Inter-batch Variability Research Articles

Natural Small Molecules Enabled Efficient Immunotherapy through Supramolecular Self-Assembly in P53-Mutated Colorectal Cancer.

Nanomedicine, constructed from therapeutics, presents an advantage in drug delivery for cancer therapies. However, nanocarrier-based treatment systems have problems such as interbatch variability, multicomponent complexity, poor drug delivery, and carrier-related toxicity. To solve these issues, the natural molecule honokiol (HK), an anticancer agent in a phase I clinical trial (CTR20170822), was used to form a self-assembly nanoparticle (SA) through hydrogen bonding and hydrophobicity. The preparation of SA needs no molecular precursors or excipients in aqueous solution, and 100% drug-loaded SA exhibited superior tumor-targeting ability due to the enhanced permeability and retention (EPR) effect. Moreover, SA significantly enhanced the antitumor immunity relative to free HK, and the mechanism has notable selectivity to the p53 pathway. Furthermore, SA exhibited excellent physiological stability and inappreciable toxicity. Taken together, this supramolecular self-assembly strategy provides a safe and "molecular economy" model for rational design of clinical therapies and is expected to promote targeted therapy of HK, especially in colorectal cancer patients with obvious p53 status.

Generating Large Numbers of Pancreatic Microtumors on Alginate-Gelatin Hydrogels for Quantitative Imaging of Tumor Growth and Photodynamic Therapy Optimization.

The emerging use of 3D culture models of cancer has provided novel insights into the therapeutic mechanisms of photodynamic therapy on a mesoscopic scale. Especially microscale tumors grown on scaffolds of extracellular matrix can provide statistically robust data on the effects of photosensitizers and photodynamic therapy by leveraging high-throughput imaging-based assays. Although highly informative, the use of such 3D cultures can be impractical due to the high costs and inter-batch variability of the extracellular matrix scaffolds that are necessary to establish such cultures. In this study, we therefore provide a protocol to generate inexpensive and defined hydrogels composed of sodium alginate and gelatin that can be used for culturing 3D microtumors in a manner that is compatible with state-of-the-art imaging assays. Our results reveal that the alginate-gelatin hydrogels can perform similarly to a commercially available ECM scaffold in terms of facilitating microtumor growth. We then applied these microtumor models to quantify the uptake and dark toxicity of benzoporphyrin derivative encapsulated in liposomes with either an anionic or a cationic surface charge. The results indicate that cationic liposomes achieve the highest level of uptake in the microtumors, yet also exert minor toxicity. Moreover, we reveal that there is typically a significant positive correlation between microtumor size and liposome uptake. In conclusion, alginate-based hydrogels are inexpensive and effective scaffolds for 3D culture models of cancer, with versatile applications in research toward photodynamic therapy.

Optimized Workflow for On-Line Derivatization for Targeted Metabolomics Approach by Gas Chromatography-Mass Spectrometry.

Using manual derivatization in gas chromatography-mass spectrometry samples have varying equilibration times before analysis which increases technical variability and limits the number of potential samples analyzed. By contrast, automated derivatization methods can derivatize and inject each sample in an identical manner. We present a fully automated (on-line) derivatization method used for targeted analysis of different matrices. We describe method optimization and compare results from using off-line and on-line derivatization protocols, including the robustness and reproducibility of the methods. Our final parameters for the derivatization process were 20 µL of methoxyamine (MeOx) in pyridine for 60 min at 30 °C followed by 80 µL N-Methyl-N-trimethylsilyltrifluoracetamide (MSTFA) for 30 min at 30 °C combined with 4 h of equilibration time. The repeatability test in plasma and liver revealed a median relative standard deviation (RSD) of 16% and 10%, respectively. Serum samples showed a consistent intra-batch median RSD of 20% with an inter-batch variability of 27% across three batches. The direct comparison of on-line versus off-line demonstrated that on-line was fit for purpose and improves repeatability with a measured median RSD of 11% compared to 17% using the same method off-line. In summary, we recommend that optimized on-line methods may improve results for metabolomics and should be used where available.

