Evaluation of various membranes at different fluxes to enable large-volume single-use perfusion bioreactors.

The growing demand for biological therapeutics has increased interest in large-volume perfusion bioreactors, but the operation and scalability of perfusion membranes remain a challenge. This study evaluates perfusion cell culture performance and monoclonal antibody (mAb) productivity at various membrane fluxes (1.5-5 LMH), utilizing polyvinylidene difluoride (PVDF), polyethersulfone (PES), or polysulfone (PS) membranes in tangential flow filtration mode. At low flux, culture with PVDF membrane maintained higher cell culture growth, permeate titer (1.06-1.34 g/L) and sieving coefficients (≥83%) but showed lower permeate volumetric throughput and higher transmembrane pressure (TMP) (>1.50 psi) in the later part of the run compared to cultures with PES and PS membrane. However, as permeate flux increased, the total mass of product decreased by around 30% for cultures with PVDF membrane, while it remained consistent with PES and PS membrane, and at the highest flux studied, PES membrane generated 12% more product than PVDF membrane. This highlights that membrane selection for large-volume perfusion bioreactors depends on the productivity and permeate flux required. Since operating large-volume perfusion bioreactors at low flux would require several cell retention devices and a complex setup, PVDF membranes are suitable for low-volume operations at low fluxes whereas PES membranes can be a desirable alternative for large-volume higher demand products at higher fluxes.

Evaluation of wakefulness electroencephalogram in OSA patients.

OBJECTIVE: Obstructive sleep apnea (OSA) has been associated with an elevated cardiovascular risk, increased daytime sleepiness, cognitive decline, and slower electroencephalographic activity (EEG). This study assesses EEG patterns during wakefulness in OSA patients compared to those without sleep-disordered breathing. MATERIALS AND METHODS: This retrospective study analyzed 30 OSA patients with an Apnea/Hypopnea Index (AHI) of 15 or higher, as well as 29 individuals without sleep-disordered breathing (AHI of 5 or lower) who underwent hospital polysomnography and met all inclusion criteria. Sociodemographic and EEG data were obtained from the sleep laboratory database. Blinded EEG analysis was conducted by two observers, assessing activity in the frontal, central, and occipital regions. RESULTS: No significant differences were observed in EEG activity between OSA and non-OSA patients. However, a weak correlation was found between decreased C3 EEG frequency and higher AHI (p = 0.033), as well as increased total sleep time and higher O2 EEG frequency (p = 0.038). Lower amplitudes in C3 (p = 0.043) and O1 (p = 0.031) were correlated with reduced average oxygen saturation. CONCLUSION: Our findings suggest that OSA-related hypoxemia may impact neuronal activity, highlighting the need to address this sleep-disordered breathing in order to potentially prevent the cognitive decline observed in OSA patients.

Pattern electroretinography response in amblyopic adults.

INTRODUCTION: Amblyopia is generally a unilateral disorder, defined by at least a difference of two lines of visual acuity between both eyes with the best-corrected visual acuity, a decrease in contrast sensitivity, and a decrease in stereopsis. Pattern electroretinogram (PERG) is a noninvasive technique that provides a retinal biopotential and is a highly sensitive indicator of changes in the macular area. Our aim was to evaluate if there are differences in the retinal response of an amblyopic eye compared with a normal eye (NE). METHODS: We evaluated twenty-four adult volunteers, twelve amblyopes (mean 43.42 ± 12.72 years old), and twelve subjects with NE (mean 35.58 ± 12.85 years old). None of the subjects in the two groups had comorbidities. A complete optometric examination was performed including parameters such as visual acuity (VA) by far and near with ETDRS chart, eye alignment with cover test, and evaluation of retinal cells response with PERG. RESULTS: The refractive error found in the NE group of subjects had a mean of - 0.95 ± 1.65D, while the amblyopic group showed a mean of - 2.03 ± 4.29D. The VA in amblyopic eyes had a mean of 0.38 ± 0.20 logMAR. Analyzing PERG data, we observed significant differences in the P50-N95 amplitudes of the amblyopic group compared with the NE group (p < 0.0001-amblyopic eye vs. NE; p = 0.039-fellow eye vs. NE). DISCUSSION: These findings suggest that amblyopic patients may also present other impairments beyond the visual cortex. PERGs seem to be an important complementary examination in the diagnosis of other impairments in amblyopia.

High-frequency repetitive magnetic stimulation rescues ischemia-injured neurons through modulation of glial-derived neurotrophic factor present in the astrocyte's secretome.

