Recent developments in membrane-based separations in biotechnology processes: review.

Membrane-based separations are the most ubiquitous unit operations in biotech processes. There are several key reasons for this. First, they can be used with a large variety of applications including clarification, concentration, buffer exchange, purification, and sterilization. Second, they are available in a variety of formats, such as depth filtration, ultrafiltration, diafiltration, nanofiltration, reverse osmosis, and microfiltration. Third, they are simple to operate and are generally robust toward normal variations in feed material and operating parameters. Fourth, membrane-based separations typically require lower capital cost when compared to other processing options. As a result of these advantages, a typical biotech process has anywhere from 10 to 20 membrane-based separation steps. In this article we review the major developments that have occurred on this topic with a focus on developments in the last 5 years.

Process analytical technology (PAT) for biopharmaceutical products: Part I. concepts and applications.

Process analytical technology (PAT) has been gaining momentum in the biotech community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. In this two part series, we address PAT as it applies to processes that produce biotech therapeutic products. In the first part, we address evolution of the underlying concepts and applications in biopharmaceutical manufacturing. We also present a literature review of applications in the areas of upstream and downstream processing to illustrate how implementation of PAT can help realize advanced approaches to ensuring product quality in real time. In the second part, we will explore similar applications in the areas of drug product manufacturing, rapid microbiology, and chemometrics as well as evolution of PAT in biotech processing.

Process analytical technology (PAT) for biopharmaceutical products: Part II. Concepts and applications.

Implementing real-time product quality control meets one or both of the key goals outlined in FDA's PAT guidance: "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." The first part of the paper presented an overview of PAT concepts and applications in the areas of upstream and downstream processing. In this second part, we present principles and case studies to illustrate implementation of PAT for drug product manufacturing, rapid microbiology, and chemometrics. We further present our thoughts on how PAT will be applied to biotech processes going forward. The role of PAT as an enabling component of the Quality by Design framework is highlighted. Integration of PAT with the principles stated in the ICH Q8, Q9, and Q10 guidance documents is also discussed.

Process analytical technology (PAT) for biopharmaceutical products.

The "Pharmaceutical Current Good Manufacturing Practices (CGMPs) for the 21st Century--A Risk Based Approach" initiative announced by the FDA in August 2002 to improve and modernize pharmaceutical manufacturing facilitated adoption of process analytical technology (PAT) by the pharmaceutical industry. The potential for improved operational control and compliance resulting from continuous real-time quality assurance was highlighted as a likely benefit that would result from PAT implementation. A considerable amount of work has been done on this topic by academic and industrial contributors in the last decade. In this paper, we will start with a brief overview of evolution of PAT concepts and a review of their application in the wider pharmaceutical industry. The rest of the paper focuses on PAT applications for biotech processes with emphasis on developments in the last five years. It is our observation that while significant advances have been accomplished with regard to our ability to analyze/monitor key process and quality attributes in the biotech industry, much more needs to be done with regard to utilizing the collected data for subsequent control of the process, to achieve optimum yield and product quality. The latter is necessary to achieve the benefits that will result from PAT implementation.

Quality and effectiveness of counselling at antiretroviral therapy centres in India: capturing counsellor and beneficiary perspectives.

BACKGROUND: Ensuring the quality and effectiveness of counselling is imperative for enabling people living with HIV to cope with treatment adherence. Countrywide assessment of antiretroviral therapy (ART) centres was undertaken to assess the quality and effectiveness of counselling. The insights gained from the assessment are expected to build an improved understanding of the counselling aspect and contribute to informing decisions strengthening the counselling provided at ART centres. METHODS: Assessment of counselling at 357 ART centres entailed interviews with counsellors and beneficiaries using a structured questionnaire administered by trained technical experts. Two counsellors and five beneficiaries at each ART centre were interviewed to assess both the quality and effectiveness of counselling. Beneficiaries were selected from different risk groups to understand their varied concerns and experiences. RESULTS: During the assessment, 618 counsellors were interviewed (45% women); also, 1785 beneficiaries were interviewed, consisting of 892 (49.9%) men, 857 (48.1%) women and 36 (2.0%) transgender. Counsellors were found to be relatively well informed on topics pertaining to pre-ART, ART preparedness and positive healthy living, and the psychosocial support extended to patients. Counsellors surveyed were not aware of critical areas such as counselling of pregnant women (44.5%) , drug adherence (44.8%) and the use of information, education and communication material during counselling, and pill count. The majority of beneficiaries reported being informed on issues pertaining to retention; however, 30-40% of beneficiaries were not informed regarding the critical elements of adherence such as counselling on ART side effects (68.5%), pill count (62.8%) and information on access to social benefit schemes (25.7%). Factors such as client volume, the training of the counsellors and adequate space for counselling affected the quality of counselling. CONCLUSION: With concerted efforts in bridging the gaps in knowledge, infrastructure and information needs, India's national AIDS control programme (NACP) can enhance the counselling services at ART centres and improve the quality of services for patient retention.

