FDA/M-CERSI Co-Processed API Workshop Proceedings.

These proceedings contain presentation summaries and discussion highlights from the University of Maryland Center of Excellence in Regulatory Science and Innovation (M-CERSI) Workshop on Co-processed API, held on July 13 and 14, 2022. This workshop examined recent advances in the use of co-processed active pharmaceutical ingredients as a technology to improve drug substance physicochemical properties and drug product manufacturing process robustness, and explored proposals for enabling commercialization of these transformative technologies. Regulatory considerations were discussed with a focus on the classification, CMC strategies, and CMC documentation supporting the use of this class of materials from clinical studies through commercialization.

An audit of pharmaceutical continuous manufacturing regulatory submissions and outcomes in the US.

Continuous manufacturing (CM) sends materials directly and continuously to the next step of a process, eliminating hold times and reducing processing times. The potential benefits of CM include improved product quality, reduced waste, lower costs, and increased manufacturing flexibility and agility. Some pharmaceutical manufacturers have been hesitant to adopt CM owing to perceived regulatory risks such as increased time to regulatory approval and market entry, more difficulty submitting postapproval changes, and higher inspectional scrutiny.

Anti-proliferative effect of levamisole on human myeloma cell lines in vitro.

Levamisole has been employed as an immunomodulatory agent in conjunction with 5-fluorouracil in the treatment of colon cancer relapse. At high doses, levamisole has been shown to have both anti-cancer and immunosuppressive activities. In vitro, levamisole has been shown to potentiate the anti-proliferative effect of 5-fluorouracil in several types of tumor cell lines; however, its mechanism of cytotoxic action and its molecular targets in cells remains to be elucidated.

Crystallization and preliminary X-ray diffraction analysis of human seminal plasma protein PSP94.

The human seminal plasma protein PSP94 is a small protein of 94 residues that contains ten cysteines. Since its discovery about 25 years ago, several potential biological functions have been reported for this protein. Many PSP94 homologues have also been identified since then from various species, but no crystal structure has been determined to date.

Anti-proliferative and cytotoxic effects of Strychnos nux-vomica root extract on human multiple myeloma cell line - RPMI 8226.

Multiple myeloma (MM) is an incurable hematological malignancy with high incidence in the elderly. The currently used chemotherapeutic drugs show severe side effects, dose-limiting toxicity and development of resistance. In search of novel plant derived anti-cancer agents, Strychnos nux-vomica L.

The co-operonic PE25/PPE41 protein complex of Mycobacterium tuberculosis elicits increased humoral and cell mediated immune response.

Background: Many of the PE/PPE proteins are either surface localized or secreted outside and are thought to be a source of antigenic variation in the host. The exact role of these proteins are still elusive. We previously reported that the PPE41 protein induces high B cell response in TB patients.

Structural study of La(0.75)Sr(0.25)CrO(3) at high temperatures.

A high-temperature neutron diffraction study has been carried out on La(0.75)Sr(0.25)CrO(3) compound in the temperature range 300-1400 K.

Investigation of diffraction line broadening due to compositional fluctuations in L-alanine-doped triglycine sulfate.

Broadening of X-ray powder diffraction peaks as a result of compositional disorder in L-alanine-doped triglycine sulfate crystals is investigated using the Williamson-Hall method. The analysis indicates that L-alanine substitution in triglycine sulfate crystals leads to anisotropic strain in the crystal.

An ELISA method to quantify the mannose 6-phosphate receptors.

Mannose 6-phosphate receptor proteins mediate transport of lysosomal enzymes to lysosomes in eukaryotes. Two receptors designated as MPR 300 and MPR 46 based on their apparent molecular mass have been well studied from human and bovine liver. In humans, it has been shown that the receptors are present in different concentrations in different tissues.

Interacting growth walk: a model for generating compact self-avoiding walks.