Due to its ability to improve the most frequent clinical sequelae left by ischemia, repetitive transcranial magnetic stimulation has been considered a promising therapeutic strategy for stroke. Those improvements are associated with changes in neurons and their synaptic liaisons. However, the hypothesis that this technique modulates astrocytes, potentiating their neuroprotective capabilities, was also raised. This study aims to identify the effects triggered by high-frequency repetitive magnetic stimulation (HF-rMS) on astrocytes that contribute to its neuroprotective effects. Neuron-glia and astrocyte cortical cultures subject to oxygen and glucose deprivation were used as an in vitro model of ischemia. Neuroprotection promoted by HF-rMS was evaluated by analysis of markers of neuronal activity and morphometric analysis of neurons. Glial reactivity was determined by immunocytochemistry. The levels of growth factors in the astrocyte-conditioned medium (CM) were assessed through a Growth Factor Array and glial-derived neurotrophic factor (GDNF) expression was analyzed by RT-PCR and Western blot. Our results show that neurons injured by ischemia can be rescued through the modulation of astrocytes by HF-rMS. This modulation helps to maintain the number and length of neurites and increases the number of neurons expressing ERK1/2 and c-Fos. Analysis of the astrocyte-CM showed that HF-rMS stimulated the release of several trophic factors by astrocytes. Moreover, GDNF was one of the released factors that contributed to the recovery mechanisms triggered by HF-rMS. Our results show that modulation of astrocytes by HF-rMS effectively rescues neurons injured by ischemia and suggest that by targeting astrocytes this approach can also be used to promote neuroprotection in other brain lesions.

Body posture as a factor determining sleep quality in patients using non-invasive ventilation.

OBJECTIVE: Sleep quality is influenced by multiple factors. Slow-wave sleep and REM sleep play a key role in homeostasis and are useful indicators of sleep quality. Studies indicate that obstructive sleep apnea (OSA) in the supine position correlates with anatomical changes that exacerbate respiratory events and influence the effectiveness of ventilation therapy. This study aimed to evaluate the correlation of body posture with polysomnographic data and adherence of patients using continuous positive airway pressure (CPAP). MATERIAL AND METHODS: This was a retrospective study of patients with OSA who had polysomnography in Rainha Santa Isabel Hospital's sleep laboratory in Torres Novas, Portugal, and met all the inclusion and exclusion criteria. Sociodemographic, polysomnographic, and ventilation therapy variables were collected from that sleep laboratory database between 2015 and 2019. RESULTS: In 30 patients with OSA, residual apnea-hypopnea index (AHIr) and arousal index were lower in the non-supine position compared to the supine position (p value 0.005 and 0.009 respectively). As measures of sleep quality, total sleep time in SWS and REM sleep were greater in the non-supine position compared to the supine position (p value of 0.002 and 0.010 respectively). CONCLUSION: The findings suggest that a supine position significantly impairs sleep quality mainly by increasing the number of respiratory events and associated sleep fragmentation. The findings also suggest that the difference in AHIr between supine and non-supine positions may contribute to non-adherence with CPAP in patients with OSA.

Eugenol ß-Amino/ß-Alkoxy Alcohols with Selective Anticancer Activity.

Eugenol, 4-allyl-2-methoxyphenol, is the main constituent of clove essential oil and has demonstrated relevant biological activity, namely anticancer activity. Aiming to increase this activity, we synthesized a series of eugenol ß-amino alcohol and ß-alkoxy alcohol derivatives, which were then tested against two human cancer cell lines, namely gastric adenocarcinoma cells (AGS) and lung adenocarcinoma cells (A549). An initial screening was performed to identify the most cytotoxic compounds. The results demonstrated that three ß-amino alcohol derivatives had anticancer activity that justified subsequent studies, having been shown to trigger apoptosis. Importantly, the most potent molecules displayed no appreciable toxicity towards human noncancer cells. Structure-activity relationships show that changes in eugenol structure led to enhanced cytotoxic activity and can contribute to the future design of more potent and selective drugs.

Theta burst stimulation in adults with symmetric and asymmetric visual acuity.