Assessment of quality of antiretroviral therapy services in India, 2014-2015.

Background: Following a decade of provision of free antiretroviral therapy (ART) in India, a nationwide assessment of ART services was conducted to review quality of care at ART centers. This paper presents the methods and defines replicable model of undertaking large scale assessments. Methods: During the period January 2014-March 2015, 357 ART centers were reviewed under four domains, namely, operations, technical, monitoring and evaluation (M&E), and logistics. Mixed methods, comprising of desk review and on-site facility assessment; random sample of records, interviews with both health-care staff and people living with HIV (PLHIV) were used. Grading for each of the domain was done on a scale of 5, with 1 (Very poor) being the lowest and 5 (Excellent) as highest. Results: 1720 health-care staff and 1762 beneficiaries were interviewed; 34 600 patient cards were reviewed. Of the 357 centers assessed 60, 169 and 128 scored Excellent, Average and Poor, respectively, in operations domain; 147, 176, 34 in Technical domain; 215, 115, 27 in M&E domain; 263, 71, 23 centers in logistics domain scored Excellent, Average and Poor, respectively. About 95% (1698/1785) of PLHIV were satisfied with the care provided at ART centers. Conclusion: The methodology used for the assessment of ART centers in India yielded insights on the different domains that impact implementation and quality of service delivery. The design of this exercise may inform other researchers and managers planning similar large-scale assessments.

Feasibility of implementing an integrated tool for improvement of treatment quality and early-warning indicators for HIV drug resistance: a pilot study of centres in India.

With the rapid scale-up in use of antiretroviral therapy (ART), monitoring the quality of care and factors that may lead to emergence of HIV drug resistance (HIVDR) is an important focus point for programme managers. The National AIDS Control Organisation of India embarked on strengthening the ART programme for continuous quality improvement (CQI), using defined quality-of-care indicators (QCIs), including World Health Organization (WHO) early-warning indicators (EWIs) for HIVDR. In this feasibility study, done during July 2014, an integrated QCI and EWI tool developed by WHO India was pilot tested across 18 purposively selected ART centres. At seven ART centres, the EWI 1 target of >90% on-time pill pick-up was achieved for adult patients, while among the paediatric age group (<15 years old) it was not achieved by any centre. EWI 2 (retention of patients in ART care at 12 months after initiation) showed that two centres had retention of both adult and paediatric patients of >85% at 12 months of ART, while 11 centres had retention between 75% and 85%. EWI 3 (pharmacy stock-out) for adult and paediatric patients showed that 11 ART centres reported a minimum of one stock-out for the first-line ART drugs in the reporting period, while EWI 4 targets (pharmacy dispensing practices) were achieved by all the centres, for both adults and children. Average retention in care at 6, 12 and 24 months after ART initiation was 82%, 77% and 71%, respectively. This feasibility study showed that EWI analyses were much simpler to conduct if information was sought only for patients receiving ART, for whom the quality of record-keeping is better and more consistent. The activity has highlighted the need for improved quality of record-keeping at the facilities and implementation of specific interventions to ensure better patient follow-up. After modifications, use of the tool will be phased in across all the ART centres in India.

The protective action of essential oils of onion and garlic in cholesterol-fed rabbits.

The effects of the essential oils of onion and garlic have been investigated in cholesterol-fed rabbits and have been compared with the effects of clofibrate. The marked rise in serum cholesterol and blood coagulability that followed 3 months of cholesterol feeding (0.2 g/kg/day) was significantly reduced by the essential oils of both onion and garlic. Fibrinolytic activity was actually increased even above the normal control levels. The essential oils of onion and garlic (equivalent to 1 g/kg/day of raw bulbs) proved more effective than clofibrate in the usual clinical dose of 33 mg/kg/day. Garlic was even more effective than onion. Cholesterol feeding increased the average lipid content of aorta from 5.95 to 13.75 mg/100 mg dry weight. With addition of clofibrate, onion or garlic the values were 7.79, 6.23 and 5.28 mg/100 ml, respectively. Thus it can be seen that the essential oils effectively prevent lipid acculation in the rabbit oarta.

