velocity was found to give an equivalent separation at low load levels and minimized solvent degassing. However, at higher loading there was a loss of resolution. A matrix of various buffers was evaluated for their effects on separation. Elevated pH resulted in a significant shift in both the elution order and relative retention times of the principal rh-IGF-1 variants, resulting in a substantial increase in effective capacity. An increase in the ionic strength further improved resolution. Several different media were evaluated with regard to particle size, shape and pore diameter using the improved mobile phase. The new conditions were scaled up 1305-fold and resulted in superimposable chromatograms, 96% recovery and > 99% purity. Thus, by optimizing the pH, ionic strength and temperature, a high-capacity preparative separation of rhIGF-1 from its related fermentation variants was obtained.
Chromatography, High Pressure Liquid↗
Incorporation of hydrophobic selectivity in capillary electrophoresis: analysis of recombinant insulin-like growth factor I variants.
A highly selective electrophoretic system employing differential hydrophobic interaction was evaluated for the quantitative determination of recombinant insulin-like growth factor I (IGF-I) variants. The system consisted of mixed aqueous-organic buffers containing suitable amounts of a zwitterionic detergent. In addition, a neutral hydrophilic coating was attached to the wall of the capillary to minimize analyte adsorption and electroosmotic flow. The zwitterionic detergent acted as a hydrophobic selector, allowing independent optimization of the electrophoretic and hydrophobic selectivities in the separation system. The extent of hydrophobic interaction was conveniently adjusted by varying the type and amount of organic modifier. Complete resolution of a mixture of IGF-I variants with closely related mass-to-charge ratios was achieved. Quantitative analysis of IGF-I process samples agreed well with HPLC results. Finally, the approach was found to be compatible with on-line capillary electrophoresis-mass spectrometry.
Amino Acid Sequence↗
Large scale, in situ isolation of periplasmic IGF-I from E. coli.
Human insulin-like growth factor I (IGF-I) accumulates in both folded and aggregated forms in the fermentation medium and cellular periplasmic space when expressed in E. coli with an endogenous secretory signal sequence. Due to its heterogeneity in form and location, low yield of IGF-I was obtained using a typical refractile body recovery strategy. To enhance recovery yield, a new procedure was developed to solubilize and extract IGF-I from cells while in fermentation broth. This method, called in situ solubilization, involves addition of chaotrope and reductant to alkaline fermentation broth and provides recovery of about 90% of all IGF-I in an isolated supernatant. To further enhance recovery, a new aqueous two-phase extraction procedure was developed which partitions soluble non-native IGF-I and biomass solids into separate liquid phases. This two-phase extraction procedure involves addition of polymer and salt to the solubilization mixture and provides about 90% recovery of solubilized IGF-I in the light phase. The performance of the solubilization and aqueous extraction procedures is reproducible at scales ranging from 10 to 1000 liters and provides a 70% cumulative recovery yield of IGF-I in the isolated light phase. The procedure provides significant initial IGF-I purification since most host proteins remain cell associated during solubilization and are enriched in heavy phase. ELISA analysis for E. coli proteins indicates that 97% of the protein in the light phase is IGF-I. Together, the techniques of in situ solubilization and aqueous two-phase extraction provide a new, high yield approach for isolating recombinant protein which is accumulated in more than one form during fermentation.
Escherichia coli↗
Consistency and stability of recombinant fermentations.
Production of proteins of consistent quality in heterologous, genetically-engineered expression systems is dependent upon identifying the manufacturing process parameters which have an impact on product structure, function, or purity, validating acceptable ranges for these variables, and performing the manufacturing process as specified. One of the factors which may affect product consistency is genetic instability of the primary product sequence, as well as instability of genes which code for proteins responsible for post-translational modification of the product. Approaches have been developed for mammalian expression systems to assure that product quality is not changing through mechanisms of genetic instability. Sensitive protein analytical methods, particularly peptide mapping, are used to evaluate product structure directly, and are more sensitive in detecting genetic instability than is direct genetic analysis by nucleotide sequencing of the recombinant gene or mRNA. These methods are being employed to demonstrate that the manufacturing process consistently yields a product of defined structure from cells cultured through the range of cell ages used in the manufacturing process and well beyond the maximum cell age defined for the process. The combination of well designed validation studies which demonstrate consistent product quality as a function of cell age, and rigorous quality control of every product lot by sensitive protein analytical methods provide the necessary assurance that product structure is not being altered through mechanisms of mutation and selection.