PURPOSE: Theta Burst Stimulation can influence adult neuro-visual response in imbalanced visual pathways, possibly by influencing cortical excitability. Our objective was to compare suppressive imbalance (SI) and visual acuity (VA) after applying repetitive Transcranial Magnetic Stimulation between groups of subjects with normal binocular vision, visual asymmetry, and amblyopia. METHODS: Thirty-five volunteers between 19 and 51 years of age were split into three groups: 6 volunteers with asymmetric VA (group A); 19 amblyopes (group B); and 10 subjects with normal binocular vision (group C). VA and SI of all groups were evaluated before and after a single session of continuous Theta Burst Stimulation (cTBS) or placebo stimulation over the right occipital cortex. RESULTS: In both groups A and B, we found a significant VA improvement in the non-dominant eye after cTBS (p = 0.04 and p = 0.01, respectively). In SI evaluation, group A and group B also revealed a significant improvement after the cTBS session (p = 0.03 and p = 0.01, respectively). Finally, in the group of volunteers with normal binocular vision and for placebo groups A and B, there were no significant differences in VA and SI after cTBS. CONCLUSIONS: Amblyopic and visually asymmetric individuals improved VA and SI of the non-dominant eye after cTBS when compared to baseline and to placebo stimulation. These enhancements were not found in the group of volunteers with normal binocular vision. We can therefore reasonably assume that cTBS may interfere with the visual system of subjects that present some kind of asymmetry, possibly by improving neuronal imbalances.

Astrocytes contribute to the neuronal recovery promoted by high-frequency repetitive magnetic stimulation in in vitro models of ischemia.

After decades of effort, there are no effective clinical treatments to induce the recovery of ischemia-injured tissues, and among the several strategies that have been explored, repetitive transcranial magnetic stimulation has proven to be one of the most promising, with beneficial effects in limb motor function, aphasia, hemispatial neglect, or dysphagia. Despite the clinical evidences, little is known about the mechanisms underlying those effects. The present study aimed to explore the cellular and molecular effects of high-frequency repetitive magnetic stimulation (HF-rMS) on an in vitro model of ischemia. Using primary cortical cultures exposed to oxygen and glucose deprivation followed by reperfusion, we observed that HF-rMS treatment prevents the ischemia-induced neuronal death by 21.2%, and the neurite degeneration triggered by ischemia. Our results also demonstrate that with this treatment there is an increase of 89.2% on the number cells expressing ERK1/2, of 20.1% on the number of cells expressing c-Fos, and a synaptogenic effect, through an increase of 62.9% in the number of synaptic puncta as well as of 49.4% in their intensity. Interestingly, our results indicate that astrocytes are crucial to the beneficial effects triggered by HF-rMS after ischemia, thus suggesting a direct effect of HF-rMS on these cells. The modulation of astrocytes with this non-invasive brain stimulation technique is a promising approach to promote the recovery of ischemia-induced injured tissues; however, it is essential to understand how these effects can be modulated in order to optimize the protocols and enhance the beneficial outcomes.

Theta-Burst Stimulation Is Able to Impact Cognitive Processing: A P300 and Neuropsychological Test Study.

INTRODUCTION: Theta-burst stimulation (TBS) is a safe non-invasive neurostimulation technique used to improve cognitive and neuropsychiatric impairments. Combined outcome evaluation using event-related potentials (ERPs) and neuropsychological tests may allow a more thorough assessment of TBS treatment efficacy; however, some mixed results have been found, and their use remains scarce. Our main objective was to evaluate whether a session of TBS to the left dorsolateral prefrontal cortex (DLPFC) can impact upon the performance of both neuropsychological and neurophysiological tests. METHODS: This double-blind sham-controlled study involved 28 healthy adults, between 18 and 30 years. Volunteers were randomly allocated to receive excitatory (intermittent [iTBS]), inhibitory (continuous TBS [cTBS]) or sham stimulation on the left DLPFC. Subjects were evaluated using ERPs (auditory oddball paradigm P300) and neuropsychological tests (Trail making test [TMT] and Stroop test of words and colours [STWC]), using a pre-post stimulation protocol. RESULTS: Inhibitory stimulation led to significantly delayed P300 peak latencies (p < 0.001), with no consistent change in N2P3 amplitudes. cTBS also significantly influenced the expected group performance in Stroop C and Stroop interference (p = 0.025) compared to the iTBS and sham groups. No significant results were found in TMT tests after TBS. CONCLUSION: Our results suggest that P300 and specific Stroop colour and words test parameters can be similarly influenced by the same TBS protocol. This emphasizes the importance of mixed evaluation using neuropsychological and neurophysiological resources in research associated with the use of transcranial magnetic stimulation and cognition.

Amino Alcohols from Eugenol as Potential Semisynthetic Insecticides: Chemical, Biological, and Computational Insights.

