Monoclonal Antibody Formulations Research Articles

Coadsorption of a Monoclonal Antibody and Nonionic Surfactant at the SiO2/Water Interface.

During the formulation of therapeutic monoclonal antibodies (mAbs), nonionic surfactants are commonly added to attenuate structural rearrangement caused by adsorption/desorption at interfaces during processing, shipping, and storage. We examined the adsorption of a mAb (COE-3) at the SiO2/water interface in the presence of pentaethylene glycol monododecyl ether (C12E5), polysorbate 80 (PS80-20EO), and a polysorbate 80 analogue with seven ethoxylates (PS80-7EO). Spectroscopic ellipsometry was used to follow COE-3 dynamic adsorption, and neutron reflection was used to determine interfacial structure and composition. Neither PS80-20EO nor C12E5 had a notable affinity for COE-3 or the interface under the conditions studied and thus did not prevent COE-3 adsorption. In contrast, PS80-7EO did coadsorb but did not influence the dynamic process or the equilibrated amount of absorbed COE-3. Near equilibration, COE-3 underwent structural rearrangement and PS80-7EO started to bind the COE-3 interfacial layer and subsequently formed a well-defined surfactant bilayer via self-assembly. The resultant interfacial layer comprised an inner mAb layer of about 70 Å thickness and an outer surfactant layer of a further 70 Å, with distinct transitional regions across the mAb-surfactant and surfactant-bulk water boundaries. Once formed, such interfacial layers were very robust and worked to prevent further mAb adsorption, desorption, and structural rearrangement. Such robust interfacial layers could be anticipated to exist for formulated mAbs stored in type II glass vials; further research is required to understand the behavior of these layers for siliconized glass syringes.

Edetate Disodium as a Polysorbate Degradation and Monoclonal Antibody Oxidation Stabilizer

Polysorbates are frequently used in biotherapeutic formulations. Interest in assessing their stability, in particular the impact of their degradation products on the stability of therapeutic proteins, has been steadily growing in the past decade. The work presented summarizes a case study of a monoclonal antibody formulation that demonstrated a simultaneous loss of polysorbate and an increase in methionine oxidation. Spiking studies were conducted to determine both the cause and a potential mitigation for the monoclonal antibody (mAb) oxidation and polysorbate 80 (PS80) loss. The results indicated that a different source material exhibited different rates of mAb oxidation and PS80 loss and that in all evaluated materials, the addition of edetate disodium to the formulation mitigated both observed issues. The mAb was assessed for the presence of lipases and lipoprotein lipase was detected at low levels. It is proposed that edetate disodium was effective in mitigating the mAb oxidation and PS80 loss by chelating calcium in the formulation and therefore decreasing the activity of the lipases.

Determination of residual poly diallyldimethylammonium chloride (pDADMAC) in monoclonal antibody formulations by size exclusion chromatography and evaporative light scattering detector

The cationic polyelectrolyte pDADMAC is widely used in biopharmaceutical industry as a flocculating agent to enhance clarification throughput and downstream filtration operations. Due to the possible toxicity, pDADMAC should be assessed for an acceptable residual level to ascertain the safety of the product to patients. The strong protein-polyelectrolyte interaction, however, can negatively affect sensitivity and accuracy of measurements. This paper reports on the application of size exclusion (SE) chromatography coupled to evaporative light scattering detector (ELSD) to the quantitative determination of pDADMAC in monoclonal antibody formulations and in process intermediates during downstream purification. The SE chromatography was performed under isocratic condition with a mobile phase consisting of 0.1% TFA in water (90%) and acetonitrile (10%) at a flow rate of 0.4 ml/min. A quantification limit (S/N = 10) of 0.85 ppm was achieved in sample matrix, which is sufficiently low for the trace analysis of this compound in protein-containing samples.

CFD Supported Investigation of Shear Induced by Bottom-Mounted Magnetic Stirrer in Monoclonal Antibody Formulation.

