CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well-known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50-62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early-temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO(2) with this product showed that the Neu5Gc levels were 46% lower at high pCO(2) conditions (140 mmHg) compared to moderate pCO(2) levels (20-80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics.
The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.
The market for protein therapeutics has grown significantly over the past two decades and the pace of development continues to increase. It is a challenge to the industry to maintain the desired quality attributes while accelerating delivery to patients, reducing the cost of goods, and providing production flexibility. Efficient manufacturing scale production of protein therapeutics is required to continue to meet the needs of the patients and stockholders. This chapter describes batch, fed-batch, and perfusion processes and their utilization in the production of monoclonal antibodies and other therapeutic proteins. In addition, we have provided detailed discussions of the ongoing challenges of lactate metabolism and the future prospects of process monitoring and control.
The insect larvae/baculovirus protein production process was dramatically simplified by expressing fusion proteins containing green fluorescent protein (GFP) and the product-of-interest. In this case, human interleukin-2 (hIL-2) and chloramphenicol acetyl-transferase (CAT) were model products. Specifically, our fusion construct was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), the UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of the product from the fusion, and the product, hIL-2 or CAT. Both the approximately 52 kDa GFPuv/hIL-2 and approximately 63 kDa GFPuv/CAT fusions were expressed in Trichoplusia ni larvae at 9.0 microg-hIL-2 and 24.1 microg-CAT per larva, respectively. The GFP enabled clear identification of the infection process, harvest time, and more importantly, the quantity of product protein. Because the GFP served as a marker, this technique obviates the need for in-process Western analyses (during expression, separation, and purification stages). As a purification marker, GFP facilitated rapid identification of product-containing elution fractions (Cha et al., 1999b), as well as product-containing waste fractions (e.g., cell pellet). Also, because the fluorescence intensity was linear with hIL-2 and CAT, we were able to select the highest-producing larvae. That is, three fold more product was found in the brightest larva compared to the average. Finally, because the GFP is attached to the product protein and the producing larvae can be selected, the infection and production processes can be made semi-continuous or continuous, replacing the current batch process. These advantages should help to enable commercialization of larvae as expression hosts.
Background: Human α 1 -proteinase inhibitor (α 1 -PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α 1 -PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α 1 -PI (r-α 1 -PI) could provide an attractive alternative. Although r-α 1 -PI has been produced in
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