Shortening the biologics clinical timeline with a novel method for generating stable, high-producing cell pools and clones.

Reducing drug development timelines is an industry-wide goal to bring medicines to patients in need more quickly. This was exemplified in the coronavirus disease 2019 pandemic where reducing development timelines had a direct impact on the number of lives lost to the disease. The use of drug substances produced using cell pools, as opposed to clones, has the potential to shorten development timelines. Toward this goal, we have developed a novel technology, GPEx® Lightning, that allows for rapid, reproducible, targeted recombination of transgenes into more than 200 Dock sites in the Chinese hamster ovary cell line genome. This allows for rapid production of high-expressing stable cell pools and clones that reach titers of 4-12 g/l in generic fed-batch production. These pools and clones are highly stable in both titer and glycosylation, showing strong similarities in glycosylation profiles.

Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II).

BACKGROUND: The ability to site-specifically conjugate a protein to a payload of interest (e.g., a fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development. For example, antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches, next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology, where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression, the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE), which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation. RESULTS: The yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen, driving enzymatic turnover. Building upon that work, here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE, which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture, as demonstrated in fed-batch cell cultures with antibody titers of 5 g · L(-1), specific cellular production rates of 75 pg · cell(-1) · d(-1), and fGly conversion yields of 95-98 %. CONCLUSIONS: We describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE, which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate.

Mammary specific transgenic over-expression of insulin-like growth factor-I (IGF-I) increases pig milk IGF-I and IGF binding proteins, with no effect on milk composition or yield.

IGF-I regulates lactation by stimulating mammary mitogenesis, inhibiting apoptosis, and partially mediating the effects of growth hormone on lactogenesis. Herein, lactation performance during first and second parity was assessed in transgenic swine (TG) that over-expressed human IGF-I in milk under the control of the bovine alpha-lactalbumin promoter, regulatory regions and signal peptide coding sequence. Milk samples were collected throughout lactation (farrowing to d24) from TG sows and non-transgenic littermates (CON) and IGF-I, IGF-II, and IGFBP determined. Colostral (<24 h postpartum) IGF-I content was 26-fold greater (p<0.001) in TG sows (949+/- 107 microg/L; range 228-1,600 microg/L) than CON (36+/-17.8 microg/L) and was 50- to 90-fold greater (p< 0.001) in mature milk (d2-24 postpartum). There was no effect of parity on milk IGF-I content. Milk IGF-II concentration was unaffected by IGF-I over-expression. Low molecular weight IGFBP (IGFBP-2 and -5) in the milk of TG sows were higher (p=0.02) than CON in the early postpartum period, but did not differ in mature milk. Milk yield, determined by weigh-suckle-weigh, was similar in TG and CON as was litter weight gain. Milk nutrient composition was not significantly affected by IGF over-expression. Thus, mammary specific transgenic over-expression of IGF-I significantly increased milk IGF-I and IGFBP content, but did not impact lactation performance in swine.