Biological pharmaceutical unit operations like homogenization or pooling of liquids are often performed in stirred vessels. Bottom-mounted magnetic stirrers are usually the system of choice in drug product manufacturing, because bottom-mounted magnetic stirrers are considered to be gentle mixing systems. Nevertheless, magnetic stirrers can cause shear stress and, thus, lead to protein damage. This study uses computational fluid dynamics (CFD), because flow and shear rates cannot easily be measured at the spot of interest. The investigation utilizes CFD models, which were checked for plausibility by comparing experimental results and model outcome. The investigators first modeled macroscopic flow across a range of vessel volume capacities. Subsequently, detailed models focusing on two locations (bearing gap (2mm - 3.5mm) and spigot gap (40μm - 80μm)) were developed. The macroscopic flow modeling showed that the direction of flow varies based on the vessel volume capacity. The detailed CFD model estimated significant flow through the bearing gap. However, the calculated shear rates in the bearing gap were always lower than the shear rates which occur directly next to the impeller tip. The CFD model calculated significantly higher shear rates in the spigot gap and flow in the lower microliter range. Shear rates at the impeller tip are typically used as parameter to characterize stirred mixing systems. Although higher shear rates were found in the spigot gap, these higher shear rates can most likely be neglected for most applications due to non-significant flow through the spigot gap.

A New Approach to Study the Physical Stability of Monoclonal Antibody Formulations—Dilution From a Denaturant

The early-stage assessment of the physical stability of new monoclonal antibodies in different formulations is often based on high-throughput techniques that suffer from various drawbacks. Accordingly, new approaches that facilitate the protein formulation development can be of high value to the industry. In this study, a dynamic light scattering plate reader is used to measure the aggregation (by means of the increase in the hydrodynamic radius [Rh]) of monoclonal antibody samples that were subject to incubation and subsequent dilution from different concentrations of a denaturing agent, that is, guanidine hydrochloride. The increase in the Rh of the protein samples is dependent not only on the denaturant concentration used but also on the buffer in which the incubation/dilution was performed. We also compare the aggregation after dilution from a denaturant with other high-throughput stability-indicating methods and find good agreement between the techniques. The proposed approach to probe the physical stability of monoclonal antibodies in different formulation conditions offers a unique combination of features—it is isothermal, probes both the resistance to denaturant-induced unfolding and the colloidal protein stability, it is entirely label-free, does not rely on complex data evaluation, and requires very short instrument measurement time on standard equipment.

Development of a Hydrodynamic Cleaning Cycle for Ultrafiltration/Diafiltration Processes Used for Monoclonal Antibody Formulation

Ultrafiltration/diafiltration (UFDF) processes are used for final formulation of high value biotherapeutics like monoclonal antibodies. Membrane fouling and cleaning are both critical factors governing the performance of these processes, but there is very limited data on either of these phenomena, particularly at the high monoclonal antibody concentrations used in current UFDF systems. The objective of this study was to compare the effectiveness of different cleaning regimens for composite regenerated cellulose membranes in Pellicon 3 tangential flow filtration cassettes after UFDF of a monoclonal antibody product. The water permeability after UFDF was 20 ± 5% smaller than the clean membrane permeability due to fouling. Membranes could be successfully cleaned with 0.5 N NaOH, but this caused an increase in permeability and a loss in solute rejection due to an opening of the membrane pores. Cleaning with DI water was not very effective under normal flow conditions but provided very good recovery of water p...

Significant Drying Time Reduction Using Microwave-Assisted Freeze-Drying for a Monoclonal Antibody

Microwave-assisted freeze-drying (MFD) is a rapid drying process well known in food technology. However, little is known about its application to biologicals. In this study, we investigated the applicability and feasibility of this technology to different monoclonal antibody formulations and the influence on the resulting product properties. Moreover, one of our main objectives was to study if significant reductions in drying times could be achieved. In addition, the effect of the drying process on the accelerated stability of a sucrose-based antibody formulation at 40°C and 25°C over 12 weeks was investigated. MFD resulted in drying time reduction >75%. For all model formulations, cake appearance and solid state properties were found to be comparable to standard lyophilized products. These formulations covered a wider range of lyophilization excipients comprising sucrose and trehalose, semi-crystalline forming solids like mannitol:sucrose mixtures and others like arginine phosphate and a mixture of 2-hydroxypropyl-β-cyclodextrin with sucrose. Moreover, comparable low changes in relative monomer content, the relative amount of soluble aggregates and cumulative particles ≥1 μm per mL were observed over 12 weeks of storage, regardless of the drying technology. This makes MFD a promising innovative alternative for the rapid production of freeze-dried biologicals while maintaining product quality.