Tip and Torque Accuracy According to the ISO 27020:2019 Norm in Currently Available Pre-Adjusted Orthodontic Brackets

Background: The precision of bracket manufacturing is fundamental to ensure the correct expression of the inbuilt information. The objective of this study was to determine the actual tip and torque values of a pool of stainless steel brackets, pre-adjusted according to the MBT prescription values, and to compare these actual values with those stated by the manufacturers in order to test their compliance with the tolerance limits reported in the ISO 27020:2019. Methods: A sample of 360 stainless steel brackets, from 12 different providers, were evaluated. All brackets had a nominal slot size of 0.022 in., belonged to the upper right central incisor, and were manufactured with the metal injection molding technique (MIM). For each provider, three different batches of the same bracket series were tested. A single-blind design was used for bracket coding. Results: Only five systems displayed torque mean values that matched the declared values (p > 0.05). Only one system did not respect the tolerance limits established in the ISO 27020:2019 norm. The tip values were different from those declared in seven of the assessed systems; however, none exceeded the tolerance limits. The inter-batch variability in most cases was not statistically significant. Conclusions: In most of the assessed systems, there can be a difference between the actual and the declared torque values, while tip information is usually accurately incorporated into the bracket slot. Lack of precision in the manufacturing process can reduce the efficacy of the appliance and force the clinician to compensate for dimensional inaccuracy through wire bending.

Compliance with the ISO 27020:2019 norm of a sample of currently available preadjusted Orthodontic bracket systems. Are the actual dimensions as expected?

BackgroundDetermine the exact slot dimension of a sample of a MBT prescription stainless steel conventional brackets from different manufacturers to compare the actual values with the nominal ones declared by the manufacturers and to verify the compliance with tolerance limits given by the ISO 27020:2019. Different batches from each manufacturer were evaluated to determine whether or not they are different in size. In addition, the geometry of the slot walls was assessed.Methods360 stainless steel preadjusted orthodontic brackets of 12 different manufacturers were assessed. All brackets had a nominal slot size of 0.022 by 0.028 inches, belonged to the right upper central incisor, and were fabricated with the metal injection molding technique (MIM). For each manufacturer, three different manufacturing batches were evaluated. Brackets were coded using a single-blind design.ResultsAll bracket systems in the study group except one displayed a statistically significant difference with the nominal declared value, although only four of the systems did not comply with the tolerance limits established by the ISO 27020:2019. In most of the systems, the slot height was oversized when compared to the nominal one. A significant interbatch variability was found in most of the evaluated systems. Most of the brackets walls were divergent.ConclusionsThe dimensional accuracy of commercially available metal brackets is not guaranteed. The respect for the norm should be enforced as well as the quality controls along the manufacturing process since orthodontic brackets are a precision medical device.

Evaluation of fast nucleic acid detection system in severe acute respiratory syndrome coronavirus 2

Objective: To evaluate the performance and application of a fast nucleic acid detection system for testing severe acute respiratory syndrome virus 2 (SARS-COV-2). Methods: Clinical samples were collected from February to July 2020 from Beijing Center for Diseases Prevention and Control and the Laboratory Department of China-Japan Friendship Hospital, to evaluate the sensitivity, specificity, anti-interference ability, precision and clinical sample coincidence rate of fast nucleic acid detection system for SARS-CoV-2. The analytical sensitivity was determined by a dilution series of 20 replications for each concentration. Analytical specificity study was performed by testing organisms whose infection produces symptoms similar to those observed at the onset of corona virus disease 2019 (COVID-19), and of the normal or pathogenic microflora that may be present in specimens collected. Potential interference substances were evaluated with different concentration in the interference study. Precision study was conducted by estimating intra-and inter-batch variability. Clinical evaluation was performed by testing 230 oropharyngeal swab specimens and 95 sputum specimens in fast nucleic acid detection system, comparing with conventional real-time fluorescent quantitative PCR (RT-qPCR) and clinical diagnostic results. Results: The analytical sensitivity of SARS-CoV-2 using fast nucleic acid detection system was 400 copies/ml. The result is negative for testing with the organisms that may likely in the circulating area or causing similar symptoms with SARS-CoV-2 and human nucleic acid, indicating that no cross reactivity with organisms. The results of precision test showed that the Coefficient of variation of Ct value of high, medium and low concentration samples was 1.90%-3.92%, and all of them were less than 5% in intra-and inter-batch testing. The results of the samples were still positive after adding the potential interfering substances, indicating that the possible interfering substances in the samples had no effect on the results. 98.46% and 97.85% diagnosis results of fast nucleic acid detection system were consistent with RT-qPCR and clinical diagnostic results, respectively. Conclusion: The fast nucleic acid detection system based on molecular parallel reaction can be used as a selection method for SARS-CoV-2 testing.