We propose an algorithm based on local growth rules for kinetically generating self-avoiding walk configurations at any given temperature. This algorithm, called the interacting growth walk (IGW) model, does not suffer from attrition on a square lattice at zero temperature, in contrast to the existing algorithms. More importantly, the IGW model facilitates growing compact configurations at lower temperatures--a feature that makes it attractive for studying a variety of processes such as the folding of proteins.

Alkaline phosphatase activity is expressed in murine splenic B-lymphocytes sensitized in vivo with Tetanus toxoid.

Alkaline phosphatase (APase) activity and proliferative response to Tetanus toxoid (TT) were measured in murine splenic lymphocytes immunized in vivo with TT. APase activity was enhanced in TT-stimulated B-lymphocytes concomitant with an increase in the proliferative response in a dose-dependent manner. Cytochemical staining for APase using beta-naphthyl phosphate also showed an increase in APase positive cells in TT-stimulated lymphocyte population.

In vitro immunization of murine lymphocytes using immobilized immunogens.

Murine splenic lymphocytes were immunized in vitro using immobilized antigens. Immobilization was achieved by covalently linking the antigens to Sepharose beads. Tetanus toxoid (TT) was used as test antigen in soluble and immobilized forms.

Isolation and characterization of intraepithelial lymphocytes from rat small intestine.

A modified procedure for isolation of intraepithelial lymphocytes (IEL) from rat small intestine was developed which makes use of a protease inhibitor-phenyl methyl sulfonyl fluoride (PMSF) in the isolation medium. Yield and viability of IEL isolated in presence of PMSF were significantly higher as compared to those isolated in absence of PMSF. IEL isolated in presence PMSF demonstrated a significantly higher proliferative response to concanavalin A (con A) compared to those isolated in absence of PMSF.

Positive regulatory role of cAMP on alkaline phosphatase activity and proliferation of mitogen stimulated B lymphocytes.

Effect of dibutyryl cyclic adenosine monophosphate (dbt cAMP) on alkaline phosphatase (APase) of mitogen stimulated murine B lymphocytes was studied. Addition of dbtcAMP to lipopolysaccharide (LPS) stimulated B cells enhanced APase activity in a dose dependent and synergistic manner. dbtcAMP also stimulated the proliferative response of LPS treated B lymphocytes.

Carboxyl group hydrogen bonding in X-ray protein structures analysed using neutron studies on amino acids.

A method is proposed to make a distinction between ionized and neutral carboxyl groups in X-ray protein structures. This is based on an analysis of the relative hydrogen bonding populations and bond-length bond-valence correlations in high-precision neutron studies of amino acids and small peptides. With the help of this method, four amino acid residues containing carboxyl groups in the refined structure of triclinic hen egg-white lysozyme have been analysed.

Alkaline phosphatase activity is expressed only in B lymphocytes committed to proliferation.

Alkaline phosphatase (APase) activity was measured in murine splenic lymphocytes stimulated with the T lymphocyte mitogens phytohemagglutinin (PHA) and Concanavalin A (Con A) and the B lymphocyte mitogens lipopolysaccharide (LPS) and anti-immunoglobulin (anti-Ig). APase activity was found to be enhanced specifically in mitogen-stimulated B lymphocytes, but not in T lymphocytes. This enhancement starts around 8 h after stimulation with a mitogen.

Refinement of triclinic lysozyme: II. The method of stereochemically restrained least squares.

Refinement of triclinic lysozyme by restrained least squares against the 2 A resolution X-ray data is described, beginning with the model from cycle 17 of the preceding paper [Hodsdon, Brown, Sieker & Jensen (1990). Acta Cryst. B46, 54-62].

Differential response of lymphocytes to free and sepharose-linked anti-immunoglobulin during different phases of the cell cycle.

During proliferation induced by anti-immunoglobulins, B lymphocytes undergo cell volume increases prior to onset of DNA synthesis. Although both Sepharose-linked and free anti-immunoglobulin evoked essentially identical increases in cell volume, the Sepharose-linked antibody induced a significantly greater DNA synthesis than free antibody as judged from [3H]thymidine incorporation studies using mass cell culture technique, as well as by using autoradiographic analysis of individual cells. These findings are considered in terms of possible differences in triggering cell volume increases and DNA synthesis by free and linked anti-immunoglobulin and/or the possible existence of B cell subpopulations responding differentially to free and to linked antibody.

Splenic B cells from CBA/N mice acquire responsiveness to anti-immunoglobulin after a brief treatment with pronase.

Although splenic B cells of CBA/N mice do not synthesize DNA in response to anti-mouse IgM (mu-chain specific), the cells respond readily to Sepharose linked anti-mu. Subsequent to a brief treatment with pronase, CBA/N splenocytes exhibited anti-mu-mediated DNA synthesis at 40 to 100% of the DNA synthetic capacity detected with Sepharose linked anti-mu. Furthermore, spleen cell populations treated with anti-Thy-1.

Enhanced phosphorylation of endogenous membrane proteins during induction of B lymphocyte proliferation.

Phosphorylation of endogenous proteins was assessed employing membrane preparations derived from splenocytes induced to proliferate in response to Sepharose linked anti-immunoglobulins. Time course studies revealed that enhanced protein phosphorylation was preceded by cell enlargement and was either followed by or closely related in time to the onset of DNA synthesis. Thus maximal enhancement of phosphorylation was initially observed at 24 h whereas cell enlargement was optimal at 16 h at a time when there was no enhancement in protein phosphorylation.

Nonresponsiveness of immature B lymphocytes to anti-immunoglobulin is reversed by pronase.

Splenic B cells are induced to proliferate upon culture with antibody having specificity for surface membrane immunoglobulins. Cells treated with pronase, washed and then cultured with antibody, exhibited a greater than 5-fold enhancement of DNA synthesis whereas pronase treatment, per se, was not mitogenic. The pronase effect exhibited specificity in that the induction of proliferation with either lipopolysaccharide or dextran sulfate was not enhanced by prior enzyme treatment.

Structure, refinement, and function of carbonic anhydrase isozymes: refinement of human carbonic anhydrase I.

The structure of human erythrocyte carbonic anhydrase I has been refined to a final R value of 19% to 2-A resolution by a combination of least squares refinement and model fitting in a three-dimensional graphics display. About 300 solvent atoms have been located bound to the protein molecule. An interesting hydrogen bond network involving Zn2+, the liganded solvent, side chain groups of Thr-199, Glu-106, Thr-7, and His-64 through two solvent molecules have been found that may be important for the catalytic mechanism of the carbonic anhydrase.

Fate of surface immunoglobulin during induction of lymphocyte proliferation.

The modulation of immunoglobulin on the surface of rabbit B lymphocytes by goat antibodies with specificity for rabbit surface membrane immunoglobulin or by such goat antibodies covalently linked to Sepharose was studied in relation to the proliferative response to these agents. Although the induction of DNA synthesis was greater in the presence of Sepharose-linked antibody than in the presence of free antibody, modulation of surface membrane immunoglobulin was induced with free but not with Sepharose-linked antibody. Thus, in the presence of free antibody the surface membrane immunoglobulin content of cells was rapidly decreased and remained at a low level throughout the culture period, whereas the surface immunoglobulin content of cells incubated with Sepharose antibody was essentially unaltered.

Reduced cAMP levels and glycogen phosphorylase activation in isoproterenol perfused SHR myocardium.

The effect of isoproterenol perfusion on cAMP levels and phosphorylase activity was investigated in the spontaneously hypertensive rat (SHR) and Kyoto Wistar normotensive control rat (WKY) heart. The basal force of contraction in physiological salt solution perfused hearts was comparable between SHR and WKY. However, the force of contraction in response to 10 nM isoproterenol perfusion was decreased approximately 20-30% in SHR heart as compared to WKY heart.