Does controlled nucleation impact the properties and stability of lyophilized monoclonal antibody formulations?

This study provides the first systematic investigation of the impact of the nucleation protocol during freeze-drying on physico-chemical properties and long-term stability of two IgG1 antibodies in sugar formulations. We hypothesized that the lower specific surface area (SSA) generated by controlled nucleation could be beneficial for the stability of interface sensitive proteins. The study compares controlled nucleated (CN) and randomly nucleated (RN) lyophilizates with high and low antibody concentrations stored at different temperatures. Formulations with and without polysorbate (PS) were included.In the “high concentration” study the formulation without PS showed reduced particle formation for CN samples compared to RN samples. PS containing formulations had an overall lower particle level with no further advantage of CN on stability. Besides the intended comparison of CN and RN samples, we observed that PS promoted sucrose crystallization in both low concentration antibody studies during storage. Additionally, our results indicate that the nucleation temperature (TN) was not the only determining factor for the resulting ice crystal size and consequently the product`s SSA.Overall, the application of CN had neither a positive nor a negative impact on the product’s physico-chemical stability. The surfactant had a much higher stabilizing effect than the reduction of the SSA by CN.

Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry: Correlation of Deuterium Uptake and Long-Term Stability of Lyophilized Monoclonal Antibody Formulations.

Solid state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) has been used to assess protein conformation and matrix interactions in lyophilized solids. ssHDX-MS metrics have been previously correlated to the formation of aggregates of lyophilized myoglobin on storage. Here, ssHDX-MS was applied to lyophilized monoclonal antibody (mAb) formulations and correlated to their long-term stability. After exposing lyophilized samples to D2O(g), the amount of deuterium incorporated at various time points was determined by mass spectrometry for four different lyophilized mAb formulations. Hydrogen-deuterium exchange data were then correlated with mAb aggregation and chemical degradation, which was obtained in stability studies of >2.5 years. Deuterium uptake on ssHDX-MS of four lyophilized mAb formulations determined at the initial time point prior to storage in the dry state was directly and strongly correlated with the extent of aggregation and chemical degradation during storage. Other measures of physical and chemical properties of the solids were weakly or poorly correlated with stability. The data demonstrate, for the first time, that ssHDX-MS results are highly correlated with the stability of lyophilized mAb formulations. The findings thus suggest that ssHDX-MS can be used as an early read-out of differences in long-term stability between formulations helping to accelerate formulation screening and selection.

Enablement of the direct analysis of excipients in monoclonal antibody formulations through the incorporation of a wide pore C18 protein trap with hydrophilic interaction liquid chromatography.

Establishing and maintaining the correct formulation composition is essential for ensuring the stability of biopharmaceutical drug products. A barrier to the routine assessment of excipient concentration is the lack of convenient and robust methods for the direct analysis of solutions containing high protein concentrations. To address this need an HPLC method was developed utilizing a wide-pore C18 guard column to trap proteins in-line with a hydrophilic interaction liquid chromatographic column to separate excipients. This method allows for a simple and direct analysis of excipients such as amino acids, carboxylic acids, simple carbohydrates, and inorganic ions across multiple drug formulations and process streams containing different therapeutic antibodies. The method was successfully validated for specificity, precision, accuracy, linearity, and robustness.

Mechanistic Investigation on Grinding-Induced Subvisible Particle Formation during Mixing and Filling of Monoclonal Antibody Formulations