Efficient pancreatic cancer detection through personalized protein corona of gold nanoparticles.

Characterization of the personalized protein corona (PC) that forms around nanomaterials upon exposure to human plasma is emerging as powerful technology for early cancer detection. However, low material stability and interbatch variability have limited its clinical application so far. Here, we present a nanoparticle-enabled blood (NEB) test that uses 120 nm gold nanoparticles (NPs) as the accumulator of blood plasma proteins. In the test, the personalized PC of gold NPs is characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. As a paradigmatic case study, pancreatic ductal adenocarcinoma (PDAC) was chosen due to the lack of effective detection strategies that lead to poor survival rate after diagnosis (<1 year) and extremely low 5-years survival rate (15-20%). Densitometric analysis of 75 protein patterns (28 from healthy subjects and 47 from PDAC patients) allowed us to distinguish nononcological and PDAC patients with good sensitivity (78.6%) and specificity (85.3%). The gold NEB test is completely aligned to affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users criteria stated by the World Health Organization for cancer screening and detection. Thus, it could be very useful in clinical practice at the first level of investigation to decide whether to carry out more invasive analyses or not.

Influence of Inter- and Intra-Batch Variability on the Sample Size Required for Demonstration of Equivalent Microstructure of Semisolid Dosage Forms

Inter- and intra-batch variability of the quality attributes contribute to the uncertainty for demonstrating equivalent microstructure of post-approval changes and generic/hybrids of semisolid topical products. Selecting a representative sample size to describe accurately the in vitro properties of semisolids and to reach enough statistical power to demonstrate similarity between two semisolid topical products is currently challenging. The objective of this work is to establish the number of batches and units per batch to be compared based on different inter-batch and intra-batch variability to demonstrate equivalence in the physical characteristics of the products that ensure a similar microstructure of the semisolid. This investigation shows that the minimum number of batches to be compared of each product is 3 and the minimum number of units per batch could be 6 in the case of low intra- and inter-batch variability. If the products are not identical, i.e., 2.5–5% differences that are expected due to differences in the manufacturing process or the suppliers of excipients, 12 units and 6 batches are needed. If intra- or inter-batch variability is larger than 10%, the number of batches and/or the number of units needs to be increased. As the interplay between inter- and intra-batch variability is complex, the sample size required for each combination of inter- and intra-batch variability and expected difference between products can be obtained in the attached tables.

Plasma and serum oxylipin, endocannabinoid, bile acid, steroid, fatty acid and nonsteroidal anti-inflammatory drug quantification in a 96-well plate format

The goal of this research was to develop a high-throughput, cost-effective method for metabolic profiling of lipid mediators and hormones involved in the regulation of inflammation and energy metabolism, along with polyunsaturated fatty acids and common over-the-counter non-steroidal anti-inflammatory drugs (NSAIDs). We describe a 96-well plate protein precipitation and filtration procedure for 50 μL of plasma or serum in the presence of 37 deuterated analogs and 2 instrument internal standards. Data is acquired in two back-to-back UPLC-MS/MS analyses using electrospray ionization with positive/negative switching and scheduled multiple reaction monitoring for the determination of 145 compounds, including oxylipins, endocannabinoids and like compounds, bile acids, glucocorticoids, sex steroids, polyunsaturated fatty acids, and 3 NSAIDs. Intra- and inter-batch variability was <25% for >70% of metabolites above the LOQ in both matrices, but higher inter-batch variability was observed for serum oxylipins and some bile acids. Results for NIST Standard Reference Material 1950, compared favorably with the 20 certified metabolite values covered by this assay, and we provide new data for oxylipins, N-acylethanolamides, glucocorticoids, and 17-hydroxy-progesterone in this material. Application to two independent cohorts of elderly men and women showed the routine detection of 86 metabolites, identified fasting state influences on essential fatty acid-derived oxylipins, N-acylethanolamides and conjugated bile acids, identified rare presence of high and low testosterone levels and the presence of NSAIDs in ∼10% of these populations. The described method appears valuable for investigations in large cohort studies to provide insight into metabolic cross-talk between the array of mediators assessed here.