Biotechnology↗
Effect of environment on insulin-like growth factor I refolding selectivity.
Recombinant human insulin-like growth factor I (IGF-I), a 70-amino-acid peptide containing three disulphide bonds, produces two monomeric and several multimeric species during refolding. To optimize production of correctly folded IGF-I, conditions which influence protein refolding, stability and solubility were systematically examined. Combinations of solution components and conditions were analysed to identify synergistic interactions which enhance or reduce refolding efficiency. IGF-I concentration had the largest effect on formation of correctly folded peptide, due to competing association reactions. Solution polarity had the next largest individual effect, both on the level of multimeric peptide and on the relative proportion of correctly folded to misfolded monomeric peptide. Salt type and concentration and chaotrope type and concentration also had large individual effects on the distribution of IGF-I forms produced. Solution polarity modulated the effects of many other conditions including chaotrope concentration, salt type and concentration, and osmolyte concentration. Simultaneously decreasing the solution polarity and increasing the salt concentration improved the yield of correctly folded IGF-I relative to either individual change. Optimum solution conditions for refolding were 2M urea, 1M NaCl and 20% (v/v) ethanol. A possible mechanism for the importance of solution polarity on IGF-I refolding is discussed.
Amino Acid Sequence↗
High resolution removal of virus from protein solutions using a membrane of unique structure.
We describe a new class of membrane that has the capability of removing particles such as viruses from solution with resolution and reproducibility superior to that of conventional membranes. This composite membrane is composed of a pre-formed microporous membrane plus a thin asymmetric, finely porous retentive layer that is quite different from conventional ultrafilters. The protein sieving characteristics of this membrane are nearly equivalent to, but slightly less than, that of conventional 100,000 Dalton cut-off ultrafiltration membranes. This membrane uniquely shows particle retention characteristics that increase monotonically from 3 to 8 logs as a function of particle diameter in the range of 28 to 93 nm. The performance of this membrane in both a single stage and a two stage system show that 4 to 6 log overall removal of virus particles in the size range 30 to 70 nm is possible with simultaneous high recovery of product protein. Clearance factors exceeding 6 logs are possible with viruses larger than 78 nm. In addition, the performance of process systems containing this membrane is predictable in accordance with the general membrane properties and equilibrium mass balance models. This membrane system is fully validatable and can be used in conjunction with other validated operations in a down-stream process to reliably achieve an over-all reduction of 12 logs of known or putative virus particles.
Bacteriophage phi X 174↗
Polyethylene glycol enhanced protein refolding.
Previous studies on the refolding of recombinant bovine carbonic anhydrase B (CAB) indicated that polyethylene glycol (PEG) significantly enhanced the recovery of active protein by reducing aggregation. To further test the ability of PEG to enhance refolding, three recombinant human proteins, deoxyribonuclease (rhDNAse), tissue plasminogen activator (rhtPA), and interferon-gamma (rhIFN-gamma) were refolded in the presence of PEG (3350 MW). rhDNAse produced from CHO cells was denatured in 7.2 M urea and refolded by rapid dilution to 4.0 M urea and 0.20 mg/ml protein. When a final PEG to rhDNAse molar ratio of 5 to 1 (0.1 milligram PEG, 3350 MW) was used in the dilution buffer, refolding was improved by 30% to yield complete recovery of active protein. Impure E. coli derived inclusion body preparations of rhDNAse were solubilized in 8 M urea and refolded by dilution to 4 M urea and 0.10 mg/ml protein. Refolding with a dilution buffer which yielded a final PEG to rhDNAse molar ratio of 10 to 1 (0.1 milligram PEG, 3350 MW) resulted in a three-fold increase in the recovery of active protein. When PEG was used in the dilution buffer, aggregation of rhDNAse did not occur during refolding in either case. rhtPA produced from CHO cells was denatured in 5 M guanidine hydrochloride (GuHCl) and refolded by rapid dilution to 0.10 M GuHCl and 0.20 mg/ml protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Downstream processing of proteins from mammalian cells.
