Meeting report on protein particles and immunogenicity of therapeutic proteins: Filling in the gaps in risk evaluation and mitigation

Carpenter, John F.; Cherney, Barry; Lubinecki, Anthony; Ma, Stacey; Marszal, Ewa; Mire‐Sluis, Anthony R.; Nikolai, Thomas J.; Novak, Jeanne M.; Ragheb, Jack A.; Šimák, Jan

doi:10.1016/j.biologicals.2010.07.002

Cited by 62 publications

(47 citation statements)

References 7 publications

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“…7(b2)). Further dilution decreased the observed concentration to 3.8×10 6 particles/mL with a mean of thermal treatment; a1-undiluted sample, a2-10-fold dilution. After 8 h thermal treatment; b1-undiluted sample, b2-10-fold dilution, b3-100-fold dilution.…”

Section: Rmmmentioning

confidence: 93%

Analyzing Subvisible Particles in Protein Drug Products: a Comparison of Dynamic Light Scattering (DLS) and Resonant Mass Measurement (RMM)

Panchal

Kotarek

Marszal

et al. 2014

AAPS J

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Abstract. Aggregation is common in protein drug manufacture, and while the effects of protein particulates are under investigation, many techniques applicable for their characterization have been recently developed. Among the methods available to characterize and quantify protein aggregates, none is applicable over the full size range and different methods often give conflicting results. The studies presented here compare two such methods: dynamic light scattering (DLS) and resonant mass measurement (RMM). The performance of each method was first characterized using polystyrene particle size standards (20, 60, 100, 200, 400, and 1,000 nm) over a range of concentrations. Standard particles were measured both singly and in binary mixtures containing 20 nm particles at a fixed concentration (10 14 particles/mL) and various concentrations of one of the other particle sizes (i.e., 60, 100, 200, 400, or 1,000 nm). DLS and RMM were then used to detect unknown aggregate content in stressed samples of IgG. Both instruments were shown to have a working range that depends on particle size and concentration. In binary mixtures and polydisperse solutions, DLS was able to resolve two species in a manner dependent on both concentration and particle size. RMM was able to resolve particles above 200 nm (150 nm for protein) at concentrations below 10 9 particles/mL. In addition, dilution was evaluated as a technique to confirm and quantify the number of particles in solution.

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Section: Rmmmentioning

confidence: 93%

Analyzing Subvisible Particles in Protein Drug Products: a Comparison of Dynamic Light Scattering (DLS) and Resonant Mass Measurement (RMM)

Panchal

Kotarek

Marszal

et al. 2014

AAPS J

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“…Protein aggregates and particles are important quality attributes of therapeutic protein formulations (1)(2)(3). Especially micron-sized aggregates (subvisible protein particles) (4) are considered as critical due to their potential risk of enhancing an immunogenic response (5).…”

Section: Introductionmentioning

confidence: 99%

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Zölls

Weinbuch

Wiggenhorn³

et al. 2013

AAPS J

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Abstract. Flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. The aim of the present study was a comparative evaluation of four of the most relevant flow imaging microscopy systems for biopharmaceuticals on the market: Micro-Flow Imaging (MFI)4100, MFI5200, Flow Cytometer And Microscope (FlowCAM) VS1, and FlowCAM PV. Polystyrene standards, particles generated from therapeutic monoclonal antibodies, and silicone oil droplets were analyzed by all systems. The performance was critically assessed regarding quantification, characterization, image quality, differentiation of protein particles and silicone oil droplets, and handling of the systems. The FlowCAM systems, especially the FlowCAM VS1, showed high-resolution images. The FlowCAM PV system provided the most precise quantification of particles of therapeutic monoclonal antibodies, also under impaired optical conditions by an increased refractive index of the formulation. Furthermore, the most accurate differentiation of protein particles and silicone oil droplets could be achieved with this instrument. The MFI systems provided excellent size and count accuracy (evaluated with polystyrene standards) especially the MFI5200 system. This instrument also showed very good performance for protein particles, also in case of an increased refractive index of the formulation. Both MFI systems were easier to use and appeared more standardized regarding measurement and data analysis as compared to the FlowCAM systems. Our study shows that the selection of the appropriate flow imaging microscopy system depends strongly on the main output parameters of interest and it is recommended to decide based on the intended application.