Effect of essential oil of onion and garlic on experimental atherosclerosis in rabbits.

The effects of the essential oils of onion (extracted from 2 g of raw onion per kg body weight) and garlic (extracted from 1 g of raw garlic per kg body weight) have been observed on experimental atherosclerosis produced by cholesterol feeding (0.5 g/kg) in rabbits. The rise in serum cholesterol and serum triglycerides was significantly reduced by both onion and garlic during the 4-month period of study. Cholesterol feeding significantly increased beta-(P less than 0.01) and pre-beta (P less than 0.001) lipoproteins while decreasing the alpha-fraction (P less than 0.001). Onion and garlic both prevented these changes. The beta/alpha ratio, which was initially 1.6 : 1, rose to 4.5 : 1 and 5.7 : 1 at the end of 2 months and 4 months of cholesterol feeding. However, this ratio did not increase significantly, both at the 2-month and 4-month period, when onion and garlic were added. Fibrinolytic activity significantly increased with onion (P less than 0.001) and garlic (P less than 0.001) while feeding only cholesterol actually decreased it (P less than 0.001). Onion and garlic reducec aortic atheroma by about half. It is suggested that the essential oils of onion and garlic protect against experimental atherosclerosis by preventing the fall in the alpha lipoprotein fraction and by enhancing fibrinolytic activity, as well as by lowering the serum cholesterol and triglyceride levels.

Displacement chromatography with on-column isomerization.

Displacement chromatography was simulated for the separation of two feed components interconverting by a reversible first order reaction and with Langmuirian adsorption behavior. The study was prompted by recent interest in the isolation of cis and trans forms of peptides containing one or more peptidyl-proline residues when the isomerization reaction interferes with the separation. The parameter values used in the simulations are similar to those found experimentally by reversed-phase chromatography and capillary electrophoresis of phenylalanine-proline dipeptide. From the concentration profiles computed by the finite difference scheme, the dependence of both the yield and production rate on the temperature, column length, flow velocity and displacer concentration was evaluated. The most important operational variable of the system is the temperature as it affects both the kinetic and adsorption parameters. The yield and production rate of the component of interest were evaluated as a function of the column length and displacer concentration under conditions that facilitate its efficient separation and the plots show an optimum. Nonetheless, optimal conditions for yield and production rate were considerably different. In the temperature range from 2 to 42 degrees C, the yield always decreases with increasing temperatures and for all the cases, optimum yield by displacement mandates the use of conditions such as pH, solvent and temperature under which the rate of interconversion is reduced to a level where it does not palpably interfere with the separation. On the other hand, under certain conditions optimal production rate can be obtained at higher temperatures.

Capillary electrochromatography: theories on electroosmotic flow in porous media.

In view of the present interest in capillary electrochromatography (CEC), theories dealing with electroosmotic flow (EOF) in porous media are reviewed with particular regard to the use of packed capillaries in CEC. Two of the models found in the pertinent literature are applicable to CEC and give simple analytical solutions. The first of the two models is based on von Smoluchowski's work as adapted and extended by Overbeek. It deals with EOF through packed capillaries under conditions of low electric field strength where the EOF varies linearly with the field strength because there is no polarization of the double layer. Overbeek's model originally developed for porous media of infinite dimensions was modified in an attempt to account for the wall effect that assumes importance in the packed capillary columns used in CEC. The second model proposed by Dukhin and his coworkers predicts EOF of at least an order of magnitude higher than that expected by classical theories. This "electroosmosis of the second kind" is believed to occur in columns packed with conductive particles like ion exchangers at high electric field strengths when the double layer is polarized and the EOF becomes a non-linear function of the applied voltage. Conditions necessary for electroosmosis of the the second kind are likely to arise upon the further development of CEC when further enhancement of the speed of analysis is brought about at electric field strength higher than that employed at present.

Cyclodextrin aided separation of peptides and proteins by capillary zone electrophoresis.

Carboxymethylated-beta-cyclodextrin (CMBCD) in the electrophoretic medium (aqueous 50 mM sodium phosphate, pH 2.5) enhanced the separation using raw fused-silica capillaries in CZE of the four standard proteins: alpha-chymotrypsinogen A, cytochrome c, lysozyme and ribonuclease A. Furthermore, with 20 mM CMBCD in the electrophoretic medium, the cis-trans isomers of angiotensin could be separated at room temperature, whereas the separation of the conformers required subambient temperatures as low as -20 degrees C without CMBCD in the electrophoretic medium [50 mM sodium phosphate (pH 2.5), containing 10% (v/v) methanol]. Addition of heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) had no effect on the separation of the above proteins and peptides. The results suggest that in microcolumn separation techniques, certain cyclodextrin additives can be useful selectivity enhancers.

