Native and Denaturing MS Protein Deconvolution for Biopharma: Monoclonal Antibodies and Antibody-Drug Conjugates to Polydisperse Membrane Proteins and Beyond.

Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early research discovery to process development. Accurate, efficient, and consistent protein MS spectral deconvolution across multiple instrument and detector platforms (time-of-flight, Orbitrap, Fourier-transform ion cyclotron resonance) is essential. When proteins are ionized during the ESI process, a distribution of consecutive multiply charged ions are observed on the m/z scale, either positive [M + nH]n+ or negative [M - nH]n- depending on the ionization polarity. The manual calculation of the neutral molecular weight (MW) of single proteins measured by ESI-MS is simple; however, algorithmic deconvolution is required for more complex protein mixtures to derive accurate MWs. Multiple deconvolution algorithms have evolved over the past two decades, all of which have their advantages and disadvantages, in terms of speed, user-input parameters (or ideally lack thereof), and whether they perform optimally on proteins analyzed under denatured or native-MS and solution conditions. Herein, we describe the utility of a parsimonious deconvolution algorithm (explaining the observed spectra with a minimum number of masses) to process a wide range of highly diverse biopharma relevant and research grade proteins and complexes (PEG-GCSF; an IgG1k; IgG1- and IgG2-biotin covalent conjugates; the membrane protein complex AqpZ; a highly polydisperse empty MSP1D1 nanodisc and the tetradecameric chaperone protein complex GroEL) analyzed under native-MS, denaturing LC-MS, and positive and negative modes of ionization, using multiple instruments and therefore multiple data formats. The implementation of a comb filter and peak sharpening option is also demonstrated to be highly effective for deconvolution of highly polydisperse and enhanced separation of a low level lysine glycation post-translational modification (+162.1 Da), partially processed heavy chain lysine residues (+128.1 Da), and loss of N-acetylglucosamine (GlcNAc; -203.1 Da).

Outcomes of Endovascular Aortic Aneurysm Repair in Kidney Transplant Recipients: Results From a National Quality Initiative.

Contrast-induced nephropathy after endovascular aortic aneurysm repair (EVAR) in kidney transplant recipients (KTRs) can have devastating consequences. The Vascular Quality Initiative (VQI) database was queried to select all KTRs who underwent EVAR between January 2003 and December 2014. Our primary outcome was renal dysfunction, defined as acute kidney injury (AKI; elevation of serum creatinine >0.5 mg/dL from baseline) or new postoperative hemodialysis requirement. Within the EVAR VQI dataset, 40 patients were KTRs (40 of 17 213, or 0.2%). Renal dysfunction occurred in five of 40 patients in the KTR group in comparison to 779 of 17 173 patients in the nontransplanted group (12.5% versus 4.5%, p < 0.01). Emergent EVAR was required in 2 (5%) patients, one of whom required dialysis after surgery and subsequently died. One-year survival after EVAR was similar in the two groups (92.9% versus 93.1%, p = 0.73). KTRs who developed renal dysfunction had significantly lower preoperative estimated glomerular filtration rates (eGFRs) (29.5 versus 54.7, p = 0.007) and a significantly higher iodine:eGFR ratio (0.78 versus 0.39, p = 0.02) despite receiving a similar volume of contrast (70.0 versus 68.8, p = 0.97). Renal dysfunction is 3 times more frequent in KTRs treated with EVAR, though overall survival did not differ between the groups. Decreased preoperative eGFR and a higher iodine:eGFR ratio are associated with postoperative renal dysfunction.

Down but not out: incidence and estimated costs to society of road casualties in Strathclyde, Scotland.

OBJECTIVES: To investigate the recent epidemiological patterns and costs of road traffic casualties (RTCs) in Strathclyde, Scotland. STUDY DESIGN: Retrospective record-linkage epidemiological study using routine data sources. METHODS: A linked police-hospital database was analysed to describe the epidemiology of RTCs from 2004 to 2009. Using UK government methodology, the costs of road casualties to the National Health Service (NHS) and society were assessed. RESULTS: RTC rates declined over the study period. Males were at higher risk than females as were those residing in more socially deprived addresses. The estimated costs of RTCs in Strathclyde amounted to £400 million annually. Of this, around one twentieth (£20 million per year), was attributable to direct NHS costs. CONCLUSIONS: Road casualties remain a major public health threat in Strathclyde, and contribute to health inequalities. RTC costs to society amount to almost a tenth of NHS revenue expenditure. Cost-effective road safety measures should be deployed more widely.

Improving Scotland's health: time for a fresh approach?