Tailoring the reactivity of printable Al/PVDF filament

Abstract Within the energetic materials and additive manufacturing (AM) communities, a number of aluminum/fluoropolymer (Al/FP) combinations have been identified for their suitability in various additive manufacturing techniques. For practical applications, such as in the case of a reactive wire or core in solid propellant, a range of selectable reactivity within a given Al/FP selection is needed. The purpose of this study was to alter the reactivity of aluminum/polyvinylidene fluoride (Al/PVDF) to produce a range of consistent burning rates, enabling the design of a printable reactive filament suitable for use as a reactive propellant core, or in other related applications. Three potential methods of tailoring the burning rate of Al/PVDF filaments were investigated: (1) selecting different aluminum fuel particles, (2) adjusting the stoichiometry of the material, and (3) changing the fuel particle size ratio from pure micro- to pure nano-aluminum. Reactive filaments consisting of PVDF and either mechanically activated aluminum-polytetrafluoroethylene (MA Al-PTFE), nanoscale aluminum (nAl), or mixtures of nano- and micro-aluminum (nAl:µAl) were tested to assess reaction speeds as well as intra- and inter-batch variability. Differential scanning calorimetry, thermogravimetric analysis, drop weight impact testing, friction testing, and porosity analysis were conducted on select materials. Filaments of 20 wt% nAl/PVDF and 32.2 wt% MA Al-PTFE/PVDF were printed using a material extrusion method into strands with dimensions, porosities, and burning rates comparable to their filament feedstock. This study determined that the selection of fuel particles and stoichiometry could reliably produce moderate burning rates between 17 and 40 mm/s. The burning rates of the mixed formulations were inconsistent in the mid-range (20–30 mm/s) with significant deviation indicating a threshold phenomenon potentially related to a shift from a slower to faster reaction mode.

Determination of the growth potential of Listeria monocytogenes in various types of Belgian artisanal cheeses by challenge tests

Cheese potentially allowing the growth of Listeria monocytogenes must be free of the pathogen in 25 g before being put on the market, while 100 cfu/g is tolerated when the pathogen is unable to grow. Challenge tests were performed in order to assess the growth potential of L. monocytogenes in at least one batch of 32 Belgian cheese varieties from 32 factories. All varieties were grouped in four categories: unripened acid-curd cheeses, mold-ripened soft cheeses, smear-ripened soft cheeses and ripened semi-hard cheeses. Associated microflora and cheese physicochemical characteristics were also studied. A cocktail of three strains was used to inoculate cheese on the first day of shelf-life, and samples were stored until the end of shelf-life at 7–9 °C. Growth potential was considered as the difference (a) between median contamination at the end and at the beginning of the test or (b) between the highest value at the end of the test and the lowest value at its beginning. L. monocytogenes always decreased in unripened acid-curd cheeses but showed extended growth in 21 out of 25 batches of ripened soft cheese. Contrasting results were obtained for semi-hard cheeses, as important intra- and inter-batch variability was observed. For the latter, the recommended method based on medians to calculate the growth potential led to erroneous food safety considerations, and it should always be advised to focus on absolute levels.

Assessment of carbon recovery from solid organic wastes by supercritical water oxidation for a regenerative life support system

The carbon recovery from organic space waste by supercritical water oxidation (SCWO) was studied to support resource recovery in a regenerative life support system. Resource recovery is of utmost importance in such systems which only have a limited total amount of mass. However, the practical waste treatment strategies for solid space wastes employed today are only storing and disposal without further recovery. This work assesses the performance of SCWO at recovering organic wastes as CO2 and water, to discuss the superiority of SCWO over most present strategies, and to evaluate the different SCWO reactor systems for space application. Experiments were carried out with a batch and a continuous reactor at different reaction conditions. The liquid and gas products distribution were analyzed to understand the conversion of organics in SCWO. Up to 97% and 93% of the feed carbon were recovered as CO2 in the continuous and the batch reactor, respectively. Residual carbon was mostly found as soluble organics in the effluent. Compared with the batch reactor, the continuous reactor system demonstrated a ten times higher capacity within the same reactor volume, while the batch reactor system was capable of handling feeds that contained particulate matter though suffering from poor heat integration (hence low-energy efficiency) and inter-batch variability. It was concluded that SCWO could be a promising technology to treat solid wastes for space applications. A continuous reactor would be more suitable for a regenerative life support system.

Freeze-dried meningococcal vaccine: Total error assessment of a near-infrared method for water content determination

Accuracy of reference values obtained by the reference method is essential to ensure the quality of a near-infrared (NIR) model. The purpose of this study was to assess the accuracy of a newly developed NIR analytical method for the determination of water content in a freeze-dried meningococcal vaccine by evaluating not only uncertainties associated with the calibration model and NIR measurement procedures but also systematic error arising from the reference Karl-Fischer method. An experimental design based on the standard addition method (SAM) in freeze-dried vaccines is proposed for total error assessment of the NIR analytical system. The accuracy profile showed that the NIR model successfully determined water content over the 1.0–6.7% range. The SAM enabled overall proportional error assessment, which yielded a slope of 1.01. The present study confirmed the suitability of the proposed NIR analytical method for the determination of water content in the freeze-dried meningococcal vaccine. Furthermore, assessment of the overall proportional error proved that the NIR analytical method could be used to accurately estimate differences in water content, therein surpassing the reference method as the method of choice for determining intra- or interbatch variability as well as stability.

Assessment of the Inter-Batch Variability of Microstructure Parameters in Topical Semisolids and Impact on the Demonstration of Equivalence.

Demonstration of similar microstructure is essential for demonstrating the equivalence of generic topical products since the microstructure of semisolids may affect the drug release. The objective of this study was to compare the microstructure-defining physical parameters of different batches of a reference ointment containing calcipotriol and betamethasone (Daivobet 50 µg/0.5 mg/g) in order to define the acceptance range that allows concluding equivalence between these batches. Being batches of the same reference product, they are expected to be clinically equivalent and possess similar microstructure. The 90% confidence intervals for the test/reference ratio of these physical parameters were calculated with parametric and non-parametric approaches. Both methods conclude that equivalent microstructure between batches cannot be demonstrated with a reasonable sample size when the acceptance range was set at ±10%, since several physical parameters exhibit inter-batch variability >10%. An acceptance range of ±10% is therefore too strict to conclude equivalence in the microstructure of semisolid dosage forms, given the inter-batch variability observed between batches of the reference product. A wider fixed acceptance range or an acceptance range widened based on the inter-batch variability of the reference product would be advisable.

The influence of fibre spatial characteristics on the flexural performance of SFRC

Research has shown that the post-peak tensile strength behaviour of Steel Fibre-Reinforced Concrete (SFRC) elements is significantly affected by the distribution of fibres in the section. The pursuit towards the effective and optimal application of SFRC in practice necessitates a thorough understanding of the influence of fibre spatial distribution on composite performance. In this research, the influence of fibre length and volume content on the spatial distribution of fibres was investigated using a photometric image analysis technique. A unique approach was developed that utilises Voronoi diagrams as the geometric descriptor quantifying fibre spatial distribution. The resulting parameters characterised the sectional dispersion of fibres as well as the degree of clustering and were related to the flexural performance of notched beams tested under three-point loading. The findings of the study highlight the role of fibre length and volume content on the spatial distribution of fibres and it is revealed that the sectional uniformity, inter-batch spatial variability, and degree of clustering are dependent on the number of fibres in the cross section. Furthermore, the results demonstrated the considerable influence of fibre distribution on the flexural performance of SFRC. It was concluded that the variability in flexural strength reduced as the variation in fibre spatial distribution reduced and that extensive clustering had an adverse effect on the effective resistance provided by fibres.

The use of solid phase microextraction for metabolomic analysis of non-small cell lung carcinoma cell line (A549) after administration of combretastatin A4

Use of solid phase microextraction (SPME) for cell culture metabolomic analysis allows for the attainment of more sophisticated data from in vitro cell cultures. Moreover, considering that SPME allows the implementation of multiple extractions from the same sample due to its non/low-depletive nature, time course studies using the same set of samples are thus facilitated via this method. Such an approach results in a reduction in the number of samples needed for analysis thus eliminates inter-batch variability related to biological variation occurring during cell culturing. The current work aims to demonstrate the capability of SPME for measurements of combretastatin A4 (CA4) effectiveness on non-small cell cancer cell line. A cultivation protocol was established in the 96-well plate, and a fiber format of SPME was selected for metabolite extraction. The extracellular metabolic pattern of cells was changed after administration of the tested drug. This suggests pharmacological activity of the administered compound towards the studied cell line model. Results support that the use of direct immersion SPME for analysis of cell cultures does not affect cells growth or contaminate sample. Consequently, SPME allows the attainment of accurate information regarding drug uptake, metabolism, and metabolomic changes in the studied cells induced by exposure to the drug simultaneously in a single experiment.

Developing global regression models for metabolite concentration prediction regardless of cell line.

Following the Process Analytical Technology (PAT) of the Food and Drug Administration (FDA), drug manufacturers are encouraged to develop innovative techniques in order to monitor and understand their processes in a better way. Within this framework, it has been demonstrated that Raman spectroscopy coupled with chemometric tools allow to predict critical parameters of mammalian cell cultures in-line and in real time. However, the development of robust and predictive regression models clearly requires many batches in order to take into account inter-batch variability and enhance models accuracy. Nevertheless, this heavy procedure has to be repeated for every new line of cell culture involving many resources. This is why we propose in this paper to develop global regression models taking into account different cell lines. Such models are finally transferred to any culture of the cells involved. This article first demonstrates the feasibility of developing regression models, not only for mammalian cell lines (CHO and HeLa cell cultures), but also for insect cell lines (Sf9 cell cultures). Then global regression models are generated, based on CHO cells, HeLa cells, and Sf9 cells. Finally, these models are evaluated considering a fourth cell line(HEK cells). In addition to suitable predictions of glucose and lactate concentration of HEK cell cultures, we expose that by adding a single HEK-cell culture to the calibration set, the predictive ability of the regression models are substantially increased. In this way, we demonstrate that using global models, it is not necessary to consider many cultures of a new cell line in order to obtain accurate models. Biotechnol. Bioeng. 2017;114: 2550-2559. © 2017 Wiley Periodicals, Inc.

Extended stability of the rituximab biosimilar CT-P10 in its opened vials and after dilution and storage in polyolefin bags

The stability of the rituximab biosimilar CT-P10, in 50mL vials at a concentration of 10mg/mL, and after dilution to final concentrations of 1 and 4mg/mL and storage in polyolefin bags at 4°C and 25°C was studied by several orthogonal and complementary methods. No significant change (as defined by a magnitude greater than the inter-batch variability) was observed, for each of the parameters characterizing physical and chemical stability studied, for the two concentrations and temperatures tested, or for any of the three batches tested. This implies that cold-chain rupture and exposure to room temperature up to 15 days both for vials and diluted bags have no deleterious consequence on the quality of the product. Moreover, this extended stability permits safe in-advance preparation, dose-banding or flat-dose, that to avoid unnecessary delays in the management of the patient, improvement of the pharmacy and nurse workload and money saving by avoiding non justified losses of this expensive drug.

Image processing in digital pathology: an opportunity to solve inter-batch variability of immunohistochemical staining

Immunohistochemistry (IHC) is a widely used technique in pathology to evidence protein expression in tissue samples. However, this staining technique is known for presenting inter-batch variations. Whole slide imaging in digital pathology offers a possibility to overcome this problem by means of image normalisation techniques. In the present paper we propose a methodology to objectively evaluate the need of image normalisation and to identify the best way to perform it. This methodology uses tissue microarray (TMA) materials and statistical analyses to evidence the possible variations occurring at colour and intensity levels as well as to evaluate the efficiency of image normalisation methods in correcting them. We applied our methodology to test different methods of image normalisation based on blind colour deconvolution that we adapted for IHC staining. These tests were carried out for different IHC experiments on different tissue types and targeting different proteins with different subcellular localisations. Our methodology enabled us to establish and to validate inter-batch normalization transforms which correct the non-relevant IHC staining variations. The normalised image series were then processed to extract coherent quantitative features characterising the IHC staining patterns.

Mammalian cell culture monitoring using in situ spectroscopy: Is your method really optimised?

In recent years, as a result of the process analytical technology initiative of the US Food and Drug Administration, many different works have been carried out on direct and in situ monitoring of critical parameters for mammalian cell cultures by Raman spectroscopy and multivariate regression techniques. However, despite interesting results, it cannot be said that the proposed monitoring strategies, which will reduce errors of the regression models and thus confidence limits of the predictions, are really optimized. Hence, the aim of this article is to optimize some critical steps of spectroscopic acquisition and data treatment in order to reach a higher level of accuracy and robustness of bioprocess monitoring. In this way, we propose first an original strategy to assess the most suited Raman acquisition time for the processes involved. In a second part, we demonstrate the importance of the interbatch variability on the accuracy of the predictive models with a particular focus on the optical probes adjustment. Finally, we propose a methodology for the optimization of the spectral variables selection in order to decrease prediction errors of multivariate regressions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:308-316, 2017.