Separation parameters via virtual migration distances in high-performance liquid chromatography, capillary zone electrophoresis and electrokinetic chromatography.

Among the various differential migration processes of separation, high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) have emerged as the two major high-performance analytical techniques for separation of complex biological substances. In both HPLC and CZE with electroendoosmotic flow, the differential migration process can be divided into a separative component, which involves selective interactions with the stationary phase or differences in the electrophoretic migration velocities, and a non-separative component representing migration by convection that does not contribute directly to separation. The introduction of virtual migration distances leads to an additivity relationship for the two components that is applicable to both of the above techniques and facilitates the recognition of the underlying similarities as well as the expression and comparison of the various separation parameters. Examination of the key migration parameters led to the characterization and the classification of the various modes of CZE with electroendoosmotic flow. The treatment was extended to the analysis of capillary electrochromatography and micellar electrokinetic chromatography; two hybrid processes which exhibit features borrowed from HPLC and CZE. The use of virtual migration distances also led to a consistent and unified description of the characteristic parameters of these separation systems.

Kinetic study on the cis-trans isomerization of peptidyl-proline dipeptides.

The equilibrium and kinetic parameters of cis-trans interconversion of dipeptides containing peptidyl-proline moiety were investigated using the in-column incubation method with both CZE and HPLC and the ad hoc dissolution method. The use of the latter was possible because the conformational make-up of the solid peptides, and consequently of their ad hoc solution, was sufficiently different from that of the solution at equilibrium. This method with 1H-NMR and CZE analyses yielded very similar results for the cis-trans isomerization of Phe Pro in aqueous sodium borate, pH 8.4, at 10 degrees C with an average value of 0.34 and 6.6 x 10(-5) s-1 for the equilibrium and rate constant, respectively. The in-column incubation method is performed by CZE or HPLC so that the conformers are separated in the first half of the column and then incubated in column where they interconvert and reach equilibrium. Subsequent separation in the second half of the column yielded four peaks. Thus by measuring the conformer composition as a function of the reaction time, the rate constant can be evaluated. The forward rate constant of 1.42 x 10(-4) s-1 determined by the CZE in-column incubation method for Phe-Pro isomerization at 10 degrees C was twice of the value obtained by the ad hoc dissolution method. It is believed that the inner wall of fused-silica capillaries has a catalytic effect on the isomerization. Computer simulation was also employed to gain further insight on the catalytic activity of the capillary inner wall on such isomerization. Whereas the experimental and simulation profiles of Phe-Pro in aqueous borate buffer, pH 8.4, with a 37 cm long capillary were in excellent agreement, a four times faster interconversion rate had to be used to match the experimental profile obtained with a 57 cm long capillary under otherwise identical conditions. The catalytic effect of the octadecyl silica stationary phase on the isomerization was confirmed by the in-column incubation method with HPLC. The overall rate of the cis-trans isomerization of Phe-Pro, which entails the reaction both on the stationary phase and in the mobility phase, was about six times faster at 0 degree C than the rate measured by NMR in free solution using the mobile phase containing 65% (v/v) sodium phosphate, pH 6.5, and 35% (v/v) methanol. The results presented here serve as a caveat that the effect of the wall in CZE or the stationary phase in HPLC on the reaction cannot be ignored.

Validatibility of a capillary isoelectric focusing method for impurity quantitation.

A strategy is presented for examining the validatability of a capillary isoelectric focusing (cIEF) method, intended for quantitation of product-related impurities in a protein drug substance, according to guidelines published by the International Conference on Harmonization (ICH). The results of this study demonstrate the suitability of cIEF as an analytical method for the quantitation of two product-related impurities in a protein drug substance: a monodeamidated degradation product and an aggregated form of the parent molecule. A range of impurity levels was generated by spiking the isolated impurity species, into a representative production lot of the drug substance. Six impurity spike levels (0.5-12% impurity for deamidated species and 0.5-8% impurity for aggregated species) were analyzed in triplicate. Measurement of impurity peak area percent in the spiked samples provided the data for computing specificity, accuracy, precision, linearity and limit of quantitation (LOQ) for the impurities. Accuracy, defined as the agreement of peak area percent for impurity species with the theoretical impurity percentage from the spike ratio, was 85-96% for the deamidated species and 73-97% for the aggregated species. A linear relationship was found between the measured area percent and the theoretical percent impurity for both impurity species (coefficient of determination, r2=0.9994 for deamidated species and =0.9827 for aggregated species). Precision (repeatability) studies demonstrated a low relative standard deviation (RSD) value (<6%) at all spike levels for both impurity species. Intermediate precision and reproducibility were evaluated by simulating many of the multivariable testing conditions expected during the life cycle of an analytical method, such as multiple equipment and laboratories. Repeated analyses of the drug substance under these varied conditions, yielded RSD values of <20%, for both impurity species. The LOQ, defined as the lowest impurity level where both accuracy and precision were achieved, was assigned at the 0.5% impurity level for both impurity species. This work illustrates a successful strategy in applying the ICH validation guidelines for impurity analytical methods to a cIEF method. Moreover, the data demonstrate the ability of cIEF to be used reliably as an analytical method for impurity quantitation.