Scotland's health remains the worst in the UK. There are several probable reasons for this. Of those that are amenable to change, health improvement policy has been excessively preoccupied with targeting individuals perceived to be 'at risk' rather than adopting a whole population perspective. Environmental as opposed to behavioural approaches to health improvement have been relatively neglected. To meet the challenge of Scotland's poor health more effectively in the future, new strategic thinking is necessary. Three initial steps are required: recognize that current approaches are inadequate and that fresh ideas are needed; identify the principles that should underlie future strategy development; translate these principles into achievable operational objectives. Five principles of a revitalized strategy to improve the health of Scotland in the future are proposed. These are start early and sustain effort; create a healthy and safe environment; reduce geographical as well as social inequalities in health; adopt an evidence-based approach to public health interventions; use epidemiology to assess need, plan interventions and monitor progress. These principles may then be translated into achievable operational policy and practice objectives.

Discovery of a novel B-Raf fusion protein related to c-Met drug resistance.

In recent years, there have been notable advances with the development of anticancer drugs including those targeting protein tyrosine kinases such as the c-Met receptor, which has been implicated in the development and progression of several cancers. However, despite such progress, drug resistance continues to be the single most important cause of cancer treatment failure, and understanding the mechanisms of drug resistance remains a major hurdle in treating patients with recurrent disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that potently inhibits c-Met-driven processes such as cell growth (proliferation and survival), motility, invasion, and morphology of a variety of tumor cells. Resistance to PF-04217903 was observed in GTL-16, a gastric carcinoma cell line with a constitutively activated c-Met receptor. In this report, mass spectrometry (MS) based quantitative phosphoproteomic analysis was used to determine changes in signaling pathways in the parental cells in response to c-Met inhibition and to investigate the changes in protein levels and related canonical pathways in both parental and PF-04217903 resistant (R3) clones in response to c-Met inhibition. The quantitative MS workflow included phosphoprotein enrichment of cell lysates from six treatment conditions: in-solution digestion, chemical labeling of peptides with a set of 6-plex isobaric tandem mass tags (TMT), HILIC fractionation, phosphopeptide enrichment, and nano LC-MS/MS on a LTQ-Orbitrap mass spectrometer. An investigation of these quantitative datasets using Ingenuity Pathways Analysis (IPA) revealed pathway changes in the various treatments that were consistent with previously observed transcriptomic and phenotypic changes. Proteomic analysis also revealed an increase in B-Raf expression in R3 clones. Expression profiling confirmed that B-Raf gene copy number was up-regulated and also indicated the presence of a mutated form of B-Raf. Using a bottom-up MS approach, SND-1 was identified as the B-Raf fusion partner. The discovery of this novel B-Raf fusion protein presents a novel target with potential clinical implications in the treatment of patients resistant to c-Met inhibitors.

Next generation Fc scaffold for multispecific antibodies.

Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3' interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies.

Steady-state kinetic and inhibition studies of the mammalian target of rapamycin (mTOR) kinase domain and mTOR complexes.

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase and a major controller of cell growth. In cells, mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2. The mTORC1 complex can phosphorylate 4EBP1 and S6K1, two key regulators of translation initiation, whereas mTORC2 phosphorylates AKT1, an event required for AKT1 activation. Here, we expressed and purified human mTORC1 and mTORC2 from HEK-293 cells using FLAG-M2 affinity chromatography. Western blotting analysis using phospho-specific antibodies indicated that recombinant mTORC1 and mTORC2 exhibit distinct substrate preferences in vitro, consistent with their roles in cells. To improve our understanding of the enzymatic properties of mTOR alone and mTOR in its complex form, steady-state kinetic profiles of truncated mTOR containing the kinase domain (residues 1360-2549) and mTORC1 were determined. The results revealed that mTORC1 is catalytically less active than truncated mTOR, as evidenced by 4.7- and 3.1-fold decreases in catalytic efficiency, k(cat)/K(m), for ATP and 4EBP1, respectively. We also found that truncated mTOR undergoes autophosphorylation through an intramolecular mechanism. Mass spectrometric analysis identified two novel mTOR autophosphorylation sites, Ser2454 and either Thr2473 or Thr2474, in addition to the previously reported Ser2481 site. Truncated mTOR and mTORC1 were completely inhibited by ATP competitive inhibitors PI103 and BEZ235 and partially inhibited by rapamycin/FKBP12 in a noncompetitive fashion toward ATP. All inhibitors tested exhibited similar inhibitory potencies between mTORC1 and truncated mTOR containing the kinase domain. Our studies presented here provide the first detailed kinetic studies of a recombinant mTOR complex.

Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network: an initial study in GSC11 glioblastoma stem cells.

Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types. Recently, a novel autocrine loop (IL-6/STAT3/HIF1alpha) has been observed in multiple tumor types (pancreatic, prostate, lung, and colon). The objective of this study was to probe perturbations of this loop in a glioblastoma cancer stem cell line (GSC11) derived from a human tumor by use of a JAK2/STAT3 phosphorylation inhibitor (WP1193), IL-6 stimulation, and hypoxia. A quantitative phosphoproteomic approach that employed phosphoprotein enrichment, chemical tagging with isobaric tags, phosphopeptide enrichment, and tandem mass spectrometry in a high-resolution instrument was applied. A total of 3414 proteins were identified in this study. A rapid Western blotting technique (<1 h) was used to confirm alterations in key protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 10% of the phosphoproteins were linked to the IL-6 pathway, and the majority of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed expected changes and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.

Injury prevention: a strategic priority for environmental health?

Injury results from the acute transfer of energy (or the acute lack of a vital element) from the environment to human tissue. It is thus, ipso facto, an 'environmental health' issue par excellence. This paper argues that injury consequently deserves consideration as a major strategic priority by environmental health professionals. Two international agreements concerning children's health and the environment have major implications for safety. The Children's Environmental Health Action Plan for Europe (CEHAPE) and the European Environmental Health Strategy make reference to the need for improved evidence and greater co-operation between the environmental and health sectors. CEHAPE is particularly relevant to safety as it focuses on four regional priority goals, the second of which refers to the prevention and reduction of health consequences from injuries by promoting safe, secure and supportive human settlements for all children. The natural strategic 'home' for injury prevention may therefore lie within environmental health, a domain from which it has generally been excluded. In support of this assertion, Scotland's recent policy initiative on the environment and human health 'Good Places, Better Health' is cited, where injury in children up to 8 years of age is one of four child health priorities being tackled during its initial implementation. An important test of the initiative may be its capacity to inform policy, practice and research in the field of injury prevention and safety promotion. If successful, it will help to validate the environmental health approach to a field that remains relatively neglected by public agencies, policy makers, practitioners and researchers.

An evaluation of police reporting of road casualties.

BACKGROUND: Under-reporting of road traffic casualties in police records has been well documented. OBJECTIVES: To investigate the extent and nature of possible under-reporting of road traffic casualties in the West of Scotland. DESIGN: A linked database comprising both police data (STATS19) and hospital in-patient records (SMR01) was created. The study period was 1997-2005 inclusive. Contrasting the number of SMR01-identified road casualties that were also recorded ("linked") in STATS19 records with those that were not ("unlinked") gives an indication of the extent and types of under-reporting of hospitalized road casualties by the police. RESULTS: 45% of hospital admissions due to road casualties were not reported to (or recorded by) the police. The STATS19 "slight casualties" that were linked to the SMR01 data was the only category that showed an increase in numbers (+4%) over the study period, whereas the numbers of STATS19 KSI (killed or seriously injured--combining fatal and serious casualties) decreased substantially (-38%). Pedal cyclists and motorcyclists were most likely to be missed by police recording. No third-party involvement, older casualties, females, length of stay in hospital (day cases), and earlier year of crash were also significantly associated with under-reporting. CONCLUSIONS: A general decline in the completeness of STATS19 is unlikely to have occurred, but there may have been an increasing tendency over time for police officers to report injuries as slight rather than serious. To improve the quality of this information, routine linkage of road casualty data derived from police and hospitalization databases should be considered.

The Yorkhill CHIRPP story: a qualitative evaluation of 10 years of injury surveillance at a Scottish children's hospital.

BACKGROUND: The Canadian Hospitals Injury Reporting and Prevention Program (CHIRPP) is an emergency department-based injury surveillance system that was devised in Canada and has been in operation since 1990. CHIRPP was imported to Glasgow's Royal Hospital for Sick Children at Yorkhill in 1996 and ran for 10 years. OBJECTIVE: To critically review CHIRPP at Yorkhill (Y-CHIRPP). The following two key questions were posed. (1) Did Y-CHIRPP fail, and, if so, why? (2) What generalisable lessons can be learned about injury surveillance? METHODS: A retrospective qualitative review of Y-CHIRPP was carried out. In gathering information, the aims were to: (a) describe the processes involved in running Y-CHIRPP; (b) identify changes made to that process over the 10 years; (c) determine the strengths and weaknesses of Y-CHIRPP. RESULTS: Taken together, and with reference to the WHO attributes of a good surveillance system, the findings suggest that Y-CHIRPP largely met the criteria of simplicity, flexibility, and acceptability. Criteria that were not, or only intermittently, met were reliability, utility, sustainability, and timeliness. CONCLUSIONS: Y-CHIRPP was, at best, a partial success. To maintain the viability of an injury surveillance system and to secure the long-term support of hospital staff, it is important that the system is perceived as an injury prevention service tool and not a research method. Experience with Y-CHIRPP suggests that injury surveillance requires three supporting posts: an emergency department staff member, a data analyst, and someone with responsibility for developing and/or lobbying for the implementation of preventive measures.