Processing equipment involving grinding of two solid surfaces has been demonstrated to induce subvisible particle formation in monoclonal antibody drug product manufacturing processes. This study elucidated potential stress types associated with grinding action to identify the stress mechanism responsible for subvisible particle formation. Several potential stress types can be associated with the grinding action, including interfacial stresses (air-liquid and liquid-solid), hydraulic/mechanical shear stress, cavitation, nucleation of stressed protein molecules, and localized thermal stress. More than one stress type can synergically affect monoclonal antibody product quality, making it challenging to determine the primary mode of stress. Our strategy was to assess and rule out some stress types through platform knowledge, rational judgments, or via small-scale models, for example, rheometer/rotator-stator homogenizer for hydraulic/mechanical shear stress, sonicator for cavitation, etc. These models may not provide direct evidence but can offer rational correlations. Cavitation, as demonstrated by sonication, proved to be quite detrimental to monoclonal antibody molecules in forming not just subvisible particles but also soluble high-molecular-weight species as well as low-molecular-weight species. This outcome was not consistent with that of grinding monoclonal antibodies between the impeller and the drive unit of a bottom-mounted mixer or between the piston and the housing of a rotary piston pump, both of which formed only subvisible particles without obvious high-molecular-weight species and low-molecular-weight species. In addition, a p-nitrophenol model suggested that cavitation in the bottom-mounted mixer is barely detectable. We attributed the grinding-induced, localized thermal effect to be the primary stress to subvisible particle formation based on a high-temperature, spray-drying model. The heat effect of spray drying also caused subvisible particles, in the absence of significant high-molecular-weight species and low-molecular-weight species, in spray-dried monoclonal antibody powders. This investigation provides a mechanistic understanding of the underlying stress mechanism leading to monoclonal antibody subvisible particle formation as the result of drug product processing involving grinding of solid surfaces.LAY ABSTRACT: Subvisible particles present in therapeutic protein formulations could adversely affect drug product safety and efficacy. We previously illustrated that grinding action of the solid surfaces in some bottom-mounted mixers and piston pump is responsible for subvisible particle formation of monoclonal antibody formulations. In this study, we delved into mechanistic understanding of the stress types associated with solid surface grinding. The approach was to employ several scale-down stress models with known stress types. Protein formulations stressed in these models were analytically characterized for subvisible particles and other degradants. Some commonly known stress types-such as air-liquid interface, mechanical stress, cavitation, nucleation, and thermal effect-were assessed in this study. The stress model yielding a degradation profile matching that of bottom-mounted mixers and piston pump warranted further assessment. Localized, thermal stress proved to be the most feasible mechanism. This study, along with previously published results, may further advance our understanding of these particular drug product manufacturing processes and benefit scientists and engineers in overcoming these development challenges.

Investigating Liquid-Liquid Phase Separation of a Monoclonal Antibody Using Solution-State NMR Spectroscopy: Effect of Arg·Glu and Arg·HCl.

Liquid-liquid phase separation (LLPS) of monoclonal antibody (mAb) formulations involves spontaneous separation into dense (protein-rich) and diluted (protein-lean) phases and should be avoided in the final drug product. Understanding the factors leading to LLPS and ways to predict and prevent it would therefore be highly beneficial. Here we describe the link between LLPS behavior of an IgG1 mAb (mAb5), its solubility, and parameters extracted using 1H NMR spectroscopy, for various formulations. We show that the formulations demonstrating least LLPS lead to the largest mAb5 NMR signal intensities. In the formulations exhibiting the highest propensity to phase-separate the mAb NMR signal intensities are the lowest, even at higher temperatures without visible phase separation, suggesting a high degree of self-association prior to distinct phase separation. Addition of arginine glutamate prevented LLPS and led to a significant increase in the observed mAb signal intensity, whereas the effect of arginine hydrochloride was only marginal. Solution NMR spectroscopy was further used to characterize the protein-lean and protein-rich phases separately and demonstrated that protein self-association in the protein-rich phase can be significantly reduced by arginine glutamate. Solution NMR spectroscopy may be useful as a tool to assess the propensity of mAb solutions to phase-separate.

A Formulation Development Approach to Identify and Select Stable Ultra–High-Concentration Monoclonal Antibody Formulations With Reduced Viscosities

High protein concentration formulations are required for low-volume administration of therapeutic antibodies targeted for subcutaneous, self-administration by patients. Ultra-high concentrations (≥150 mg/mL) can lead to dramatically increased solution viscosities, which in turn can lead to stability, manufacturing, and delivery challenges. In this study, various categories and individual types of pharmaceutical excipients and other additives (56 in total) were screened for their viscosity reducing effects on 2 different mAbs. The physicochemical stability profile, as well as viscosity ranges, of several candidate antibody formulations, identified and designed based on the results of the excipient screening, were evaluated over a 6-month time period under accelerated and real-time storage conditions. In addition to reducing the solution viscosities to acceptable levels for parenteral administration (using currently available and acceptable delivery devices), the candidate formulations did not result in notable losses of physicochemical stability of the 2 antibodies on storage for 6 months at 25°C. The experiments described here demonstrate the feasibility of a formulation development and selection approach to identify candidate high-concentration antibody formulations with viscosities within pharmaceutically acceptable ranges that do not adversely affect their physicochemical storage stability.