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“…2,[12][13][14] Because a serious concern has recently been raised that visible and subvisible particles, in addition to soluble aggregates, may potentially induce immunogenicity [15][16][17] elucidating the mechanism of aggregate formation is becoming increasingly important.…”

Section: 7)mentioning

confidence: 99%

“…2,[8][9][10][11] Aggregate formation varies depending on cell line characteristics, physical properties of mAbs, medium compositions, and culture conditions. 2,[12][13][14] Because a serious concern has recently been raised that visible and subvisible particles, in addition to soluble aggregates, may potentially induce immunogenicity [15][16][17] elucidating the mechanism of aggregate formation is becoming increasingly important.…”

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confidence: 99%

Comparison of Antibody Molecules Produced from Two Cell Lines with Contrasting Productivities and Aggregate Contents

Ishii

Imamoto

Yamamoto

et al. 2015

Biological & Pharmaceutical Bulletin

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Cell culture processes that produce therapeutic antibodies with high productivity (titer) and low aggregate content reduce the risk of adverse effects and expense to patients. To elucidate the mechanism of aggregate formation, we compared trastuzumab samples produced from two contrasting cell lines: cell line A, which exhibits high titer and low aggregate content, and cell line B, which exhibits low titer and high aggregate content. Cell line B produced significantly fewer (approximately 1/3) antibodies compared with cell line A and contained higher (approximately 3-fold) percentages of aggregates. The aggregates of antibodies found in the protein A-purified samples of cell line B were associated mostly with noncovalent interactions. Cell line B exhibited a low content of monomers/dimers of light chains in the medium and within cells. Because light chains are essential for the correct folding of heavy chains and secretion of mature antibodies, the characteristics of cell line B may be attributed to low levels of light chain production. In addition, protein A-purified antibodies from cell line B (but not those from cell line A) contained fragments that are expected to expose the hydrophobic C H 3 domain, which may serve as nuclei for aggregation.Key words antibody aggregate; light chain production; cell line; antibody fragment; covalent aggregate; noncovalent aggregate Therapeutic monoclonal antibodies (mAbs) have been widely used because of their high antigenic specificity, long serum half-life, and low incidence of undesirable side effects. The global market for therapeutic mAbs is growing annually. 1)Because large doses of therapeutic mAbs are usually required to achieve clinical success, mAb production processes with high productivity (titer) are required to reduce the expense to patients. The quality of therapeutic mAbs expressed is also critical because mAbs produced from Chinese hamster ovary (CHO) cells are heterogeneous, owing to aggregation 2,3) and various post-translational modifications. 4,5) This aggregation and modification of antibodies may lead to a reduction in biological activity and/or an increase in undesirable side effects under certain circumstances. The content of aggregated species is one of the most critical sources of heterogeneity because protein aggregates may induce immunogenic responses and result in adverse events following administration. 6,7)Therefore, the aggregate content of mAbs must be precisely controlled. Because protein aggregates are heterogeneous, they are classified on the basis of characteristics such as size, solubility, covalence, reversibility, structure (native/denatured), and formation mechanisms.2,8-11) Aggregate formation varies depending on cell line characteristics, physical properties of mAbs, medium compositions, and culture conditions. 2,[12][13][14] Because a serious concern has recently been raised that visible and subvisible particles, in addition to soluble aggregates, may potentially induce immunogenicity [15][16][17] elucidating the mechanism of aggre...

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Meeting report on protein particles and immunogenicity of therapeutic proteins: Filling in the gaps in risk evaluation and mitigation

Cited by 62 publications

References 7 publications

Analyzing Subvisible Particles in Protein Drug Products: a Comparison of Dynamic Light Scattering (DLS) and Resonant Mass Measurement (RMM)

Analyzing Subvisible Particles in Protein Drug Products: a Comparison of Dynamic Light Scattering (DLS) and Resonant Mass Measurement (RMM)

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Comparison of Antibody Molecules Produced from Two Cell Lines with Contrasting Productivities and Aggregate Contents

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