Discovery, characterization, and remediation of a C-terminal Fc-extension in proteins expressed in CHO cells.

Protein-based biotherapeutics are produced in engineered cells through complex processes and may contain a wide variety of variants and post-translational modifications that must be monitored or controlled to ensure product quality. Recently, a low level (~1-5%) impurity was observed in a number of proteins derived from stably transfected Chinese hamster ovary (CHO) cells using mass spectrometry. These molecules include antibodies and Fc fusion proteins where Fc is on the C-terminus of the construct. By liquid chromatography-mass spectrometry (LC-MS), the impurity was found to be ~1177 Da larger than the expected mass. After tryptic digestion and analysis by LC-MS/MS, the impurity was localized to the C-terminus of Fc in the form of an Fc sequence extension. Targeted higher-energy collision dissociation was performed using various normalized collision energies (NCE) on two charge states of the extended peptide, resulting in nearly complete fragment ion coverage. The amino acid sequence, SLSLSPEAEAASASELFQ, obtained by the de novo sequencing effort matches a portion of the vector sequence used in the transfection of the CHO cells, specifically in the promoter region of the selection cassette downstream of the protein coding sequence. The modification was the result of an unexpected splicing event, caused by the resemblance of the commonly used GGU codon of the C-terminal glycine to a consensus splicing donor. Three alternative codons for glycine were tested to alleviate the modification, and all were found to completely eliminate the undesirable C-terminal extension, thus improving product quality.

Anti-obesity effects of GIPR antagonists alone and in combination with GLP-1R agonists in preclinical models.

Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic ß-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.

High-resolution mass spectrometry confirms the presence of a hydroxyproline (Hyp) post-translational modification in the GGGGP linker of an Fc-fusion protein.

Flexible and protease resistant (G4S)n linkers are used extensively in protein engineering to connect various protein domains. Recently, several groups have observed xylose-based O-glycosylation at linker Ser residues that yield unwanted heterogeneity and may affect product quality. Because of this, an engineering effort was implemented to explore different linker sequence constructs. Here, we demonstrate the presence of an unexpected hydroxylation of a prolyl residue in the linker, made possible through the use of high-resolution mass spectrometry (HR-MS) and MSn. The discovery started with the detection of a poorly resolved ∼+17 Da mass addition at the reduced protein chain level of an Fc-fusion construct by liquid chromatography-MS. Upon further investigation at the peptide level using HR-MS, the mass increase was determined to be +15.99 Da and was localized to the linker peptide SLSLSPGGGGGPAR [210-223]. This peptide corresponds to the C-terminus of Fc [210-216], the G4P linker [217-221], and first 2 amino acids of a growth factor [222-223]. The linker peptide was first subjected to MS2 with collision-induced dissociation (CID) activation. The fragmentation profile localized the modification to the GGGPA [218-222] portion of the peptide. Accurate mass measurement indicated that the modification is an addition of an oxygen and cannot be CH4, thus eliminating several possibilities such as Pro→Leu. However, other possibilities cannot be ruled out. Higher-energy collision-induced dissociation (HCD)-MS2 and MS3 using CID/CID were both unable to differentiate between Ala222→ Ser222 or Pro221→ Hyp221. Finally, MS3 using high-resolution CID/HCD confirmed the mass increase to be a Pro221→Hyp221 post-translational modification.

Protein-inhibitor complexes analyzed by alkaline capillary LC-MS.

Liquid chromatography-mass spectrometry (LC-MS) has been used extensively in determination of the molecular weights of proteins, as well as covalent protein-ligand complexes. We have successfully developed LC-MS method for protein molecular weight measurement using small-bore and capillary LC-MS under acidic and basic conditions. A high pH method was critical in studying complexes that were unstable under acidic conditions. Microgram sensitivity was achieved using both methods. A protocol to study the binding mode of protein-ligand complexes under denaturing conditions was developed. These methods were applied to CP88 (a proprietary cysteine protease) inhibitors and revealed different binding modes of inhibitors to proteins that had similar non-reversible behavior in biochemical activity assays. The method also confirmed that one inhibitor studied binds to CP88 in a reversible covalent manner.

