Introduction to the application of QbD principles for the development of monoclonal antibodies.

Quality by Design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody.

QbD implementation and Post Approval Lifecycle Management (PALM).

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11) [1-3]. An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody.

Establishing a control system using QbD principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody.

Recombinant protein expression for therapeutic applications.

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Many of these applications involve complex glycoproteins and antibodies with relatively high production needs. These demands have driven the development of a variety of improvements in protein expression technology, particularly involving mammalian and microbial culture systems.

Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention: characterization of separation efficiency and impact of perfusion on product quality.

Several small-scale Chinese hamster ovary (CHO) suspension cultures were grown in perfusion mode using a new acoustic filtration system. The separation performance was evaluated at different cell concentrations and perfusion rates for two different CHO cell lines. It was found that the separation performance depends inversely on the cell concentration and perfusion rate.

Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.

A two-dimensional protein map of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical protein products. As part of our effort to better understand CHO cell physiology and protein expression changes caused by modified culture conditions, we have begun to map CHO cell polypeptides. A parental cell line reference map was established using two-dimensional gel electrophoresis with immobilized pH gradients (pH 3-10) in the first dimension and a linear acrylamide gradient (9-18%T) in the second dimension.

Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant DNase in batch culture with increased sialic acid.

Under some cell culture conditions, recombinant glycoprotein therapeutics expressed in Chinese hamster ovary (CHO) cells lose sialic acid during the course of the culture (Sliwkowski et al., 1992; Munzert et al., 1996).

Inhibin gene expression in a large cell calcifying Sertoli cell tumour and serum inhibin and activin levels.

Inhibin is a potential tumour suppressor gene product in the gonads. While inhibin gene products may have a role in tumourigenesis, serum inhibin levels can be used as a marker for ovarian tumours derived from granulosa cells. Tumours derived from Sertoli cells, testicular counterparts of granulosa cells, are rare.

Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders.

Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis associated with tumors and other pathological conditions, including proliferative diabetic retinopathy and age-related macular degeneration. The murine anti-human VEGF monoclonal antibody (muMAb VEGF) A.4.

Development of cancer cachexia-like syndrome and adrenal tumors in inhibin-deficient mice.

Activins and inhibins, members of the type beta transforming growth factor superfamily of growth regulatory proteins, are produced in multiple tissues and affect diverse physiologic processes. Using embryonic stem cell technology, we previously demonstrated that inhibin can function as a gonadal tumor suppressor. In this study, we show that development of gonadal tumors is rapidly followed by a cancer cachexia-like wasting syndrome.

Localization of inhibin and activin binding sites in the testis during development by in situ ligand binding.

Inhibin and activin are related proteins thought to be potential paracrine regulators of testicular development and maintenance of spermatogenesis. Messenger RNA and proteins immunologically related to both factors have been identified in the adult testis. However, the role(s) of these factors in paracrine regulation of testicular function is poorly understood.

Inhibins, activins, their binding proteins and receptors: interactions underlying paracrine activity in the testis.

The inhibin-related peptides are present in the testis from early gestation through adulthood. They are produced from multiple testicular sites in a highly regulated manner, suggesting important paracrine roles. Similarly, receptors for these peptides are located in specific stages of the seminiferous tubule and on particular cell types, and an additional level of control is afforded by specific binding proteins, such as follistatin, which may regulate bioavailability.

In situ ligand binding of recombinant human [125I] activin-A and recombinant human [125I]inhibin-A to the adult rat ovary.

Inhibin and activin are hormones produced by ovarian follicles. Specific ovarian cells that bind iodinated recombinant human (rh)-activin-A and rh-inhibin-A were identified by in situ ligand binding. Iodinated rh-molecules were incubated with tissue sections from ovaries, uterus, and oviduct, collected on the mornings of metestrus, diestrus, proestrus, and estrus and the evening of proestrus.

Quantitative two-site enzyme-linked immunosorbent assays for inhibin A, activin A and activin B.

We have developed three specific enzyme-linked immunosorbent assay (ELISA) formats which quantitate inhibin A in conditioned media and serum. The assays are sensitive in a range 0.078-5.

A widely expressed transmembrane serine/threonine kinase that does not bind activin, inhibin, transforming growth factor beta, or bone morphogenic factor.

Molecular cloning of complementary DNAs (cDNA) whose expression products bind activin and transforming growth factor beta (TGF-beta 1 and -beta 2) suggests that transmembrane serine/threonine kinases constitute a new class of signaling molecules. A human liver cell cDNA which codes for a new serine/threonine kinase receptor (SKR1) was identified using degenerate oligonucleotide primers complementary to coding sequence for mouse activin and Caenorhabditis elegans daf-1 serine/threonine receptor kinase subdomains VI and VIII in the polymerase chain reaction. The deduced 509-amino acid product consisted of a cysteine-rich extracellular domain and a cytoplasmic serine/threonine kinase domain which are 10-20 and 40% homologous to the respective domains in the activin and transforming growth factor beta receptor kinases.

Recombinant human inhibin A and recombinant human activin A regulate pituitary and ovarian function in the adult female rat.

The roles of recombinant human inhibin A (rh-inhibin A) and rh-activin A in regulating the pituitary and ovary of the adult female rat were examined. Serum and pituitary FSH and LH and serum estradiol and progesterone concentrations were evaluated at 1, 2, and 6-12 h after sc hormone administration on metestrus and on proestrus. A second study examined the effect of the hormones 24 h after injection at 1000 h on each day of the cycle.

Follistatin modulates activin activity in a cell- and tissue-specific manner.

The high affinity activin-binding protein, follistatin, has recently been shown to block activin-stimulated activities in several in vitro systems. In the present study we sought to extend these observations and investigate the effects of follistatin on the activity of activin in stimulating the re-aggregation of Sertoli cell monolayers and proliferation of testicular germ cells, as measured by incorporation of [3H]-thymidine in vitro. Germ-Sertoli cell cocultures prepared from 21 day old rats were treated with media alone or media containing recombinant human (rh) activin A or rh activin B with or without follistatin, the low affinity activin-binding protein, alpha 2 macroglobulin, or a monoclonal antibody (mAB) known to block activin B activity.

Development of a specific and sensitive two-site enzyme-linked immunosorbent assay for measurement of inhibin-A in serum.

A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme-linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 +/- 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 +/- 6 pg/ml in a 10% serum matrix, with intra-and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-beta 1 or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-beta 1.

Pharmacokinetic profile of recombinant human (rh) inhibin A and activin A in the immature rat. II. Tissue distribution of [125I]rh-inhibin A and [125I]rh-activin A in immature female and male rats.

The tissue distribution of recombinant human inhibin A (rh-inhibin A) and rh-activin A was determined in immature female Sprague Dawley-derived rats after iv administration of radiolabeled proteins. [125I]rh-Inhibin A and [125I]rh-activin A diverge in their distribution to tissues of the immature female rat as examined histologically (whole body autoradiography and thin section analysis) and by computing the percent dose and tissue to blood ratios for individual tissues. [125I]rh-inhibin A accumulated in the spleen, adrenal, bone marrow, and ovary after iv injection.

Pharmacokinetic profile of recombinant human (rh) inhibin A and activin A in the immature rat. I. Serum profile of rh-inhibin A and rh-activin A in the immature female rat.

The serum pharmacokinetics of recombinant human inhibin A (rh-inhibin A) and rh-activin A were examined in immature female Sprague Dawley-derived rats after iv and sc injection of the drugs. After iv administration of rh-inhibin A (120 micrograms/kg), the serum concentrations were described by a biexponential equation. The weight-normalized clearance was 21.

Identification and characterization of binding proteins for inhibin and activin in human serum and follicular fluids.

Inhibins and activins are produced by a variety of tissues and may have important endocrine and paracrine roles in development, reproduction, and hematopoiesis. However, little is known regarding the physical properties or concentrations of inhibin and activin in biological fluids. Binding proteins for inhibin or activin in serum or at production or target sites may have important implications for restricting the bioactivity of these hormones and may alter the immunoreactivity of these molecules in biological fluids.

Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol.

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.