Multi-criteria manufacturability indices for ranking high-concentration monoclonal antibody formulations.

ABSTRACTThe need for high‐concentration formulations for subcutaneous delivery of therapeutic monoclonal antibodies (mAbs) can present manufacturability challenges for the final ultrafiltration/diafiltration (UF/DF) step. Viscosity levels and the propensity to aggregate are key considerations for high‐concentration formulations. This work presents novel frameworks for deriving a set of manufacturability indices related to viscosity and thermostability to rank high‐concentration mAb formulation conditions in terms of their ease of manufacture. This is illustrated by analyzing published high‐throughput biophysical screening data that explores the influence of different formulation conditions (pH, ions, and excipients) on the solution viscosity and product thermostability. A decision tree classification method, CART (Classification and Regression Tree) is used to identify the critical formulation conditions that influence the viscosity and thermostability. In this work, three different multi‐criteria data analysis frameworks were investigated to derive manufacturability indices from analysis of the stress maps and the process conditions experienced in the final UF/DF step. Polynomial regression techniques were used to transform the experimental data into a set of stress maps that show viscosity and thermostability as functions of the formulation conditions. A mathematical filtrate flux model was used to capture the time profiles of protein concentration and flux decay behavior during UF/DF. Multi‐criteria decision‐making analysis was used to identify the optimal formulation conditions that minimize the potential for both viscosity and aggregation issues during UF/DF. Biotechnol. Bioeng. 2017;114: 2043–2056. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Perodicals, Inc.

Dipicolinic acid as a novel spore-inspired excipient for antibody formulation

Ionic excipients are commonly used in aqueous therapeutic monoclonal antibody (mAb) formulations. Novel excipients are of industrial interest, with a recent focus on Arg salt forms and their application as viscosity reducing and stabilizing additives. Here, we report that the calcium salt of dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid), uniquely present in nature in the core of certain bacterial spores, reduces the viscosity of a mAb formulated at 150mg/mL, below that achieved by Arg hydrochloride at the same concentration (10mM). DPA also reduced the reversible phase separation of the same formulation, which characteristically occurs for this mAb upon cooling to 4°C. Differential scanning calorimetry and differential scanning fluorimetry did not reveal a conformation destabilisation of the mAb in the presence of 10mM DPA, or by the related quinolinic acid (QA, pyridine-2,3-dicarboxylic acid). However, fluorescence spectrophotometry did reveal localised (aromatic) conformational changes to the mAb attributed to DPA, dependent on the salt form. While precise mechanisms of action remain to be identified, our preliminary data suggest that these DPA salts are worthy of further investigation as novel ionic excipient for biologics formulation.

Lyophilization Cycle Design for Dual Chamber Cartridges and a Method for Online Process Control: The “DCC LyoMate” Procedure

Freeze-drying process design is a challenging task that necessitates a profound understanding of the complex interrelation among critical process parameters (e.g., shelf temperature and chamber pressure), heat transfer characteristics of the involved materials (e.g., product containers and holder devices), and critical quality attributes of the product (e.g., collapse temperatures). The Dual Chamber Cartridge “(DCC) LyoMate” (from lyophilization and automated) is a manometric temperature measurement–based process control strategy that was developed within this study to streamline this complicated task. It was successfully applied using 5% sucrose formulations with 0.5 and 1 mL fill volumes. The system was further challenged using 2, 20, and 100 mg/mL monoclonal antibody formulations. The DCC LyoMate method did not only produce pharmaceutically acceptable cakes but was also able to maintain the desired product temperature irrespective of formulation and protein content. It enabled successful process design even at high protein concentrations and aided the design and online control of the lyophilization process for drying in DCCs within a single development run. Thus, it helps to reduce development cost and the DCC LyoMate can also be easily installed on every freeze-dryer capable of performing a manometric temperature measurement, without the need for hardware modification.