Estimated prevalence of glaucomatous blindness in the Negev region of Israel.

An estimate of the prevalence of glaucomatous blindness in the Negev region of Israel was obtained by pooling two sources of available data: a state-run regional registry of blind people and the records of the glaucoma clinic of the Soroka Medical Centre, Beer Sheva. The denominator was the total population insured with the Kupat Holim (Sick Fund) of the Histadrut (General Federation of Labour). Glaucoma was the cause of blindness in only 10% of registered cases. Ninety-five individuals fulfilling the blindness criteria (3/60 or less, or a reduction of the visual field to 20 degrees or less in the better eye) were identified from both sources: this represents a total population prevalence of 39 per 100 000 population and 153 per 100 000 for those aged 41 and over. Glaucomatous blindness was more frequent in males than females, but the risk appeared to increase exponentially with age in both sexes. These data provide a previously lacking quantitative estimate of the prevalence of glaucomatous blindness in the Negev region.

Apolipoprotein J inhibits the migration and adhesion of endothelial cells.

BACKGROUND: Apolipoprotein J (ApoJ) is expressed after vascular injury and remodeling and may inhibit endothelial cell activation in the vascular wall. Recently, ApoJ was identified as upregulated in hyperplastic lesions after prosthetic arterial grafting. This study analyzed the effect of ApoJ on human umbilical vein endothelial cell (HUVEC) migration, adhesion, and proliferation. METHODS: Cell migration towards ApoJ + fetal bovine serum (FBS) or vascular endothelial growth factor (VEGF) was evaluated with the use of a microchemotaxis chamber with or without a fibronectin-coated membrane. For migration that involved fibronectin, cells were exposed to ApoJ before or after placement on the membrane. Cell adhesion to fibronectin was studied similarly but without stimulant. The vital dye alamar blue assessed proliferation of ApoJ + FBS- or VEGF-stimulated HUVECs. RESULTS: ApoJ alone did not cause migration or proliferation of HUVECs. Without fibronectin, ApoJ decreased the migration of HUVECs towards FBS or VEGF. When fibronectin was introduced, ApoJ decreased cell migration toward FBS or VEGF and decreased adhesion only when HUVECs in solution were exposed to ApoJ before the placement on fibronectin. ApoJ had no effect on FBS- or VEGF-induced proliferation. CONCLUSIONS: ApoJ inhibits HUVEC migration and adhesion. By altering endothelial function during vascular injury, ApoJ appears to regulate, in part, the early development of intimal hyperplasia after prosthetic arterial grafting.

Research on injury prevention: time for an international agenda?

OBJECTIVE: To propose an initial agenda for a systematic international research strategy designed to meet the information needs of injury prevention worldwide. CRITERIA FOR INCLUSION AND EXCLUSION OF ARTICLES: The world literature since 1977 was surveyed to obtain an overview of the current research effort on injury epidemiology and prevention. Articles were cited to illustrate the breadth and nature of work published on the topic with special reference to the prevention of home and traffic injuries. CONCLUSIONS: There has been a recent upsurge of interest in injury prevention, although much of the work has been descriptive rather than analytical or evaluative. The implementation of existing knowledge has been patchy. To meet the information needs of effective injury prevention, several elements of an international research agenda are proposed. These include: the achievement of a consensus on terminology, definition, and classification; clarification of the roles of social deprivation, gender, risk taking behaviour, personality, stress, alcohol, drugs, chronic illness, and disability in the aetiology of injury; the development of multi-agency models of good injury prevention practice; the evaluation of counter-measures; the development and evaluation of routine injury surveillance systems.

An approach to the analysis of health care needs and resources.

A semi-quantitative method of analysing the relationship between health care resources and need is described. It utilises the Donabedian model which expresses needs and resources in equivalent units in order to estimate the ratio of resources to need. The optimum resource/need ratio is regarded as that pertaining to the reference population; deviation from this optimum ratio in the subunits of the reference population is interpreted as a manifestation of inequitable resource distribution. An example is presented of the application of the method to the Greater Glasgow Health Board and its five constituent districts for the years 1974-77. It is argued that this method might, without undermining the principle of geographical equity, meet some of the objections to the more rigid 'formula' approach in the report of the Resource Allocation Working Party (RAWP) and in the Scottish Health Authorities Revenue Equalisation (SHARE) report.