A series of ß-amino alcohols were prepared by the reaction of eugenol epoxide with aliphatic and aromatic amine nucleophiles. The synthesized compounds were fully characterized and evaluated as potential insecticides through the assessment of their biological activity against Sf9 insect cells, compared with a commercial synthetic pesticide (chlorpyrifos, CHPY). Three derivatives bearing a terminal benzene ring, either substituted or unsubstituted, were identified as the most potent molecules, two of them displaying higher toxicity to insect cells than CHPY. In addition, the most promising molecules were able to increase the activity of serine proteases (caspases) pivotal to apoptosis and were more toxic to insect cells than human cells. Structure-based inverted virtual screening and molecular dynamics simulations demonstrate that these molecules likely target acetylcholinesterase and/or the insect odorant-binding proteins and are able to form stable complexes with these proteins. Encapsulation assays in liposomes of DMPG and DPPC/DMPG (1:1) were performed for the most active compound, and high encapsulation efficiencies were obtained. A thermosensitive formulation was achieved with the compound release being more efficient at higher temperatures.

Wide-surface pore microfiltration membrane drastically improves sieving decay in TFF-based perfusion cell culture and streamline chromatography integration for continuous bioprocessing.

Although several compelling benefits for bioprocess intensification have been reported, the need for a streamlined integration of perfusion cultures with capture chromatography still remains unmet. Here, a robust solution is established by conducting tangential flow filtration-based perfusion with a wide-surface pore microfiltration membrane. The resulting integrated continuous bioprocess demonstrated negligible retention of antibody, DNA, and host cell proteins in the bioreactor with average sieving coefficients of 98 ± 1%, 124 ± 28%, and 109 ± 27%, respectively. Further discussion regarding the potential membrane fouling mechanisms is also provided by comparing two membranes with different surface pore structures and the same hollow fiber length, total membrane area, and chemistry. A cake-growth profile is reported for the narrower surface pore, 0.65-µm nominal retention perfusion membrane with final antibody sieving coefficients ≤70%. Whereas the sieving coefficient remained ≥85% during 40 culture days for the wide-surface pore, 0.2-µm nominal retention rating membrane. The wide-surface pore structure, confirmed by scanning electron microscopy imaging, minimizes the formation of biomass deposits on the membrane surface and drastically improves product sieving. This study not only offers a robust alternative for integrated continuous bioprocess by eliminating additional filtration steps while overcoming sieving decay, but also provides insight into membranes' fouling mechanism.

Impact of micro and macroporous TFF membranes on product sieving and chromatography loading for perfusion cell culture.

Bioprocess intensification can be achieved through high cell density perfusion cell culture with continuous protein capture integration. Protein passage and cell retention are commonly accomplished using tangential flow filtration systems consisting of microporous membranes. Significant challenges, including low efficiency and decaying product sieving over time, are commonly observed in these cell retention devices. Here, we demonstrate that a macroporous membrane overcomes the product sieving challenges when comparing to several other membrane chemistries and pore sizes within the microporous range. This way, variable chromatography column loading is avoided. The macroporous membrane yielded a 13,000 L/m2 volumetric throughput. The membrane's cut-off size results in an increased permeate turbidity due to particles passage, such as cell debris, through pores ranging from 1 to 4 µm. In addition, successful chromatography column plugging mitigation was achieved by employing depth filtration before the chromatographic step. Depth filtration volumetric throughputs were between 600 and 1,000 L/m2 . Combing a macroporous cell retention device with a depth filter not only provided an alternative to address the challenge of undesired long protein residence times in the bioreactor due to product sieving decay, but also exhibited a throughput increase, making the integration of multicolumn capture chromatography with a perfusion cell culture a more robust process.

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system.

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20-25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.

Transcranial Magnetic Stimulation in Adults With Amblyopia.

BACKGROUND: Through transcranial magnetic stimulation (TMS) it is possible to change cortical excitability of the visual cortex, and to influence binocular balance. The main goal of our study is to assess the effect of transcranial magnetic stimulation, specifically theta burst stimulation (TBS), in a group of amblyopic volunteers measuring several visual parameters: visual acuity, suppressive imbalance, and stereoacuity. METHODS: Thirteen volunteers aged 19 to 24 years, randomly split in 2 groups, underwent 1 session of continuous TBS, stimulating the right occipital lobe. The first group with 8 volunteers was exposed to active stimulation with cTBS, and the other group with 5 volunteers was exposed to placebo stimulation. RESULTS: Significant improvements in visual acuity, suppressive imbalance, and stereoacuity were found in the amblyopic eye after cTBS. The average value of amblyopia in visual acuity before stimulation was 0.32 ± 0.20 logMar and after cTBS was 0.19 ± 0.17 logMar. The mean value for the control group before placebo stimulation was 0.28 ± 0.17 and after placebo stimulation was 0.28 ± 0.16. The suppressive imbalance in the group of amblyope subjects stimulated before cTBS was 0.26 ± 0.18 and after was 0.12 ± 0.12; the suppressive imbalance of the control group before the placebo stimulation was 0.34 ± 0.37 and after was 0.32 ± 0.40. CONCLUSIONS: Visual acuity, suppressive imbalance, and stereoacuity had significant enhancements compared with baseline after cTBS over the right occipital lobe in an ambliopic population.