Less than a decade ago, the use of continuous mammalian cell lines for the production of cloned proteins was considered strictly a research tool. At that time, few thought it possible to allay the many safety concerns associated with transformed cells. It soon became clear that mammalian expression systems had numerous advantages over bacteria for production of therapeutic proteins, initiating a multidisciplinary effort to address these concerns in a thorough and reliable manner. The success of these efforts is exemplified by the emergence of product molecules into the market. Today, there are seven recombinant human therapeutics that have received FDA approval. Almost half of them (OKT3, t-PA, and EPO) are produced in mammalian cells, with the remainder produced in bacteria (insulin, growth hormone, and alpha-interferon) or yeast (hepatitis vaccine). At least a dozen more recombinant cell culture products are in advanced human clinical trials. With the accumulation of data and experience, continuous mammalian cell lines will no doubt be the preferred hosts for many future products of biotechnology.
Animals↗
Process validation for cell culture-derived pharmaceutical proteins.
Principles of process validation are extremely powerful tools in assurance of product quality. They are especially useful for reducing those risks not easily measured routinely during production. When combined with effective process and facility design principles, characterization of cell banks and products, appropriate lot release tests, and adherence to cGMP, safe cell culture biologicals can be prepared in a reliable manner.
Animals↗
Downstream processing of proteins: recent advances.
This review on the downstream processing of proteins describes innovations that have occurred in the field since 1983. Several areas have seen particularly high levels of achievement, and are accorded expanded coverage relative to our previous review [1]. As an example, the increasing integration of downstream operations with upstream technologies, such as molecular biology and fermentation, has led to the development of some very powerful processes. The degree to which organizations understand that there needs to be one unified process, rather than the independent steps of cloning, fermentation and recovery, seems directly related to the ultimate speed and success of the development effort. In 1983 one of the most active development areas was chromatography, especially affinity chromatography. This is still true today, and this topic has been expanded to include biospecific adsorptions that would not traditionally be classified as chromatography. With more proteins being developed for human administration, there has been an increased emphasis on all aspects of process hygiene. In addition, there has been much discussion about the impact of regulatory demands on the design and development of the manufacturing processes. Therefore, a section has been added which covers several of the regulatory issues that have been raised for products of the new biotechnology. Finally, as some of the early process development achievements are now beginning to bear fruit in the form of patents, we have increased our citation of this area of the literature.
Journal Article↗
The properties of recombinant chicken growth hormone and its effects on growth, body composition, feed efficiency, and other factors in broiler chickens.
Recombinant chicken GH (rcGH) was produced and characterized. Comparison of protein sequence, amino acid composition, mol wt, purity, and immunocross-reactivity showed that except for the N-terminal methionyl group arising from the bacterial expression system, the recombinant and pituitary-derived cGHs were identical. When tested in a hypophysectomized rat growth assay, the recombinant and pituitary materials had the same specific bioactivity. Within 60 min after sc injection of rcGH (480-960 micrograms/kg) in chickens, plasma GH levels increased 4- to 6-fold and remained significantly elevated for at least 5 h. Thrice-daily injections from age 2-24 days had little effect on growth or feed consumption in either male or female broiler chicks. Plasma levels of insulin and triglycerides were significantly elevated by rcGH in 24-day-old females, but not in males. Injection of rcGH counteracted a reduction of tibia length observed in saline-injected controls. The rcGH had no effect on carcass protein, ash content, or nitrogen retention. It is important to note that exogenous GH can be a productivity-enhancing factor in other commercially important species. Administration of bovine GH to cows has been shown to induce a significant increase in milk production (28). This study shows that administration of rcGH to chickens can lead to some significant metabolic effects. However, it is the conclusion of this report that the level of circulating GH is not the limiting factor in the growth of this highly selected species.
Amino Acid Sequence↗
Large-scale production and recovery of human immune interferon.
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Cell Separation↗
Characterization studies of human tissue-type plasminogen activator produced by recombinant DNA technology.
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Amino Acid Sequence↗
Laboratory and clinical experience with recombinant plasminogen activator.
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Amino Acid Sequence↗
Downstream processing of proteins.
This review focuses on the recovery of proteins from fermented starting materials, covering developments that have appeared in the literature since 1979. The major subjects discussed include cell disruption and extraction, solid/liquid separations, chromatography, separations in solution, and finishing operations. While advances have occurred in every area, the greatest diversity of innovation has taken place in the fields of chromatography and tangential-flow filtration.
Journal Article↗