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A methodical development approach was deployed in a novel portable manufacturing (Pharmacy on Demand) unit to purify ciprofloxacin hydrochloride hydrate within assay, water content, and impurity specifications described by the United States Pharmacopeia (USP) monograph and ICH Q3A(R2) guidelines for new impurities in drug substances. A series of design-of-experiment (DOE) and one-factor at a time (OFAT) experiments led to the optimization and control of a continuous two-stage crystallization that increased both the purity and yield of ciprofloxacin hydrochloride hydrate. Additionally, a statistically significant linear model was derived in batch within a 20 °C range that tracked the level of a difficult-to-purge impurity in stage 1 of the purification. This model was tested in continuous flow and predicted the impurity removal within 5% accuracy. With parametric control of process parameters, determined by optimization and modeling work, continuous flow isolations produced an active pharmaceutical ingredient (API) which had no individual impurities above 0.07%, with an isolated yield of 74%. In addition, acceptance criteria for assay (between 98 and 102%) and water content (between 4.7 and 6.7%) were met per the USP monograph for ciprofloxacin hydrochloride hydrate for the first time in the novel POD system.

Abstract : A strategy for combining theoretical and empirical model predictions to enhance process monitoring and disturbance detection in continuous pharmaceutical manufacturing is investigated using the first two steps of ciprofloxacin. The first-principles component is a dynamic model that reads in process parameter data and returns a concentration prediction for each species in the system using well-established equations and numerical discretization. The input data for the dynamic model comes from low-cost and reliable sensors that are already commonly deployed in manufacturing scenarios, such as flowmeters and thermocouples, making the approach amenable to potential uniform deployment across numerous manufacturing sites. The empirical component is infrared spectra collected from an inline flow cell that feeds to a partial least squares regression model for product concentration. Process parameter disturbances were introduced while continuously collecting the outlet stream infrared spectra, reagent flowrates, reactor temperature, and running the theoretical and empirical prediction models. Post-processing included the application of changepoint analysis, which is a statistical method of determining changes in the mean of a given time-series dataset. Both types of disturbances were captured as changepoints in the theoretical and empirical model predictions and could be obtained more rapidly by analyzing the residuals between the two predictions. This indicates that the deployment of theoretical models along with empirical is a robust approach for rapidly detecting deviations in the process health, reducing the time that potentially out of specification material is sent downstream. Additionally, by comparing trends in the models with the process parameter data, root-cause analysis can be rapidly carried out for a given disturbance. This places emphasis on holistic process monitoring by incorporating characterization knowledge and understanding into the process along with applying all available data sources to ensure product quality. Highlights: • First-principles and empirical concentration predictions were analyzed and compared for a multi-step continuous flow reaction. Graphical Abstract:

The strategies and experimental methods for implementation of process analytical technology (PAT) on the mobile pharmaceutical manufacturing system, Pharmacy on Demand (PoD), are discussed. With multiple processes to be monitored on the PoD end-to-end continuous manufacturing process, PAT and its real-time process monitoring capability play a significant role in ensuring final product quality. Here, we discuss PAT implementation for real-time monitoring of an intermediate and API concentrations with in-line Fourier-transformed infrared and Raman spectroscopy for the five-step continuous synthesis of ciprofloxacin on the PoD synthesis unit. Two partial least squares regression models were built and verified with flow chemistry experiments to obtain a root-mean-square error of prediction (RMSEP) of 2.2 mg/mL with a relative error of 2.8% for the step 2 FlowIR model and a RMSEP of 0.9 mg/mL with a relative error of 2.8% for the step 5 Raman model. These models were deployed during an 11 h step 1–3 and a 5 h step 4–5 continuous ciprofloxacin synthesis run performed on the PoD system. In these runs, the real-time prediction of intermediate and product concentration was achieved with an online model processing software (Solo_Predictor) and a PAT data collection and management software (synTQ).

B cell-restricted immunotoxins were constructed by conjugating anti-B monoclonal antibodies to saporin, the major ribosome inactivating protein from the seeds of the plant Saponaria officinalis. HD37-SAP is directed against CD19, the broadest B cell-specific determinant. HD39-SAP and HD6-SAP recognize two different epitopes on the CD22 molecule, an antigen present on the cell surface of B cells at late stages of differentiation. All three immunotoxins inhibited DNA synthesis and protein synthesis in target B lymphoma cells with a dose-related effect, in short incubation times and in the absence of potentiators. A clonogenic assay demonstrated that all immunotoxins could eliminate more than two logs of clonogenic malignant B cells with a two-hour incubation at concentrations not toxic to cells not bearing target antigens. The immunotoxin activity was evaluated by DNA synthesis inhibition in fresh B-chronic lymphocytic leukemia cells (B-CLL) stimulated to proliferate by incubation with an antibody specific for the receptor of C3b complement component (CR1) plus B cell growth factor. B-CLL cell DNA synthesis was actively inhibited by treatment at low immunotoxin concentration without need of potentiators. Immunotoxins exerted their effect also in whole blood of CLL patients under conditions achievable in vivo. We conclude that B cell-restricted immunotoxins HD37-SAP, HD39-SAP, and HD6-SAP are good candidates for in vivo therapy of B-cell malignancies.

We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50 (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC500.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.

The authors conjugated, by a disulphide bond, the antihuman T-lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis.The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen-induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC500.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomologous monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2α= 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2β= 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double-antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1 -SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans. © 1988 by Grune & Stratton, Inc.

The experimental approach taken and challenges overcome in developing a high-purity production (>100 g) scale process for the telescoped synthesis of the antibiotic ciprofloxacin is outlined. The process was first optimized for each step sequentially with regard to purity and yield, with necessary process changes identified and implemented before scaling for longer runs. These changes included implementing a continuous liquid–liquid extraction (CLLE) step and eliminating and replacing the base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) initially used in the ring-closure step due to DBU plausibly forming a decomposition side product that negatively impacted the final product purity. Process conditions were scaled 1.5–2-fold in order to enable the ultimate project goal of producing enough crude ciprofloxacin within 24 h to manufacture 1000 250 mg tablets. Working toward this goal, several production-scale runs were carried out to assess the reproducibility and robustness of the finalized process conditions, with the first three steps being run continuously up to 22 h and the last two steps being run continuously up to 10 h. The end result is a process with a throughput of ∼29 g/h (∼700 g/24 h) with a crude product stream profile of 94 ± 2% and 34 ± 3 mg/mL after five chemical transformations across four reactors and one continuous CLLE unit operation with each intermediate step maintaining a purity >95% by HPLC.

A monoclonal antibody reactive with the immunoglobulin heavy chain (TEC IgM) has been conjugated to saporin-6 (SAP), which is the major ribosome-inactivating protein from the seeds of the plant Saponaria officinalis. Studies with Burkitts lymphoma cell line Bjab 113 demonstrate that this immunotoxin is capable of killing 3 logs (99.9%) of clonogenic lymphoma cells after a 2-hour incubation. The presence of human bone marrow inhibits the activity of the conjugate. However, full potency of TEC IgM-SAP immunotoxin is restored by adding 1 mM amantadine to the incubation medium. The reaction is highly specific and is inhibited by the presence of excess anti-µ-antibody or human serum. Clonal growth of other Burkitts lymphoma cell lines is inhibited to a lesser extent by the immunotoxin. The presence of surface IgM on the different cell lines is directly correlated to target cell killing by TEC IgM-SAP. Isolation of Bjab 113 clones surviving treatment demonstrates that only a minority are truly resistant and that the others randomly escape the treatment. The highly potent and specific activity of this conjugate in the presence of bone marrow buffy coat and its exceptionally rapid onset of action make this conjugate a good candidate for the ex vivo elimination of neoplastic cells from the bone marrow of non-Hodgkins lymphoma patients.