Healthcare professionals' assumptions as barriers to LGBTI healthcare.

Lesbian, gay, bisexual, trans and intersex (LGBTI) people experience significant healthcare inequalities and barriers to healthcare services. Contextualised within six Member States of the European Union (EU), this paper discusses efforts to identify and explore the nature of barriers to healthcare as part of Health4LGBTI, a 2-year pilot project funded by the EU. Data were generated through focus groups and interviews with LGBTI people and healthcare professionals and analysed using thematic analysis. Findings reveal that barriers to healthcare are underpinned by two related assumptions held by healthcare professionals: first, the assumption that patients are heterosexual, cisgender and non-intersex by default; second, the assumption that LGBTI people do not experience significant problems (and therefore that their experience is mostly irrelevant to healthcare). On the other hand, it is notable that responding healthcare professionals were broadly 'LGBTI-friendly'. Thus, we argue that efforts to improve LGBTI healthcare should not be limited to engaging with healthcare professionals with negative views of LGBTI people. Rather, such efforts should also tackle these assumptions amongst LGBTI-friendly healthcare professionals.

Co-producing knowledge of lesbian, gay, bisexual, trans and intersex (LGBTI) health-care inequalities via rapid reviews of grey literature in 27 EU Member States.

BACKGROUND: The health inequalities experienced by lesbian, gay, bisexual, trans and intersex (LGBTI) people are well documented with several reviews of global research summarizing key inequalities. These reviews also show how the health-care needs of LGBTI people are often poorly understood whilst suggesting that targeted initiatives to reduce inequalities should involve LGBTI people. OBJECTIVES: To determine what is known about the health-care inequalities faced by LGBTI people? What are the barriers faced by LGBTI people whilst accessing health care, and health professionals when providing care? What examples of promising practice exist? DESIGN: Rapid reviews of grey literature were co-produced with LGBTI people in 27 countries followed by a thematic analysis and synthesis across all data sets. The review included grey literature from each country that might not otherwise be accessible due to language barriers. MAIN RESULTS: Rapid reviews showed that LGBTI people faced various inequalities and barriers whilst accessing health care. Where heterosexuality, binary gender and assumed male/female sex characteristics were upheld as the norm, and where LGBTI people differed from these norms, discrimination could result. In consultations where LGBTI people feared discrimination and did not disclose their LGBTI status, health professionals lacked the information required for appropriate assessments. CONCLUSION: With greater understanding of sexual orientation (LGB people), gender identity (trans people) and sex characteristics (intersex people), combined with access to contemporary knowledge and training, health professionals can work in collaboration with researchers, policymakers and LGBTI people to develop systems that are better attuned to the needs of all service users.

A review of lesbian, gay, bisexual, trans and intersex (LGBTI) health and healthcare inequalities.

BACKGROUND: Lesbian, gay, bisexual, trans and intersex (LGBTI) people experience significant health inequalities. Located within a European Commission funded pilot project, this paper presents a review of the health inequalities faced by LGBTI people and the barriers health professionals encounter when providing care. METHODS: A narrative synthesis of 57 papers including systematic reviews, narrative reviews, meta-analyses and primary research. Literature was searched in Cochrane, Campbell Collaboration, Web of Science, CINAHL, PsychINFO and Medline. The review was undertaken to promote understanding of the causes and range of inequalities, as well as how to reduce inequalities. RESULTS: LGBTI people are more likely to experience health inequalities due to heteronormativity or heterosexism, minority stress, experiences of victimization and discrimination, compounded by stigma. Inequalities pertaining to LGBTI health(care) vary depending on gender, age, income and disability as well as between LGBTI groupings. Gaps in the literature remain around how these factors intersect to influence health, with further large-scale research needed particularly regarding trans and intersex people. CONCLUSION: Health inequalities can be addressed via changes in policy, research and in practice through health services that accommodate the needs of LGBTI people. With improved training to address gaps in their knowledge of LGBTI health and healthcare, health professionals should work in collaboration with LGBTI people to address a range of barriers that prevent access to care. Through structural change combined with increased knowledge and understanding, services can potentially become more inclusive and equally accessible to all.

On-Line Ion Exchange Liquid Chromatography as a Process Analytical Technology for Monoclonal Antibody Characterization in Continuous Bioprocessing.

Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co-elution of impurities.

Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc.