Assessment of chemical modifications of sites in the CDRs of recombinant antibodies

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.     

Low pKa of Lys promotes glycation at one complementarity-determining region of a bispecific antibody

Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. Cation exchange chromatography and liquid chromatography-mass spectrometry were used to characterize glycation at a lysine residue within a heavy chain (HC) CDR (HC-CDR3-Lys98) of a bispecific antibody. Thermodynamic analysis revealed that this reaction is reversible and can occur under physiological conditions with an apparent affinity of 8–10 mM for a glucose binding to HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5°C–45°C and can be well predicted in vitro and in a non-human primate. In addition, this glycation reaction was found to be driven by an unusually low pK_a on the ε-amino group of HC-CDR3-Lys98. Van't Hoff analysis and homology modeling suggested that this reaction is enthalpically driven, with this lysine residue surrounded by a microenvironment with low polarity. This study provides, to our knowledge, new insights toward a mechanistic understanding of protein glycation and strategies to mitigate the impact of protein glycation during pharmaceutical development.     

Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.     

Symmetry of Fv architecture is conducive to grafting a second antibody binding site in the Fv region.

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single-chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovered an architectural basis common to many Fv regions that permits grafting a chi-site onto the Fv surface that diametrically opposes the normal combining site. By using molecular graphics analysis, chimeric complementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an overall geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions for inserting chi CDRs are very highly conserved, as are the corresponding chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply this approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins.     

A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.     

A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.     

从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础     

Functional assessment of antibody oxidation by native mass spectrometry

Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (huFcRn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultra-performance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/huFcRn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis.

In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mAb3/huFcRn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to huFcRn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics.     

A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior

The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.     

A novel TNFalpha antagonizing peptide-Fc fusion protein designed based on CDRs of TNFalpha neutralizing monoclonal antibody

The variable regions of antibody molecules bind antigens with high affinity and specificity. The binding sites are imparted largely to the hypervariable portions (i.e., CDRs) of the variable region. Peptides derived from CDRs can bind antigen with similar specificity acting as mimic of antibody and become drug-designing core, although with markedly lower affinity. In order to increase the affinity and bioactivity, in this study, a novel peptide (PT) designed on CDRs of a TNFalpha neutralizing monoclonal antibody Z12 was linked with Fc fragment of human IgG1. The interaction mode of PT-linker-Fc (PLF) with TNFalpha was analyzed with computer-guided molecular modeling method. After expression in Escherichia coli and purification, recombinant PT-linker-Fc could bind directly with the TNFalpha coated on the ELISA plates. Furthermore, PLF could competitively inhibit the binding of Z12 to TNFalpha and also inhibit the TNFalpha-induced cytotoxicity on L929 cells. The TNFalpha antagonizing activity of PLF was significantly higher than that of the free peptide. This study highlights the potential of human Fc to enhance the potency of peptides designed on the CDRs of antibodies and could be useful in developing new TNFalpha antagonists.     

DnaK expression in response to heat shock of Streptococcus mutans

Abstract The oral pathogen, Streptococcus mutans , persistently colonizes human hosts and initiates oral disease despite extreme variations in environmental conditions. To begin to investigate the role of the stress protein, DnaK (Hsp70), in environmental stress responses by S. mutans , pulse—chase experiments were initially used to establish that a functional heat shock response existed in this organism. A C-terminal fragment of the S. mutans dnaK gene was cloned and engineered to be expressed with a histidine tag. Using the recombinant DnaK protein that had been purified by nickel affinity chromatography, an antibody specific for the S. mutans DnaK protein was generated to analyse DnaK expression under homeostatic and heat shock conditions. Western blot analysis indicated that the anti-recombinant DnaK antibody specifically recognized a protein (molecular mass approx. 68 kDa) which was induced in response to thermal stress. Elucidating the role of DnaK in responses by S. mutans to various environmental Stressors will provide a better understanding of how DnaK is involved in survival of extreme environments and the contribution of the DnaK protein to the virulence of S. mutans .     

Identification of potential sites for tryptophan oxidation in recombinant antibodies using tert-butylhydroperoxide and quantitative LC-MS

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.     

Identification and characterization of major flavonoids and caffeoylquinic acids in three Compositae plants by LC/DAD-APCI/MS

In this study, a liquid chromatography/diode array detector-atmospheric pressure chemical ionization/mass spectrometry (LC/DAD-APCI/MS) was successfully developed to identify and characterize the main flavonoids and caffeoylquinic acids (CQAs) of three common Compositae plants (Chrysanthemum morifolium Raman, Artemisia annua, and Chrysanthemum coronarium) which have been used as herbal medicine. Identifications were performed by comparing the retention time, UV and mass spectra of samples with standards or/and earlier publications. The crude methanolic extracts of these plants were assayed directly using LC/MS without any further pretreatment. The proposed method is rapid and reproducible and is useful for characterization and evaluation of different plant flavonoids and CQAs. A total of 41 different flavonoids and 6 CQAs were identified and confirmed by APCI-MS. The main components of three Compositae plants were also compared. Although there exist some similarities in the flavonoidic content of the leaf and flower of C. morifolium, significant variations in their varieties and concentrations were observed. Artemisia annua processes substantial amount of alkylated derivatives of flavones and Chrysanthemum coronarium contains only CQAs. These findings suggest that although all the plants studied are from the same Compositae family, their flavonoids and phenolic compositions are markedly different. The proposed method is useful for further chromatographic fingerprinting of plant flavonoids.     

Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation.

A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.     

Controlling glycation of recombinant antibody in fed-batch cell cultures

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.     

Characterization of a Monoclonal Antibody Cell Culture Production Process Using a Quality by Design Approach

The goal of quality by design (QbD) in cell culture manufacturing is to develop manufacturing processes which deliver products with consistent critical quality attributes (CQAs). QbD approaches can lead to better process understanding through the use of process parameter risk ranking and statistical design of experiments (DOE). The QbD process starts with an analysis of process parameter risk with respect to CQAs and key performance indicators (KPIs). Initial DOE study designs and their factor test ranges are based on the outcomes of the process parameter risk ranking exercises. Initial DOE studies screen factors for significant influences on CQAs as well as characterize responses for process KPIs. In the case study provided here, multifactor process characterization studies using a scale-down model resulted in significant variation in charge heterogeneity of a monoclonal antibody (MAb) as measured by ion-exchange chromatography (IEC). Iterative DOE studies, using both screening and response surface designs, were used to narrow the operating parameter ranges so that charge heterogeneity could be controlled to an acceptable level. The data from the DOE studies were used to predict worst-case conditions, which were then verified by testing at those conditions. Using the approach described here, multivariate process parameter ranges were identified that yield acceptable CQA levels and that still provide operational flexibility for manufacturing.     

Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress

In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions.     

Structural consensus among antibodies defines the antigen binding site

The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ~20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification.     

Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain

Peptide display in antibody complementarity determining regions (CDRs) offers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human kappa-light chain CDR1 (kappa-CDR1) and CDR2 (kappa-CDR2), we performed alignments of 1330 kappa-light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into kappa-CDR1 and kappa-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into kappa-CDR1 and kappa-CDR2, and analyzing the expression and ability of the modified antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies.     

Raman based chemometric model development for glycation and glycosylation real time monitoring in a manufacturing scale CHO cell bioreactor process

The Quality by Design (QbD) approach to the production of therapeutic monoclonal antibodies (mAbs) emphasizes an understanding of the production process ensuring product quality is maintained throughout. Current methods for measuring critical quality attributes (CQAs) such as glycation and glycosylation are time and resource intensive, often, only tested offline once per batch process. Process analytical technology (PAT) tools such as Raman spectroscopy combined with chemometric modeling can provide real time measurements process variables and are aligned with the QbD approach. This study utilizes these tools to build partial least squares (PLS) regression models to provide real time monitoring of glycation and glycosylation profiles. In total, seven cell line specific chemometric PLS models; % mono-glycated, % non-glycated, % G0F-GlcNac, % G0, % G0F, % G1F, and % G2F were considered. PLS models were initially developed using small scale data to verify the capability of Raman to measure these CQAs effectively. Accurate PLS model predictions were observed at small scale (5 L). At manufacturing scale (2000 L) some glycosylation models showed higher error, indicating that scale may be a key consideration in glycosylation profile PLS model development. Model robustness was then considered by supplementing models with a single batch of manufacturing scale data. This data addition had a significant impact on the predictive capability of each model, with an improvement of 77.5% in the case of the G2F. The finalized models show the capability of Raman as a PAT tool to deliver real time monitoring of glycation and glycosylation profiles at manufacturing scale.