Utility of High Throughput Screening Techniques to Predict Stability of Monoclonal Antibody Formulations During Early Stage Development

Selecting optimal formulation conditions for monoclonal antibodies for first time in human clinical trials is challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long-term stability. Accelerated stability studies are considered to be the gold standard for excipient screening, but they are relatively low throughput and time consuming. High throughput screening (HTS) techniques allow for large amounts of data to be collected quickly and easily, and can be used to screen solution conditions for early formulation development. The utility of using accelerated stability compared to HTS techniques (differential scanning light scattering and differential scanning fluorescence) for early formulation screening was evaluated along with the impact of excipients of various types on aggregation of monoclonal antibodies from multiple IgG subtypes. The excipient rank order using quantitative HTS measures was found to correlate with accelerated stability aggregation rate ranking for only 33% (by differential scanning fluorescence) to 42% (by differential scanning light scattering) of the antibodies tested, due to the high intrinsic stability and minimal impact of excipients on aggregation rates and HTS data. Also explored was a case study of employing a platform formulation instead of broader formulation screening for early formulation development.

Evaluation of aggregate and silicone-oil counts in pre-filled siliconized syringes: An orthogonal study characterising the entire subvisible size range

Characterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routinely extended to the sub-visible size-range (0.1–10μm). Additionally, with the increased use of pre-filled syringes (PFS), particle differentiation is required between proteinaceous and non-proteinaceous particles such as silicone-oil droplets. Here, three orthogonal techniques: Raster Image Correlation Spectroscopy (RICS), Resonance Mass Measurements (RMM) and Micro-Flow Imaging (MFI), were evaluated with respect to their sub-visible particle measurement and characterisation capabilities. Particle formation in mAb PFS solutions was evaluated with increasing polysorbate-20 (PS-20) concentrations. All three techniques provided complementary but distinct information on protein aggregate and silicone-oil droplet presence. PS-20 limited the generation of mAb aggregates during agitation, while increasing the number of silicone-oil droplets (PS-20 concentration dependant). MFI and RMM revealed PS-20 lead to the formation of larger micron-sized droplets, with RICS revealing an increase in smaller sub-micron droplets. Subtle differences in data sets complicate the apparent correlation between silicone-oil sloughing and mAb aggregates’ generation. RICS (though the use of a specific dye) demonstrates an improved selectivity for mAb aggregates, a broader measurement size-range and smaller sample volume requirement. Thus, RICS is proposed to add value to the currently available particle measurement techniques and enable informed decisions during mAb formulation development.

Effects of Histidine and Sucrose on the Biophysical Properties of a Monoclonal Antibody.

Histidine is a commonly used buffer in formulation of monoclonal antibodies (mAb), often with excipients like sucrose. The objective of this study was to examine the effects of both histidine and sucrose on the biophysical characteristics of a mAb. The hydrodynamic radius of the mAb was determined by dynamic light scattering and confirmed by size exclusion chromatography. Data were also obtained for the osmotic virial coefficients (from osmotic pressure measurements), the solution viscosity, and the mAb thermal stability (using differential scanning calorimetry) at selected conditions. There were no significant changes in mAb conformation / stability as determined by DSC. The hydrodynamic radius initially increased with increasing histidine concentration, going through a maximum at a histidine concentration of about 20mM. The addition of sucrose increased the mAb hydrodynamic radius at all histidine concentrations by about 0.5nm. The observed effects of histidine and sucrose on the hydrodynamic radius were also reflected in changes in the osmotic pressure and solution viscosity. These results provide important insights into the effects of both histidine and sucrose on the behavior of concentrated mAb solutions, including the potential impact on ultrafiltration / diafiltration processes.

Knockout of a difficult-to-remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations.

While the majority of host cell protein (HCP) impurities are effectively removed in typical downstream purification processes, a small population of HCPs are particularly challenging. Previous studies have identified HCPs that are challenging for a variety of reasons. Lipoprotein lipase (LPL)-a Chinese hamster ovary (CHO) HCP that functions to hydrolyze esters in triglycerides-was one of ten HCPs identified in previous studies as being susceptible to retention in downstream processing. LPL may degrade polysorbate 80 (PS-80) and polysorbate 20 (PS-20) in final product formulations due to the structural similarity between polysorbates and triglycerides. In this work, recombinant LPL was found to have enzymatic activity against PS-80 and PS-20 in a range of solution conditions that are typical of mAb formulations. LPL knockout CHO cells were created with CRISPR and TALEN technologies and resulting cell culture harvest fluid demonstrated significantly reduced polysorbate degradation without significant impact on cell viability when compared to wild-type samples. Biotechnol. Bioeng. 2017;114: 1006-1015. © 2016 Wiley Periodicals, Inc.