A correlative study of seminal biochemistry and testicular histology in infertile males.

Two androgen-dependent constituents of the seminal plasma, fructose and acid phosphatase, have been estimated in 50 infertile males along with a testicular biopsy. Azoospermics, as a group, showed a very wide range of fructose (16-600 mg%) as compared to 210-397 mg% in healthy fertile males. Oligospermics tended to have low values with a mean of 218 +/- 75.1 mg%. Acid phosphatase in the controls was 1927 +/- 164.6 K.A. unit/ml and was generally higher in the infertile groups. The state of spermatogenesis, as revealed by testicular biopsy, bore no consistent relationship with the seminal fructose or acid phosphatase. It appears that there may be no inter-relationship between the activity of the germinal epithelium and the secretion of the accessory glands and, although both are androgen-dependent, they can be affected separately by a multitude of factors in human infertility.

Domiciliary health counselling in patients with sexually transmitted diseases.

To improve treatment compliance, follow-up and attendance of STD patients and their contacts, a programme, based on domiciliary visits was attempted. The programme was limited to male defaulters not responding to written and telephone calls. House visits to 79 out of 169 defaulters were made. Approximately half the number of houses were found locked. Fourteen defaulting patients reported for completion of their treatment and follow-up. Of these 11 were directly interviewed. None of the 14 patients agreed to disclose the addresses of their extramarital partners. During the study period 51 female sex contacts reported for treatment as compared to 27 in the control group. Though the numbers were small the study revealed that direct contact with the patient yielded the best results. It is suggested that to make the system cost-effective the health workers should spend time with STD patients in the clinic to mark out the correct location and time for meeting the patient either in the work place or at home in case of a follow-up.

Periodic syphilis profile in a New Delhi hospital.

The study reports the prevalence of syphilis determined at intervals of ten years amongst attendees of the Sexually Transmitted Diseases Centre (STD), in New Delhi, from 1954, to 1994. It was observed that the number of STD cases increased by about eight times in 1984 as compared to 1954. The prevalence of STDs had risen from 5.5 per cent in 1964 to 14.7 per cent in 1994. However, the syphilis load has been decreasing since 1974. Childhood syphilis has decreased from 12.6 per cent in 1954 to 0.5 per cent in 1994. However in adult males it increased from 62.3 per cent in 1954 to 80.4 per cent in 1984. Cardiovascular syphilis was last reported in 1954. The prevalence of syphilis was approximately three times higher in males than in females.

Breast feeding practices among rural mothers of Delhi.

PIP: Between October 1991 and September 1992, in six villages of the Najafgarh block in West Delhi, India, nursing students in community health interviewed 600 mothers aged 16-40 to determine the prevalence of breast feeding. 72.3% were 20-30 years old. All the women had breast fed their infants. The mean family size was 2.2. 52 (9%) had thrown away the colostrum because they believed it would harm their newborns and cause constipation, vomiting, and other gastrointestinal problems. Eight mothers threw out the colostrum because older women told them to, and without knowing its harmful or beneficial effects. 52.6% of the mothers began breast feeding within 6 hours. 22.7% began between 6 and 24 hours of delivery. Among the 47.4% of cases who delayed the first breast feed beyond 6 hours, 2.3%, 3%, 18.75%, and 37.3%, respectively, gave cow's milk, Ganga water, sugar solution, or honey as the first feed to their newborn. 28.7% of mothers were illiterate. Most mothers thought that breast feeding should continue as long as the child wants it. Women with three children were more likely to breast feed their third child for more than one year than primiparous women (22.6% vs. 5.2%). 81.6% thought that weaning should start before the infant reaches 6 months of age. Thus, most mothers understood the benefits of early weaning.^ieng