Stuart E. Builder

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.

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.

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.

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.

RECOMBINANT HUMAN TISSUE-PLASMINOGEN ACTIVATOR: BIOCHEMISTRY, PHARMACOLOGY, AND PROCESS DEVELOPMENT§

Tissue plasminogen activator (t-PA) is an enzyme that regulates the dissolution of blood clots via the activation of plasminogen. By increasing the concentration of t-PA in the bloodstream, it is possible to accelerate blood clot dissolution from days to minutes. Administration of t-PA can dissolve blood clots causing heart attack or pulmonary embolism, thus limiting the damage caused by these diseases. Because of its low natural abundance, t-PA must be produced by recombinant DNA technology to obtain therapeutically useful amounts. This paper discusses process design and regulatory considerations associated with the manufacture of recombinant therapeutics.

[11] Large-scale production and recovery of human immune interferon

Publisher Summary This chapter discusses the development of a method for large-scale production of human immune interferon based on induction scheme with the addition of a cell fractionation process and a novel method for product recovery. A 10- to 15-fold enhancement in interferon yield and a 50-fold increase in purity of crude product were accomplished by nylon wool column fractionation of lymphocytes combined with simultaneous reduction of autologous plasma protein levels prior to induction. The production method described is suitable for large-scale preparation of natural human immune interferon. Its principal advantages include greater interferon yield and purity without substantial increase in process costs. The process provides a method for the production of both higher and more consistent yields of interferon. A major factor in the higher yield from nylon wool fractionated buffy coat cells is the prolongation of the active production period compared to unfractionated cell preparations. Higher purity without concomitant yield loss results from removal of about 70% of buffy coat plasma, a step which results in reduced yields when using unfractionated cells. This sequential application of nylon wool fractionation and protein removal steps combines to provide 50-fold improvement in initial purity, which leads to higher purification yields and a reduction in purification costs.